Research ArticleProtective Effects of Hydrogen Sulfide in the Ageing Kidney
Cui-Lan Hou Ming-Jie Wang Chen Sun Yong Huang Sheng Jin Xue-PanMuYing Chen and Yi-Chun Zhu
Research Center on Aging and Medicine Fudan University Shanghai Key Laboratory of Bioactive Small MoleculesDepartment of Physiology and Pathophysiology Shanghai Medical College Fudan University Shanghai China
Correspondence should be addressed to Yi-Chun Zhu yczhushmueducn
Received 4 June 2016 Revised 22 September 2016 Accepted 3 October 2016
Academic Editor Claudio Cabello-Verrugio
Copyright copy 2016 Cui-Lan Hou et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Aims The study aimed to examine whether hydrogen sulfide (H2S) generation changed in the kidney of the ageing mouse and
its relationship with impaired kidney function Results H2S levels in the plasma urine and kidney decreased significantly in
ageing mice The expression of two known H2S-producing enzymes in kidney cystathionine 120574-lyase (CSE) and cystathionine-
120573-synthase (CBS) decreased significantly during ageing Chronic H2S donor (NaHS 50120583molkgday 10 weeks) treatment could
alleviate oxidative stress levels and renal tubular interstitial collagen depositionThese protective effects may relate to transcriptionfactor Nrf2 activation and antioxidant proteins such as HO-1 SIRT1 SOD1 and SOD2 expression upregulation in the ageing kidneyafter NaHS treatment Furthermore the expression of H
2S-producing enzymes changed with exogenous H
2S administration and
contributed to elevated H2S levels in the ageing kidney Conclusions Endogenous hydrogen sulfide production in the ageing kidney
is insufficient Exogenous H2S can partially rescue ageing-related kidney dysfunction by reducing oxidative stress decreasing
collagen deposition and enhancing Nrf2 nuclear translocation Recovery of endogenous hydrogen sulfide production may alsocontribute to the beneficial effects of NaHS treatment
1 Introduction
Population ageing is a global phenomenon and exerts heavydemands on the healthcare system and society The agedpopulation 65 years or older will reach 1 billion peopleaccounting for 13 of the total worldwide population in2030 [1] Ageing is a natural process accompanied by gradualdeclining in physiological functions Impaired renal functionin ageing people is of great clinical relevance and usuallyassociates with cardiovascular diseases and even mortalityThe characteristics of the ageing kidney include nephroscle-rosis nephron hypertrophy cortical volume reduction andcyst formation [2] On the other hand the high frequency ofunderlying diseases among ageing people such as concurrentdiabetes complicates the treatment of nephropathy There-fore understanding the process of kidney ageing might helpto improve the quality of life of the ageing population and toprovide precise treatment for senile nephropathy
Hydrogen sulfide (H2S) is a gasotransmitter generated
endogenously by cystathionine-120574-lyase (CSE) cystathionine-120573-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase
(3-MST) H2S has diverse physiological functions such as
relaxing blood vessels lowering blood pressure [3 4] anti-apoptosis [5] anti-inflammation [6] and antioxidative stress[7] In recent years emerging studies have focused on thepossibility of life span elongation by H
2S Miller and Roth
firstly reported the regulatory role of H2S (50 ppm) in C
elegans ageing [8] and Wei and Kenyon recently confirmedthis effect [9]
The beneficial effects of H2S on lifespan elongation
involve both direct and indirect mechanisms Some keyregulatory molecules such as Sirtuins [8] and Klotho [10]contribute to the direct effects of H
2S whereas the antioxida-
tive nature ofH2S protects the ageing heart or brain indirectly
[11 12] Organ-specific mechanisms are of great clinicalvalue for treating ageing-related diseases as well as pursuinghealthy ageing Our previous study showed that heart H
2S
levels in long-term fructose-fed ageing mice decreased from0020120583molg protein to 0013120583molg protein whichmay playsome roles in the pathogenesis of diabetic cardiomyopathy[13] The aim of this study was to investigate the endogenousproduction of H
2S in the ageing kidney and the effect of
Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 7570489 13 pageshttpdxdoiorg10115520167570489
2 Oxidative Medicine and Cellular Longevity
chronic H2S supplements in protecting the kidney from
ageing-related damage
2 Materials and Methods
21 Animals and NaHS Administration Eight-week-oldmaleC57BL6 mice were purchased from Department of Labora-tory Animal Science of Fudan University and raised undercontrolled conditions (22 plusmn 2∘C 45ndash55 relative humidityand 12 h dark-light cycle) with unrestricted access to diet andwater until 16 months of age (old group) Another group of8-week-old male C57BL6 mice were purchased and raiseduntil 3 months of age (young control group) We furtherdivided the old mice into four groups old control withnormal saline old with low-dose NaHS (10 120583molkgday)old withmedium-dose NaHS (50120583molkgday) and old withhigh-dose NaHS (100 120583molkgday) The treatment whichconsisted of intraperitoneal injection of NaHS or normalsaline once a day lasted for 10 weeks All animal studies wereapproved by the Ethics Committee of Experimental ResearchFudan University Shanghai Medical College
22 Metabolism and Biochemical Analyses Mice were placedinmetabolic cages (Tecniplast Italy) separately formetabolismevaluation After 3 daysrsquo acclimation 24-hour water andfood intake were measured and urine was collected Glucosestrips (OneTouch Johnson) were used to determine fastingplasma glucose with blood collected from the tail vein beforeeuthanasia Plasma was obtained by centrifuging a bloodsample at 3000 rpm 4∘C for 15 minutes (min) Plasma levelsof creatinine (Crea) blood urea nitrogen (BUN) total choles-terol (CHOL) triglycerides (TG) low-density lipoproteincholesterol (LDL-C) and high-density lipoprotein choles-terol (HDL-C) were determined by automatic biochemicalanalyser (Cobas 6000 Roche Basel Switzerland)
23 Detection of Reactive Oxygen Species (ROS) Levels ROSlevels in the kidney were measured using dihydroethidium(DHE) staining (Sigma-Aldrich) [14] Briefly DHE powderwas dissolved in dimethyl sulfoxide and further dilutedwith phosphate-buffered saline (PBS) at 55∘C until fullydissolution Mice were injected with DHE solution (100 120583L27mgkg)maintained at 37ndash40∘C and reinjected after 30minEighteen hours later mice were anesthetized and the kidneytissues were embedded in optimum cutting temperaturecompound (OCT) Mice kidney tissue sections (7120583m) wereobtained by using a frozen tissue slicer and observed under alaser confocal microscope (Zeiss LSM710) at the wavelengthof 488610 nm Florescence values were normalized to the oldgroups
24 Morphological and Histological Analyses The kidneytissues were excised fixed in 10 formalin and embedded inparaffin Kidney sections (4120583m) were stained with Massonrsquoshematoxylin and eosin (HE) and TUNEL stain according tothe manufacturerrsquos instructions Renal pathological changeswere observed under an optical microscope Apoptosis wasdetermined through TUNEL staining
25 Measurement of H2S Levels and the Activity of H2S-Producing Enzymes H
2S levels in plasma urine and kidney
tissues were determined as previously described [15] Theactivity of CSECBS in the kidney tissues was measuredby the method described by Tao et al [16] Briefly 260 120583Lhomogenized kidney tissues were incubated together with20120583L L-cysteine (10mmolL) and 20120583L pyridoxal-51015840 phos-phate (2mmolL) in an EP tube for 30min at 37∘C Then30 120583L supernatant obtained after centrifugation (10min at12000 rpm) was incubated with 80 120583L monobromobimane(MBB) for 40min on a shaker at room temperature Reactionwas terminated by adding 20 formic acid and tested by gaschromatography-mass spectrometry (GC-MS) It should beborne inmind that thismethod can only test the overall activ-ity of CSE and CBS because they share the same substrates
26 Enzymatic Activity Assay SIRT1 activity was determinedby using the SIRT1 Fluorometric Drug Discovery Kit (BML-AK555-0001 Enzo) according to the manufacturerrsquos proto-col [17] Briefly for cell-free measurement of the reactionbetween H
2S and recombinant SIRT1 10 120583L SIRT1 protein
(025U) and 5 120583L NaHS (0 125 25 50 and 100 120583molL)were incubated with 5 120583L substrate (025mmolL) and 5120583LNAD (025mmolL) plus 25 120583L assay buffer For tissue SIRT1activity measurement the reaction system contains 10120583Lkidney tissue homogenate 5 120583L substrate (025mmolL)and 5 120583L NAD (025mmolL) plus 30 120583L assay buffer Bothreactions were carried out at 37∘C for 40min and stopped byaddition of 1x Fluor de Lys Developer II plus nicotinamide(50 120583L per well) (every 1mL stop solution contains 760 120583Lassay buffer 40120583L 50mmolL nicotinamide and 200 120583L 5xDeveloper II) Fifteen mins later fluorescence values weremeasured on a fluorometric reader (SynergyMxUSA)withexcitation at 360 nm and emission at 460 nm
27 Western Blot Analysis The kidney cytoplasm nuclearproteins were collected under the kit protocol (Nuclear andCytoplasmic Protein Extraction Kit Beyotime Biotechnol-ogy Nanjing) and quantified using a BCA reagent (ShenNeng Bo Cai Corp Shanghai) The proteins were resolvedon a sodium dodecyl sulfate 10 polyacrylamide gel andtransferred onto polyvinylidene fluoride membrane (Mil-lipore Bedford MA USA) and incubated with primaryantibodies (1 1000 dilution) against Bcl-2 Bax CSE CBS3-MST SIRT1 (Santa Cruz CA USA) Collagen I (ColI) Collagen III (Col III) Fibronectin (FN) SOD1 SOD2(AbcamCompany USA) or Nrf2 HO-1 (Proteintech China)at 4∘C overnight The blots were washed with phosphatebuffer saline (TBST) for three times and then incubated withhorseradish peroxidase-conjugated secondary antibodies foranother 1 hour at room temperature After washing theblots were visualized by using chemiluminescent substrate(ECL) The densities of immunoblot bands were analysedusing a scanning densitometer (model GS-800 Bio-RadLaboratories Hercules CA USA) coupled with Bio-Radpersonal computer analysis software
28 Statistical Analysis Results are expressed as mean plusmnSEM Statistical analysis was performed using SPSS software
Oxidative Medicine and Cellular Longevity 3
Table 1 Results are means plusmn SE Old groups were treated with a variety of NaHS (0 10 50 and 100120583molkgday) treatments for 10 weekslowast119875 lt 005 and lowastlowast119875 lt 001 compared with old control (young group119873 = 14 old groups119873 = 18)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
P = 0039
0
2
4
6
8
10
Wat
er in
take
(mL
day)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(b)
P = 0002P = 0040
00
02
04
06
08
Urin
e vol
ume (
mL
day)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(c)
P = 0034P = 0041
0
500
1000
1500
2000
2500
Urin
e pro
tein
leve
l (m
gL)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(d)
Figure 1 Twenty-four-hour metabolic characteristics of young and old mice The old groups were treated with different doses of NaHS orsaline as the control for 10 weeks (a) Food intake (b) Water intake (c) Urine volume (d)The level of urinary protein Values are mean plusmn SE119875 lt 005 was considered significant (young groups119873 = 12 old groups119873 = 14)
version 210 (SPSS Inc Chicago IL USA) Comparisonsamong groups were performed by one-way ANOVA Paireddata were evaluated by two-tailed Studentrsquos 119905-test Statisticalsignificance was considered when 119875 lt 005
3 Results
31 Hydrogen Sulfide Donor NaHS Has a Protective Effect inAgeing Mice Metabolism There was no significant differencein fasting blood-glucose between the young and old groupsand NaHS (10 50 and 100 120583molkgday) treatment changedneither the fasting blood-glucose nor the body weight among
old groups (Table 1) Compared with young mice foodand water intake were decreased whereas the urine volumeincreased in old control mice Chronic NaHS treatmentfor 10 weeks did not change the food and water intake(Figures 1(a) and 1(b)) but could decrease the 24-hour urinevolume and the contents of urine protein (Figures 1(c) and1(d)) The maximum effect of NaHS in decreased urinevolumewas achieved in the 100 120583molkgdayNaHS treatmentgroup whereas the 50 120583molkgday NaHS treatment groupdecreased urine protein contents the most (Figures 1(c) and1(d)) The blood level of Crea and LDL-C did not change(Figures 2(a) and 2(e)) whereas the contents of BUN CHOL
4 Oxidative Medicine and Cellular Longevity
P = 0022
0
5
10
15Cr
ea le
vel (
mm
olL
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
P = 0033 P = 0045
0
5
10
15
BUN
leve
l (m
mol
L)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(b)
P = 0045P = 0015
00
05
10
15
20
CHO
L le
vel (
mm
olL
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(c)
P = 0042 P = 0002
00
02
04
06
08
10
TG le
vel (
mm
olL
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(d)
000
005
010
015
020
025
LDL-
C le
vel (
mm
olL
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(e)
P = 0047P = 0026
00
02
04
06
08
10
HD
L-C
leve
l (m
mol
L)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(f)
Figure 2 Blood biochemical results for young and old mice (a) Crea creatinine (b) BUN blood urea nitrogen (c) CHOL total cholesterol(d) TG triglycerides (e) LDL-C low-density lipoprotein (f)HDL-C high-density lipoprotein Values aremeanplusmn SE119875 lt 005was consideredsignificant (young groups119873 = 12 old groups119873 = 14)
TG and HDL-C increased dramatically in old control micecomparedwith youngmice (Figures 2(b) 2(c) 2(d) and 2(f))Ten weeks of 50120583molkgday NaHS treatment significantlyalleviated the increase of Crea BUN CHOL and HDL-C (Figures 2(a) 2(b) 2(c) and 2(f)) and 100 120583molkgdayNaHS therapy decreased the TG contents (Figure 2(d)) Ourresults indicate that there is impaired kidney function duringageing and exogenous H
2S treatment could partially reverse
such impairment
32 NaHS Alleviates the Level of Oxidative Stress in the AgeingKidney ROS levels in the kidney were examined after 10weeks of NaHS therapy DHE fluorescence intensity andmal-ondialdehyde (MDA) levels were elevated significantly in theold control group compared with young mice and chronicNaHS treatment could mitigate these changes (Figures 3(a)3(b) and 3(c)) Accordingly SOD activity and glutathioneperoxidase (GPx) levels were decreased in old mice and50 120583molkgday NaHS treatment could partially rescue these
Oxidative Medicine and Cellular Longevity 5
Young Old
200120583m200120583m200120583m
Old-50120583m NaHS
(a)
00
05
10
15
DH
E flu
ores
cenc
e (fo
lds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
P = 0042P = 0025
(b)
P lt 0001
P = 0030
P = 0004
00
05
10
15
MD
A le
vel (
fold
s of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(c)
P lt 0001
P = 0001
P = 0016
00
02
04
06
08
SOD
in k
idne
y (U
mg
prot
ein)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(d)
P = 0010 P = 0018
00
05
10
15
20
GPx
leve
l (fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(e)
Figure 3 H2S donor NaHS protected the ageing kidney from oxidative stress ((a) and (b)) DHE staining and the DHE fluorescence in the
kidney tissue (c) Malondialdehyde (MDA) levels in the kidney (d) Total SOD activity renal tissue (e) Glutathione peroxidase (GPx) levelsin the kidney Values are mean plusmn SE 119875 lt 005 was considered significant (young groups119873 = 6 old groups119873 = 8)
changes (Figures 3(d) and 3(e)) Our results indicated that theH2S donor could protect the ageing kidney from oxidative
stress
33 Ageing Mice Exhibit Kidney Remodeling and Apopto-sis and Chronic NaHS Treatment Mitigates These ProcessesMasson staining showed significantly increased interstitialfibrosis compared with young groups and 10 weeks of
50 120583molkgday NaHS treatment could partially reduce col-lagen deposition (Figures 4(a) and 4(b)) There was higherexpression of Col III and FN in the ageing kidney and thistrend became less significant with chronic NaHS treatment(Figures 4(d) and 4(e)) Col I expressionwas also increased inthe ageing kidney but with no significant difference betweenthe NaHS-treated and nontreated groups (Figure 4(c)) InadditionmRNA levels of Col I Col III and FN also increased
6 Oxidative Medicine and Cellular Longevity
Young
100120583m100120583m100120583m
Old-50120583m NaHSOld
(a)
Old Old-50YoungNaHS (120583molkgday)
00
05
10
15
Col
lage
n co
nten
t (fo
lds o
f old
cont
rol)
P = 0034P = 0024
(b)
GAPDH
Col I
Young Old-100Old-50Old-10OldNaHS (120583molkgday)
00
05
10
15
Col
IG
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P = 0025
(c)
GAPDH
Col III
00
05
10
15
Col
IIIG
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P = 0037
P = 0001 P = 0025
Young Old-100Old-50Old-10OldNaHS (120583molkgday)
(d)
GAPDH
FN
00
05
10
15
FNG
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P lt 0001P lt 0001
Young Old-100Old-50Old-10OldNaHS (120583molkgday)
(e)
Figure 4 Renal pathological changes of ageing mice ((a) and (b)) Masson staining of the kidney tissue (c) Col I and (d) Col III expressionin ageing mice (119873 = 8) (e) FN expression in ageing mice (119873 = 10) 119875 lt 005 was considered significant
Oxidative Medicine and Cellular Longevity 7
during ageing (Figure S1 in SupplementaryMaterial availableonline at httpdxdoiorg10115520167570489)
The ageing kidney appeared to have a higher apoptosislevel compared with that of the young group evidencedby increased expression of proapoptotic Bax and decreasedexpression of antiapoptotic Bcl-2 (Figure 5(b)) TUNELstaining revealed the same changes (Figure 5(a)) HE stainingshowed tubular dilation and inflammatory infiltration amongageing mice whereas chronic NaHS therapy could mitigatethis damage (Figure 5(c)) Our results indicated that kidneyremodeling and apoptosis had occurred in ageing mice andchronic NaHS treatment could mitigate these processes
34 Endogenous Hydrogen Sulfide Production Is Decreasedin Ageing Mice NaHS Treatment Alleviates the Reduction byIncreasing the Expression and Activity of Hydrogen Sulfide-Producing Enzymes Compared with young groups ageingmice showed lower CSE and CBS expression although3-MST expression remained unchanged Ten weeks ofNaHS treatments (50 120583molkgday) significantly increasedthe expression of CSE and CBS but not the 3-MST (Figures6(a1) 6(a2) and 6(a3)) Plasma urine and kidney H
2S levels
decreased significantly during ageing (Figures 6(b1) 6(b2)and 6(b3)) whereas 10 weeks of NaHS (50 120583molkgday)treatment alleviated the plasma and urine H
2S levels (Figures
6(b2) and 6(b3)) H2S levels in ageing kidney tissues were also
increased to some extent after NaHS treatment but withoutstatistical significance (Figure 6(b1)) CSECBS activity inthe ageing kidney decreased and 100 120583molkgday NaHStreatment diminished this reduction (Figure 6(c))
35 NaHSActivates SIRT1 in the Ageing Kidney To determinewhether H
2S protects the ageing kidney through the SIRT1-
mediated pathway the protein and transcriptional levelsof SIRT1 were examined Both the protein expression andmRNA transcription of SIRT1 were decreased in ageing micecompared with young ones and NaHS (50120583molkgday)treatment could improve SIRT1 protein expression but notthe mRNA levels (Figure 7(a)) SIRT1 is a deacetylase and10 weeks of NaHS treatment did not affect total deacetylaseactivity in the ageing kidney (Figure 7(b)) but 25120583molLNaHS could directly increase the activity of recombinantSIRT1 protein in vitro (Figure 7(c)) Measurement of SIRT1activity in the ageing kidney will help to make clear whetherchronic NaHS treatment selectively influences SIRT1 activitywhile total deacetylase activity remains the same
36 Effects of Chronic NaHS Treatment on the Expressionof Antioxidant-Related Proteins in the Ageing Kidney Theexpression of antioxidant proteins in kidney tissue wasexamined by western blot analysis at the end of chronicNaHS treatment Ten weeks of NaHS (50 120583molkgday) treat-ment could increase the Nrf2 expression and improve itsdownstream antioxidative proteins such as HO-1 SOD1 andSOD2 (Figure 8(a)) Moreover compared with young groupsboth the nuclear and cytosol Nrf2 levels were decreased andNaHS (50120583molkgday) treatment could selectively increasenuclear Nrf2 (Figure 8(b)) As shown in Figure S2A theNrf2 nuclear translation in the kidney tissue was insufficient
As shown in Figure S2B Nrf2 translocation in the NRK-52E cells treated with NaHS (50 120583molL) was significantlyinduced from cytosol to nucleus and peaked at 60min Thetranslocation ofNrf2 from cytoplasm into nuclearmay be oneof the protective mechanisms of H
2S against ageing-related
oxidative stress
4 Discussion
In this study we employed an ageing mouse model toinvestigate chronic NaHS treatment in the process of kidneysenescence Our work reveals two important findings (1)lower plasma urine and kidney H
2S levels and reduction of
kidneyCSE andCBS expression and activity are accompaniedwith ageing (2) exogenous administration of H
2S donor
NaHS mitigates ageing-related kidney dysfunction and theprotective effect of NaHS may at least partially relate toimproved endogenous H
2S production and its antioxidative
natureDuring ageing an elevated amount of ROS generated
from glycolysis specifically caused by the defects in thepolyol pathway uncoupling of nitric oxide synthase xanthineoxidase and advanced glycation results in the progressivedeterioration of renal function [18] Emerging data suggestthat ROS are related to the pathophysiology of glomerulardysfunction interstitial fibrosis and glomerulosclerosis [1920] In streptozotocin-induced diabetic rats H
2S therapy
(14 120583molkgday) improved renal function and decreasedglomerular basement thickening mesangial expansion andinterstitial fibrosis [18] Our previous study showed thatchronic NaHS treatment (30 60 and 120 120583molkgday) sig-nificantly reduced ROS levels in the kidney of GK rats [21]Consistently we found that chronic NaHS treatment couldreduce ROS levels (50 120583molkgday) and MDA contents (50and 100 120583molkgday) and increase GPx levels and SODactivity in the kidney of ageingmice H
2S takes part in a great
variety of physiological and pathophysiological processesbecause of its antiapoptotic antioxidative anti-inflammatoryand proangiogenic activities in mammals and the reduc-tion of endogenous H
2S levels has been related to various
diseases Zhou et al reported that endogenous H2S gen-
eration and CSE protein expression decreased significantlyin the streptozotocin-induced diabetic rat model and thatexogenous H
2S (14 120583molkgday) protected against diabetic
nephropathy [18] Our previous study also showed thatchronic NaHS (30 120583molkgday) treatment might amelioratediabetic complications of the kidney [21]
In this study we confirmed intensified interstitial fibrosisin the ageing kidney and observed decreased accumulation ofCol III (50 and 100 120583molkgday) and FN (at 50120583molkgday)with exogenous NaHSWe also observed increased apoptosisin the ageing kidney and chronic NaHS treatment could par-tially reverse this deterioration Age-related kidney damagemight reversely correlate with endogenous H
2S production
We found that both H2S levels in the plasma urine and
kidney and the kidney expression of CSE and CBS decreasedsignificantly with ageing As part of our bodyrsquos antioxidativedefense an insufficient H
2S system may decrease the overall
ability of ROS scavenging and render the kidney to be
8 Oxidative Medicine and Cellular Longevity
Young Old Old-50120583m NaHS
100120583m 100120583m 100120583m
(a)
GAPDH GAPDH
BAX Bcl-2
Young Old-50Old
00
05
10
15
BAX
GA
PDH
(fol
ds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
NaHS (120583molkgday)Young Old-50Old
NaHS (120583molkgday)
Old Old-50YoungNaHS (120583molkgday)
00
05
10
15
20
25
Bcl-2
GA
PDH
(fol
ds o
f old
cont
rol) P = 0001 P = 0029
P = 0034P = 0012
(b)
Young Old Old-50120583m NaHS
100120583m100120583m100120583m
(c)
Figure 5 Renal pathological changes of ageing mice (a) TUNEL staining of the kidney tissue (b) Bax (119873 = 6) and Bcl-2 (119873 = 7) expressionin ageing mice (c) HE staining of the kidney tissue Values are means plusmn SE 119875 lt 005 was considered significant
Oxidative Medicine and Cellular Longevity 9
GAPDH
GAPDH
CBS
CSE
Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old
GAPDH
MST
Young Old-100Old-50Old-10Old
NaHS (120583molkgday)P = 0008
P = 0029 P = 0028
P = 0020
P = 0048
P = 0007
P = 0029
P = 0001 P = 0005
P = 0043
P = 0014Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
(a1)
(a2)
(a3)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
Kidn
ey H
2S
(120583m
olg
)(fo
lds o
f old
cont
rol)
(b1)
(b2)
(b3)
(c)
00
05
10
15
Plas
ma H
2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
5
Urin
e H2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
(fold
s of o
ld co
ntro
l)CS
ECB
S ac
tivity
(120583m
olg
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
CSE
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
20
25
CBS
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
MST
GA
PDH
(fold
s of o
ld co
ntro
l)
Figure 6 Exogenous H2S alleviated ageing-related decreasing of H
2S production (a1)ndash(a3) Kidney expression of CSE (119873 = 11) and CBS
decreased significantly whereas 3-MST kept constant during ageing (119873 = 10) (b1)ndash(b3) Plasma urine and kidney tissueH2S levels decreased
significantly during ageing and rose again after chronic NaHS treatment (c) The activity of CSECBS in the ageing kidney declined whereas100 120583molkgday NaHS treatment could mitigate the reduction Values are means plusmn SE 119875 lt 005 was considered significant (119873 = 12)
10 Oxidative Medicine and Cellular Longevity
GAPDH
SIRT1
Young Old-100Old-50Old-10Old
P = 0007 P = 0015 P = 0023
NaHS (120583molkgday)
00
05
10
15
20
SIRT
1G
APD
H (f
olds
of o
ld co
ntro
l)
0
1
2
3
SIRT
1 m
RNA
(fol
ds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
P = 0049
00
05
10
15
Tota
l hist
one d
eace
tyla
se ac
tivity
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
P = 0001
P lt 0001
00
05
10
15
20
25
SIRT
1 ac
tivat
ion
in v
itro
(fold
s of c
ontro
l)
125 25 50 100ControlNaHS (120583molL)
(c)
Figure 7 SIRT1 was involved in NaHS-mediated ageing kidney protection (a) SIRT1 protein and mRNA expressions in the kidney afterchronic NaHS treatment for 10 weeks (119873 = 8) (b) Total deacetylase activity exchangeable in the kidney (119873 = 10) (c) NaHS-activatedrecombinant protein SIRT1 activity in vitro (119873 = 10) Values are mean plusmn SE 119875 lt 005 was considered significant
under oxidative stress Reasonably chronic NaHS treatmentprotects the ageing kidney by ROS scavenging and enhancingendogenous H
2S production in kidney tissue as reported
previously inmyocardium [22] DecreasedCBS activity in theageing kidney may also lead to homocysteine accumulationBecause the latter plays an important role in chronic renalfailure [23 24] further study is needed to explore whetherhomocysteine metabolism disorder is involved in ageing-related kidney damage and whether exogenous NaHS couldhelp restore the normal metabolic pathway
Christopher Hine reported that sulfur amino acid restric-tion could alter H
2S production and protect the liver from
ischemia-reperfusion injury [25] indicating the beneficialrole of endogenous H
2S SIRT1 which emerges as a major
life span regulator has been widely explored in the car-diovascular system and nervous system but it is rare in
the urinary system SIRT1 has robust biological effects andcan affect metabolic homeostasis and ageing Consistentwith previous studies [26 27] we found that both theexpression and activity of SIRT1 decreased rapidly in theageing kidney The reduction of NAD+ biosynthesis maybe responsible for reduced SIRT1 activity [26] Here wereport an interesting finding that a low concentration ofNaHS (25120583molL) could directly induce SIRT1 activationin a cell-free system whereas chronic treatment of NaHS(50 120583molkgday) selectively improves the expression but notthe activity of SIRT1 in the ageing kidney SIRT1 also regulateslipid metabolism by manipulating PPAR-120572 LXR FXR andSREBP signals [28 29] For example SIRT1 positively regu-lates LXR proteins which act as cholesterol sensor and adjustwhole body cholesterol and lipid homeostasis [30] Our bloodbiochemical tests showed that chronic NaHS treatment could
Oxidative Medicine and Cellular Longevity 11
Nrf2
GAPDH
GAPDH
SOD2
GAPDH
SOD1
GAPDH
HO-1
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday) NaHS (120583molkgday)
00
05
10
15
Nrf2
GA
PDH
(fol
ds o
f old
cont
rol)
00
05
10
15
20
HO
-1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
2G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
NS
GAPDH
Cyto Nrf2
Young Old-50OldNaHS (120583molkgday)
P lt 0001
00
05
10
15
20
Cyto
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
Nuclear Nrf2
Lamin B
Young Old-50Old
P = 0010 P = 0044
NaHS (120583molkgday)
00
05
10
15
20
Nuc
lear
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
Figure 8 The protective effects of the H2S donor on the expression of antioxidant-related proteins in the ageing kidney tissue (a) The
expression change of Nrf2 (119873 = 11) HO-1 (119873 = 10) SOD1 (119873 = 11) and SOD2 (119873 = 10) after 10 weeks of exogenous H2S donor (b) Nrf2
was translocated from cytosol to nucleus Lamin B was used as nuclear control and GAPDH was used as cytosol control (119873 = 6) Values aremean plusmn SE 119875 lt 005 was considered significant
12 Oxidative Medicine and Cellular Longevity
decrease CHOL and TG Therefore H2S may modulate liver
and renal lipidmetabolism through an enhanced SIRT1 signaland contribute to the prevention of renal disease progression
Nrf2 is an important antioxidative molecule that mod-ulates the expression of antioxidant genes [30] and thusprevents age-related oxidative stress [31 32] Here we showedthat the kidney Nrf2 expression decreased during age-ing and that chronic NaHS treatment could rescue Nrf2expression (10 and 50 120583molkgday) and enhance its nucleartranslocation (50120583molkgday) The precise mechanism ofexcessive oxidative stress in ageing is not clear but thedecreased expression of Nrf2antioxidant stress element-linked antioxidant genes is thought to play a role [32ndash34]We found that chronic NaHS treatment could activate Nrf2downstream genes such as HO-1 SOD1 and SOD2 whichconsequently enhanced resistance to oxidative stress in theageing kidney Taken together our data support the idea thatH2S plays a crucial role in protecting the ageing kidney from
antifibrosis and antiapoptosis through the regulation of redoxhomeostasis
5 Conclusion
In summary we demonstrated that 3-MST was expressed inthe ageing kidney and that endogenous H
2S levels decreased
because of the impaired expression ofH2S-producing enzymes
Chronic exogenous H2S treatment could protect the ageing
kidney by reducing oxidative stress decreasing collagendeposition and enhancing Nrf2 nuclear translocation aswell as increasing endogenous H
2S production
Competing Interests
The authors declare no conflict of interests regarding thepublication of this paper
Authorsrsquo ContributionsCui-Lan Hou and Ming-Jie Wang contributed equally to thiswork
AcknowledgmentsThe authors thank Bo Tan for his excellent technical assis-tance in H
2S measurements This present study was sup-
ported by the National Natural Science Foundation of China(NSFC) (no 81230003 to Yi-Chun Zhu no 81670248 toMing-Jie Wang and no 81402917 to Bo Tan) the Ministryof Education (20110071130009 to Yi-Chun Zhu) the Shang-hai Pujiang Program (15PJ1400700 to Ming-Jie Wang) theResearch Center on Ageing and Medicine Fudan University(13dz2260700 to Ming-Jie Wang) and Key Laboratory Pro-gramof the EducationCommission of ShanghaiMunicipality(ZDSYS14005 to Yi-Chun Zhu)
References
[1] J Volkert H SchulzMHarter OWlodarczyk and S AndreasldquoThe prevalence of mental disorders in older people inWesterncountriesmdasha meta-analysisrdquo Ageing Research Reviews vol 12no 1 pp 339ndash353 2013
[2] A Denic R J Glassock and A D Rule ldquoStructural andfunctional changes with the aging kidneyrdquo Advances in ChronicKidney Disease vol 23 no 1 pp 19ndash28 2016
[3] G Yang L Wu B Jiang et al ldquoH2S as a physiologic vasore-
laxant hypertension in mice with deletion of cystathionine 120574-lyaserdquo Science vol 322 no 5901 pp 587ndash590 2008
[4] Y Sun Y Huang R Zhang et al ldquoHydrogen sulfide upreg-ulates KATP channel expression in vascular smooth musclecells of spontaneously hypertensive ratsrdquo Journal of MolecularMedicine vol 93 no 4 pp 439ndash455 2015
[5] C Guo F Liang W Shah Masood and X Yan ldquoHydrogen sul-fide protected gastric epithelial cell from ischemiareperfusioninjury by Keap1 s-sulfhydration MAPK dependent anti-apoptosis and NF-120581B dependent anti-inflammation pathwayrdquoEuropean Journal of Pharmacology vol 725 no 1 pp 70ndash782014
[6] J Du Y Huang H Yan et al ldquoHydrogen sulfide suppresses oxi-dized low-density lipoprotein (Ox-LDL)-stimulated monocytechemoattractant protein 1 generation frommacrophages via thenuclear factor 120581b (NF-120581B) pathwayrdquo The Journal of BiologicalChemistry vol 289 no 14 pp 9741ndash9753 2014
[7] J Zheng T Zhao Y Yuan N Hu and X Tang ldquoHydrogensulfide (H
through oxidative stress and inflammatory response via Nrf2-NF-120581B pathwaysrdquo Chemico-Biological Interactions vol 242 no1 pp 353ndash362 2015
[8] D L Miller and M B Roth ldquoHydrogen sulfide increasesthermotolerance and lifespan in Caenorhabditis elegansrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 104 no 51 pp 20618ndash20622 2007
[9] Y Wei and C Kenyon ldquoRoles for ROS and hydrogen sulfidein the longevity response to germline loss in Caenorhabditiselegansrdquo Proceedings of the National Academy of Sciences vol113 no 20 pp e2832ndashe2841 2016
[10] Y Zhang Z-H Tang Z Ren et al ldquoHydrogen sulfide thenext potent preventive and therapeutic agent in aging and age-associated diseaserdquo Molecular and Cellular Biology vol 33 no6 pp 1104ndash1113 2013
[11] C Wei Y Zhao L Wang et al ldquoH2S restores the cardiopro-
tection from ischemic post-conditioning in isolated aged ratheartsrdquo Cell Biology International vol 39 no 10 pp 1173ndash11762015
[12] R E Sandu A Buga A T Balseanu M Moldovan and APopa-Wagner ldquoTwenty-four hours hypothermia has temporaryefficacy in reducing brain infarction and inflammation in agedratsrdquo Neurobiology of Aging vol 38 no 1 pp 127ndash140 2016
[13] S Jin S X Pu C L Hou et al ldquoCardiac H2S generation
is reduced in ageing diabetic micerdquo Oxidative Medicine andCellular Longevity vol 2015 Article ID 758358 14 pages 2015
[14] K L Quick and L L Dugan ldquoSuperoxide stress identifiesneurons at risk in a model of ataxia-telangiectasiardquo Annals ofNeurology vol 49 no 5 pp 627ndash635 2001
[15] X Shen C B Pattillo S Pardue S C Bir R Wang and C GKevil ldquoMeasurement of plasma hydrogen sulfide in vivo and invitrordquo Free Radical Biology andMedicine vol 50 no 9 pp 1021ndash1031 2011
[16] B-B Tao S-Y Liu C-C Zhang et al ldquoVEGFR2 functionsas an H
2S-targeting receptor protein kinase with its novel
Cys1045-Cys1024 disulfide bond serving as a specific molecularswitch for hydrogen sulfide actions in vascular endothelialcellsrdquo Antioxidants amp Redox Signaling vol 19 no 5 pp 448ndash464 2013
Oxidative Medicine and Cellular Longevity 13
[17] K J Bitterman R M Anderson H Y Cohen M Latorre-Esteves and D A Sinclair ldquoInhibition of silencing and accel-erated aging by nicotinamide a putative negative regulatorof yeast Sir2 and human SIRT1rdquo The Journal of BiologicalChemistry vol 277 no 47 pp 45099ndash45107 2002
[18] X Zhou Y Feng Z Zhan and J Chen ldquoHydrogen sulfidealleviates diabetic nephropathy in a streptozotocin-induceddiabetic ratmodelrdquoThe Journal of Biological Chemistry vol 289no 42 pp 28827ndash28834 2014
[19] P Wang C K Isaak Y L Siow and K O ldquoDownregulationof cystathionine 120573-synthase and cystathionine 120574-lyase expres-sion stimulates inflammation in kidney ischemiamdashreperfusioninjuryrdquo Physiological Reports vol 24 no 12 article e12251 2014
[20] F Yi and P-L Li ldquoMechanisms of homocysteine-inducedglomerular injury and sclerosisrdquo American Journal of Nephrol-ogy vol 28 no 2 pp 254ndash264 2008
[21] R Xue D-D Hao J-P Sun et al ldquoHydrogen sulfide treat-ment promotes glucose uptake by increasing insulin receptorsensitivity and ameliorates kidney lesions in type 2 diabetesrdquoAntioxidants amp Redox Signaling vol 19 no 1 pp 5ndash23 2013
[22] N Li M-J Wang S Jin et al ldquoThe H2S donor NaHS changes
the expression pattern ofH2S-producing enzymes aftermyocar-
dial infarctionrdquo Oxidative Medicine and Cellular Longevity vol2016 Article ID 6492469 11 pages 2016
[23] U Sen W E Rodriguez N Tyagi M Kumar S Kundu andS C Tyagi ldquoCiglitazone a PPAR120574 agonist ameliorates diabeticnephropathy in part through homocysteine clearancerdquo Ameri-can Journal of PhysiologymdashEndocrinology and Metabolism vol295 no 5 pp E1205ndashE1212 2008
[24] W E Rodriguez U Sen N Tyagi et al ldquoPPAR gammaagonist normalizes glomerular filtration rate tissue levels ofhomocysteine and attenuates endothelial-myocyte uncouplingin alloxan induced diabetic micerdquo International Journal ofBiological Sciences vol 4 no 4 pp 236ndash244 2008
[25] C Hine E Harputlugil Y Zhang et al ldquoEndogenous hydrogensulfide production is essential for dietary restriction benefitsrdquoCell vol 160 no 1-2 pp 132ndash144 2015
[26] S-I Imai ldquoDissecting systemic control ofmetabolism and agingin the NAD World the importance of SIRT1 and NAMPT-mediated NAD biosynthesisrdquo FEBS Letters vol 585 no 11 pp1657ndash1662 2011
[27] N Braidy G J Guillemin H Mansour T Chan-Ling APoljak and R Grant ldquoAge related changes inNAD+metabolismoxidative stress and sirt1 activity in wistar ratsrdquo PLoS ONE vol6 no 4 Article ID e19194 pp 1ndash18 2011
[28] A PurushothamT T SchugQXu S Surapureddi XGuo andX Li ldquoHepatocyte-specific deletion of SIRT1 alters fatty acidmetabolism and results in hepatic steatosis and inflammationrdquoCell Metabolism vol 9 no 4 pp 327ndash338 2009
[29] A K Walker F Yang K Jiang et al ldquoConserved roleof SIRT1 orthologs in fasting-dependent inhibition of thelipidcholesterol regulator SREBPrdquoGenes and Development vol24 no 13 pp 1403ndash1417 2010
[30] X Li S Zhang G Blander J G Tse M Krieger and L Guar-ente ldquoSIRT1 deacetylates and positively regulates the nuclearreceptor LXRrdquoMolecular Cell vol 28 no 1 pp 91ndash106 2007
[31] M M Sachdeva M Cano and J T Handa ldquoNrf2 signaling isimpaired in the aging RPE given an oxidative insultrdquo Experi-mental Eye Research vol 119 no 1 pp 111ndash114 2014
[32] A Loboda M Damulewicz E Pyza A Jozkowicz and JDulak ldquoRole of Nrf2HO-1 system in development oxidative
stress response and diseases an evolutionarily conservedmech-anismrdquo Cellular and Molecular Life Sciences vol 73 no 17 pp3221ndash3247 2016
[33] G Yang K Zhao Y Ju et al ldquoHydrogen sulfide protects againstcellular senescence via S-sulfhydration of Keap1 and activationof Nrf2rdquo Antioxidants amp Redox Signaling vol 18 no 15 pp1906ndash1919 2013
[34] A J Done M J Gage N C Nieto and T TraustadottirldquoExercise-induced Nrf2-signaling is impaired in agingrdquo FreeRadical Biology and Medicine vol 96 no 1 pp 130ndash138 2016
chronic H2S supplements in protecting the kidney from
ageing-related damage
2 Materials and Methods
21 Animals and NaHS Administration Eight-week-oldmaleC57BL6 mice were purchased from Department of Labora-tory Animal Science of Fudan University and raised undercontrolled conditions (22 plusmn 2∘C 45ndash55 relative humidityand 12 h dark-light cycle) with unrestricted access to diet andwater until 16 months of age (old group) Another group of8-week-old male C57BL6 mice were purchased and raiseduntil 3 months of age (young control group) We furtherdivided the old mice into four groups old control withnormal saline old with low-dose NaHS (10 120583molkgday)old withmedium-dose NaHS (50120583molkgday) and old withhigh-dose NaHS (100 120583molkgday) The treatment whichconsisted of intraperitoneal injection of NaHS or normalsaline once a day lasted for 10 weeks All animal studies wereapproved by the Ethics Committee of Experimental ResearchFudan University Shanghai Medical College
22 Metabolism and Biochemical Analyses Mice were placedinmetabolic cages (Tecniplast Italy) separately formetabolismevaluation After 3 daysrsquo acclimation 24-hour water andfood intake were measured and urine was collected Glucosestrips (OneTouch Johnson) were used to determine fastingplasma glucose with blood collected from the tail vein beforeeuthanasia Plasma was obtained by centrifuging a bloodsample at 3000 rpm 4∘C for 15 minutes (min) Plasma levelsof creatinine (Crea) blood urea nitrogen (BUN) total choles-terol (CHOL) triglycerides (TG) low-density lipoproteincholesterol (LDL-C) and high-density lipoprotein choles-terol (HDL-C) were determined by automatic biochemicalanalyser (Cobas 6000 Roche Basel Switzerland)
23 Detection of Reactive Oxygen Species (ROS) Levels ROSlevels in the kidney were measured using dihydroethidium(DHE) staining (Sigma-Aldrich) [14] Briefly DHE powderwas dissolved in dimethyl sulfoxide and further dilutedwith phosphate-buffered saline (PBS) at 55∘C until fullydissolution Mice were injected with DHE solution (100 120583L27mgkg)maintained at 37ndash40∘C and reinjected after 30minEighteen hours later mice were anesthetized and the kidneytissues were embedded in optimum cutting temperaturecompound (OCT) Mice kidney tissue sections (7120583m) wereobtained by using a frozen tissue slicer and observed under alaser confocal microscope (Zeiss LSM710) at the wavelengthof 488610 nm Florescence values were normalized to the oldgroups
24 Morphological and Histological Analyses The kidneytissues were excised fixed in 10 formalin and embedded inparaffin Kidney sections (4120583m) were stained with Massonrsquoshematoxylin and eosin (HE) and TUNEL stain according tothe manufacturerrsquos instructions Renal pathological changeswere observed under an optical microscope Apoptosis wasdetermined through TUNEL staining
25 Measurement of H2S Levels and the Activity of H2S-Producing Enzymes H
2S levels in plasma urine and kidney
tissues were determined as previously described [15] Theactivity of CSECBS in the kidney tissues was measuredby the method described by Tao et al [16] Briefly 260 120583Lhomogenized kidney tissues were incubated together with20120583L L-cysteine (10mmolL) and 20120583L pyridoxal-51015840 phos-phate (2mmolL) in an EP tube for 30min at 37∘C Then30 120583L supernatant obtained after centrifugation (10min at12000 rpm) was incubated with 80 120583L monobromobimane(MBB) for 40min on a shaker at room temperature Reactionwas terminated by adding 20 formic acid and tested by gaschromatography-mass spectrometry (GC-MS) It should beborne inmind that thismethod can only test the overall activ-ity of CSE and CBS because they share the same substrates
26 Enzymatic Activity Assay SIRT1 activity was determinedby using the SIRT1 Fluorometric Drug Discovery Kit (BML-AK555-0001 Enzo) according to the manufacturerrsquos proto-col [17] Briefly for cell-free measurement of the reactionbetween H
2S and recombinant SIRT1 10 120583L SIRT1 protein
(025U) and 5 120583L NaHS (0 125 25 50 and 100 120583molL)were incubated with 5 120583L substrate (025mmolL) and 5120583LNAD (025mmolL) plus 25 120583L assay buffer For tissue SIRT1activity measurement the reaction system contains 10120583Lkidney tissue homogenate 5 120583L substrate (025mmolL)and 5 120583L NAD (025mmolL) plus 30 120583L assay buffer Bothreactions were carried out at 37∘C for 40min and stopped byaddition of 1x Fluor de Lys Developer II plus nicotinamide(50 120583L per well) (every 1mL stop solution contains 760 120583Lassay buffer 40120583L 50mmolL nicotinamide and 200 120583L 5xDeveloper II) Fifteen mins later fluorescence values weremeasured on a fluorometric reader (SynergyMxUSA)withexcitation at 360 nm and emission at 460 nm
27 Western Blot Analysis The kidney cytoplasm nuclearproteins were collected under the kit protocol (Nuclear andCytoplasmic Protein Extraction Kit Beyotime Biotechnol-ogy Nanjing) and quantified using a BCA reagent (ShenNeng Bo Cai Corp Shanghai) The proteins were resolvedon a sodium dodecyl sulfate 10 polyacrylamide gel andtransferred onto polyvinylidene fluoride membrane (Mil-lipore Bedford MA USA) and incubated with primaryantibodies (1 1000 dilution) against Bcl-2 Bax CSE CBS3-MST SIRT1 (Santa Cruz CA USA) Collagen I (ColI) Collagen III (Col III) Fibronectin (FN) SOD1 SOD2(AbcamCompany USA) or Nrf2 HO-1 (Proteintech China)at 4∘C overnight The blots were washed with phosphatebuffer saline (TBST) for three times and then incubated withhorseradish peroxidase-conjugated secondary antibodies foranother 1 hour at room temperature After washing theblots were visualized by using chemiluminescent substrate(ECL) The densities of immunoblot bands were analysedusing a scanning densitometer (model GS-800 Bio-RadLaboratories Hercules CA USA) coupled with Bio-Radpersonal computer analysis software
28 Statistical Analysis Results are expressed as mean plusmnSEM Statistical analysis was performed using SPSS software
Oxidative Medicine and Cellular Longevity 3
Table 1 Results are means plusmn SE Old groups were treated with a variety of NaHS (0 10 50 and 100120583molkgday) treatments for 10 weekslowast119875 lt 005 and lowastlowast119875 lt 001 compared with old control (young group119873 = 14 old groups119873 = 18)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
P = 0039
0
2
4
6
8
10
Wat
er in
take
(mL
day)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(b)
P = 0002P = 0040
00
02
04
06
08
Urin
e vol
ume (
mL
day)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(c)
P = 0034P = 0041
0
500
1000
1500
2000
2500
Urin
e pro
tein
leve
l (m
gL)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(d)
Figure 1 Twenty-four-hour metabolic characteristics of young and old mice The old groups were treated with different doses of NaHS orsaline as the control for 10 weeks (a) Food intake (b) Water intake (c) Urine volume (d)The level of urinary protein Values are mean plusmn SE119875 lt 005 was considered significant (young groups119873 = 12 old groups119873 = 14)
version 210 (SPSS Inc Chicago IL USA) Comparisonsamong groups were performed by one-way ANOVA Paireddata were evaluated by two-tailed Studentrsquos 119905-test Statisticalsignificance was considered when 119875 lt 005
3 Results
31 Hydrogen Sulfide Donor NaHS Has a Protective Effect inAgeing Mice Metabolism There was no significant differencein fasting blood-glucose between the young and old groupsand NaHS (10 50 and 100 120583molkgday) treatment changedneither the fasting blood-glucose nor the body weight among
old groups (Table 1) Compared with young mice foodand water intake were decreased whereas the urine volumeincreased in old control mice Chronic NaHS treatmentfor 10 weeks did not change the food and water intake(Figures 1(a) and 1(b)) but could decrease the 24-hour urinevolume and the contents of urine protein (Figures 1(c) and1(d)) The maximum effect of NaHS in decreased urinevolumewas achieved in the 100 120583molkgdayNaHS treatmentgroup whereas the 50 120583molkgday NaHS treatment groupdecreased urine protein contents the most (Figures 1(c) and1(d)) The blood level of Crea and LDL-C did not change(Figures 2(a) and 2(e)) whereas the contents of BUN CHOL
4 Oxidative Medicine and Cellular Longevity
P = 0022
0
5
10
15Cr
ea le
vel (
mm
olL
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
P = 0033 P = 0045
0
5
10
15
BUN
leve
l (m
mol
L)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(b)
P = 0045P = 0015
00
05
10
15
20
CHO
L le
vel (
mm
olL
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(c)
P = 0042 P = 0002
00
02
04
06
08
10
TG le
vel (
mm
olL
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(d)
000
005
010
015
020
025
LDL-
C le
vel (
mm
olL
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(e)
P = 0047P = 0026
00
02
04
06
08
10
HD
L-C
leve
l (m
mol
L)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(f)
Figure 2 Blood biochemical results for young and old mice (a) Crea creatinine (b) BUN blood urea nitrogen (c) CHOL total cholesterol(d) TG triglycerides (e) LDL-C low-density lipoprotein (f)HDL-C high-density lipoprotein Values aremeanplusmn SE119875 lt 005was consideredsignificant (young groups119873 = 12 old groups119873 = 14)
TG and HDL-C increased dramatically in old control micecomparedwith youngmice (Figures 2(b) 2(c) 2(d) and 2(f))Ten weeks of 50120583molkgday NaHS treatment significantlyalleviated the increase of Crea BUN CHOL and HDL-C (Figures 2(a) 2(b) 2(c) and 2(f)) and 100 120583molkgdayNaHS therapy decreased the TG contents (Figure 2(d)) Ourresults indicate that there is impaired kidney function duringageing and exogenous H
2S treatment could partially reverse
such impairment
32 NaHS Alleviates the Level of Oxidative Stress in the AgeingKidney ROS levels in the kidney were examined after 10weeks of NaHS therapy DHE fluorescence intensity andmal-ondialdehyde (MDA) levels were elevated significantly in theold control group compared with young mice and chronicNaHS treatment could mitigate these changes (Figures 3(a)3(b) and 3(c)) Accordingly SOD activity and glutathioneperoxidase (GPx) levels were decreased in old mice and50 120583molkgday NaHS treatment could partially rescue these
Oxidative Medicine and Cellular Longevity 5
Young Old
200120583m200120583m200120583m
Old-50120583m NaHS
(a)
00
05
10
15
DH
E flu
ores
cenc
e (fo
lds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
P = 0042P = 0025
(b)
P lt 0001
P = 0030
P = 0004
00
05
10
15
MD
A le
vel (
fold
s of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(c)
P lt 0001
P = 0001
P = 0016
00
02
04
06
08
SOD
in k
idne
y (U
mg
prot
ein)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(d)
P = 0010 P = 0018
00
05
10
15
20
GPx
leve
l (fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(e)
Figure 3 H2S donor NaHS protected the ageing kidney from oxidative stress ((a) and (b)) DHE staining and the DHE fluorescence in the
kidney tissue (c) Malondialdehyde (MDA) levels in the kidney (d) Total SOD activity renal tissue (e) Glutathione peroxidase (GPx) levelsin the kidney Values are mean plusmn SE 119875 lt 005 was considered significant (young groups119873 = 6 old groups119873 = 8)
changes (Figures 3(d) and 3(e)) Our results indicated that theH2S donor could protect the ageing kidney from oxidative
stress
33 Ageing Mice Exhibit Kidney Remodeling and Apopto-sis and Chronic NaHS Treatment Mitigates These ProcessesMasson staining showed significantly increased interstitialfibrosis compared with young groups and 10 weeks of
50 120583molkgday NaHS treatment could partially reduce col-lagen deposition (Figures 4(a) and 4(b)) There was higherexpression of Col III and FN in the ageing kidney and thistrend became less significant with chronic NaHS treatment(Figures 4(d) and 4(e)) Col I expressionwas also increased inthe ageing kidney but with no significant difference betweenthe NaHS-treated and nontreated groups (Figure 4(c)) InadditionmRNA levels of Col I Col III and FN also increased
6 Oxidative Medicine and Cellular Longevity
Young
100120583m100120583m100120583m
Old-50120583m NaHSOld
(a)
Old Old-50YoungNaHS (120583molkgday)
00
05
10
15
Col
lage
n co
nten
t (fo
lds o
f old
cont
rol)
P = 0034P = 0024
(b)
GAPDH
Col I
Young Old-100Old-50Old-10OldNaHS (120583molkgday)
00
05
10
15
Col
IG
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P = 0025
(c)
GAPDH
Col III
00
05
10
15
Col
IIIG
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P = 0037
P = 0001 P = 0025
Young Old-100Old-50Old-10OldNaHS (120583molkgday)
(d)
GAPDH
FN
00
05
10
15
FNG
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P lt 0001P lt 0001
Young Old-100Old-50Old-10OldNaHS (120583molkgday)
(e)
Figure 4 Renal pathological changes of ageing mice ((a) and (b)) Masson staining of the kidney tissue (c) Col I and (d) Col III expressionin ageing mice (119873 = 8) (e) FN expression in ageing mice (119873 = 10) 119875 lt 005 was considered significant
Oxidative Medicine and Cellular Longevity 7
during ageing (Figure S1 in SupplementaryMaterial availableonline at httpdxdoiorg10115520167570489)
The ageing kidney appeared to have a higher apoptosislevel compared with that of the young group evidencedby increased expression of proapoptotic Bax and decreasedexpression of antiapoptotic Bcl-2 (Figure 5(b)) TUNELstaining revealed the same changes (Figure 5(a)) HE stainingshowed tubular dilation and inflammatory infiltration amongageing mice whereas chronic NaHS therapy could mitigatethis damage (Figure 5(c)) Our results indicated that kidneyremodeling and apoptosis had occurred in ageing mice andchronic NaHS treatment could mitigate these processes
34 Endogenous Hydrogen Sulfide Production Is Decreasedin Ageing Mice NaHS Treatment Alleviates the Reduction byIncreasing the Expression and Activity of Hydrogen Sulfide-Producing Enzymes Compared with young groups ageingmice showed lower CSE and CBS expression although3-MST expression remained unchanged Ten weeks ofNaHS treatments (50 120583molkgday) significantly increasedthe expression of CSE and CBS but not the 3-MST (Figures6(a1) 6(a2) and 6(a3)) Plasma urine and kidney H
2S levels
decreased significantly during ageing (Figures 6(b1) 6(b2)and 6(b3)) whereas 10 weeks of NaHS (50 120583molkgday)treatment alleviated the plasma and urine H
2S levels (Figures
6(b2) and 6(b3)) H2S levels in ageing kidney tissues were also
increased to some extent after NaHS treatment but withoutstatistical significance (Figure 6(b1)) CSECBS activity inthe ageing kidney decreased and 100 120583molkgday NaHStreatment diminished this reduction (Figure 6(c))
35 NaHSActivates SIRT1 in the Ageing Kidney To determinewhether H
2S protects the ageing kidney through the SIRT1-
mediated pathway the protein and transcriptional levelsof SIRT1 were examined Both the protein expression andmRNA transcription of SIRT1 were decreased in ageing micecompared with young ones and NaHS (50120583molkgday)treatment could improve SIRT1 protein expression but notthe mRNA levels (Figure 7(a)) SIRT1 is a deacetylase and10 weeks of NaHS treatment did not affect total deacetylaseactivity in the ageing kidney (Figure 7(b)) but 25120583molLNaHS could directly increase the activity of recombinantSIRT1 protein in vitro (Figure 7(c)) Measurement of SIRT1activity in the ageing kidney will help to make clear whetherchronic NaHS treatment selectively influences SIRT1 activitywhile total deacetylase activity remains the same
36 Effects of Chronic NaHS Treatment on the Expressionof Antioxidant-Related Proteins in the Ageing Kidney Theexpression of antioxidant proteins in kidney tissue wasexamined by western blot analysis at the end of chronicNaHS treatment Ten weeks of NaHS (50 120583molkgday) treat-ment could increase the Nrf2 expression and improve itsdownstream antioxidative proteins such as HO-1 SOD1 andSOD2 (Figure 8(a)) Moreover compared with young groupsboth the nuclear and cytosol Nrf2 levels were decreased andNaHS (50120583molkgday) treatment could selectively increasenuclear Nrf2 (Figure 8(b)) As shown in Figure S2A theNrf2 nuclear translation in the kidney tissue was insufficient
As shown in Figure S2B Nrf2 translocation in the NRK-52E cells treated with NaHS (50 120583molL) was significantlyinduced from cytosol to nucleus and peaked at 60min Thetranslocation ofNrf2 from cytoplasm into nuclearmay be oneof the protective mechanisms of H
2S against ageing-related
oxidative stress
4 Discussion
In this study we employed an ageing mouse model toinvestigate chronic NaHS treatment in the process of kidneysenescence Our work reveals two important findings (1)lower plasma urine and kidney H
2S levels and reduction of
kidneyCSE andCBS expression and activity are accompaniedwith ageing (2) exogenous administration of H
2S donor
NaHS mitigates ageing-related kidney dysfunction and theprotective effect of NaHS may at least partially relate toimproved endogenous H
2S production and its antioxidative
natureDuring ageing an elevated amount of ROS generated
from glycolysis specifically caused by the defects in thepolyol pathway uncoupling of nitric oxide synthase xanthineoxidase and advanced glycation results in the progressivedeterioration of renal function [18] Emerging data suggestthat ROS are related to the pathophysiology of glomerulardysfunction interstitial fibrosis and glomerulosclerosis [1920] In streptozotocin-induced diabetic rats H
2S therapy
(14 120583molkgday) improved renal function and decreasedglomerular basement thickening mesangial expansion andinterstitial fibrosis [18] Our previous study showed thatchronic NaHS treatment (30 60 and 120 120583molkgday) sig-nificantly reduced ROS levels in the kidney of GK rats [21]Consistently we found that chronic NaHS treatment couldreduce ROS levels (50 120583molkgday) and MDA contents (50and 100 120583molkgday) and increase GPx levels and SODactivity in the kidney of ageingmice H
2S takes part in a great
variety of physiological and pathophysiological processesbecause of its antiapoptotic antioxidative anti-inflammatoryand proangiogenic activities in mammals and the reduc-tion of endogenous H
2S levels has been related to various
diseases Zhou et al reported that endogenous H2S gen-
eration and CSE protein expression decreased significantlyin the streptozotocin-induced diabetic rat model and thatexogenous H
2S (14 120583molkgday) protected against diabetic
nephropathy [18] Our previous study also showed thatchronic NaHS (30 120583molkgday) treatment might amelioratediabetic complications of the kidney [21]
In this study we confirmed intensified interstitial fibrosisin the ageing kidney and observed decreased accumulation ofCol III (50 and 100 120583molkgday) and FN (at 50120583molkgday)with exogenous NaHSWe also observed increased apoptosisin the ageing kidney and chronic NaHS treatment could par-tially reverse this deterioration Age-related kidney damagemight reversely correlate with endogenous H
2S production
We found that both H2S levels in the plasma urine and
kidney and the kidney expression of CSE and CBS decreasedsignificantly with ageing As part of our bodyrsquos antioxidativedefense an insufficient H
2S system may decrease the overall
ability of ROS scavenging and render the kidney to be
8 Oxidative Medicine and Cellular Longevity
Young Old Old-50120583m NaHS
100120583m 100120583m 100120583m
(a)
GAPDH GAPDH
BAX Bcl-2
Young Old-50Old
00
05
10
15
BAX
GA
PDH
(fol
ds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
NaHS (120583molkgday)Young Old-50Old
NaHS (120583molkgday)
Old Old-50YoungNaHS (120583molkgday)
00
05
10
15
20
25
Bcl-2
GA
PDH
(fol
ds o
f old
cont
rol) P = 0001 P = 0029
P = 0034P = 0012
(b)
Young Old Old-50120583m NaHS
100120583m100120583m100120583m
(c)
Figure 5 Renal pathological changes of ageing mice (a) TUNEL staining of the kidney tissue (b) Bax (119873 = 6) and Bcl-2 (119873 = 7) expressionin ageing mice (c) HE staining of the kidney tissue Values are means plusmn SE 119875 lt 005 was considered significant
Oxidative Medicine and Cellular Longevity 9
GAPDH
GAPDH
CBS
CSE
Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old
GAPDH
MST
Young Old-100Old-50Old-10Old
NaHS (120583molkgday)P = 0008
P = 0029 P = 0028
P = 0020
P = 0048
P = 0007
P = 0029
P = 0001 P = 0005
P = 0043
P = 0014Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
(a1)
(a2)
(a3)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
Kidn
ey H
2S
(120583m
olg
)(fo
lds o
f old
cont
rol)
(b1)
(b2)
(b3)
(c)
00
05
10
15
Plas
ma H
2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
5
Urin
e H2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
(fold
s of o
ld co
ntro
l)CS
ECB
S ac
tivity
(120583m
olg
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
CSE
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
20
25
CBS
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
MST
GA
PDH
(fold
s of o
ld co
ntro
l)
Figure 6 Exogenous H2S alleviated ageing-related decreasing of H
2S production (a1)ndash(a3) Kidney expression of CSE (119873 = 11) and CBS
decreased significantly whereas 3-MST kept constant during ageing (119873 = 10) (b1)ndash(b3) Plasma urine and kidney tissueH2S levels decreased
significantly during ageing and rose again after chronic NaHS treatment (c) The activity of CSECBS in the ageing kidney declined whereas100 120583molkgday NaHS treatment could mitigate the reduction Values are means plusmn SE 119875 lt 005 was considered significant (119873 = 12)
10 Oxidative Medicine and Cellular Longevity
GAPDH
SIRT1
Young Old-100Old-50Old-10Old
P = 0007 P = 0015 P = 0023
NaHS (120583molkgday)
00
05
10
15
20
SIRT
1G
APD
H (f
olds
of o
ld co
ntro
l)
0
1
2
3
SIRT
1 m
RNA
(fol
ds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
P = 0049
00
05
10
15
Tota
l hist
one d
eace
tyla
se ac
tivity
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
P = 0001
P lt 0001
00
05
10
15
20
25
SIRT
1 ac
tivat
ion
in v
itro
(fold
s of c
ontro
l)
125 25 50 100ControlNaHS (120583molL)
(c)
Figure 7 SIRT1 was involved in NaHS-mediated ageing kidney protection (a) SIRT1 protein and mRNA expressions in the kidney afterchronic NaHS treatment for 10 weeks (119873 = 8) (b) Total deacetylase activity exchangeable in the kidney (119873 = 10) (c) NaHS-activatedrecombinant protein SIRT1 activity in vitro (119873 = 10) Values are mean plusmn SE 119875 lt 005 was considered significant
under oxidative stress Reasonably chronic NaHS treatmentprotects the ageing kidney by ROS scavenging and enhancingendogenous H
2S production in kidney tissue as reported
previously inmyocardium [22] DecreasedCBS activity in theageing kidney may also lead to homocysteine accumulationBecause the latter plays an important role in chronic renalfailure [23 24] further study is needed to explore whetherhomocysteine metabolism disorder is involved in ageing-related kidney damage and whether exogenous NaHS couldhelp restore the normal metabolic pathway
Christopher Hine reported that sulfur amino acid restric-tion could alter H
2S production and protect the liver from
ischemia-reperfusion injury [25] indicating the beneficialrole of endogenous H
2S SIRT1 which emerges as a major
life span regulator has been widely explored in the car-diovascular system and nervous system but it is rare in
the urinary system SIRT1 has robust biological effects andcan affect metabolic homeostasis and ageing Consistentwith previous studies [26 27] we found that both theexpression and activity of SIRT1 decreased rapidly in theageing kidney The reduction of NAD+ biosynthesis maybe responsible for reduced SIRT1 activity [26] Here wereport an interesting finding that a low concentration ofNaHS (25120583molL) could directly induce SIRT1 activationin a cell-free system whereas chronic treatment of NaHS(50 120583molkgday) selectively improves the expression but notthe activity of SIRT1 in the ageing kidney SIRT1 also regulateslipid metabolism by manipulating PPAR-120572 LXR FXR andSREBP signals [28 29] For example SIRT1 positively regu-lates LXR proteins which act as cholesterol sensor and adjustwhole body cholesterol and lipid homeostasis [30] Our bloodbiochemical tests showed that chronic NaHS treatment could
Oxidative Medicine and Cellular Longevity 11
Nrf2
GAPDH
GAPDH
SOD2
GAPDH
SOD1
GAPDH
HO-1
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday) NaHS (120583molkgday)
00
05
10
15
Nrf2
GA
PDH
(fol
ds o
f old
cont
rol)
00
05
10
15
20
HO
-1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
2G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
NS
GAPDH
Cyto Nrf2
Young Old-50OldNaHS (120583molkgday)
P lt 0001
00
05
10
15
20
Cyto
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
Nuclear Nrf2
Lamin B
Young Old-50Old
P = 0010 P = 0044
NaHS (120583molkgday)
00
05
10
15
20
Nuc
lear
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
Figure 8 The protective effects of the H2S donor on the expression of antioxidant-related proteins in the ageing kidney tissue (a) The
expression change of Nrf2 (119873 = 11) HO-1 (119873 = 10) SOD1 (119873 = 11) and SOD2 (119873 = 10) after 10 weeks of exogenous H2S donor (b) Nrf2
was translocated from cytosol to nucleus Lamin B was used as nuclear control and GAPDH was used as cytosol control (119873 = 6) Values aremean plusmn SE 119875 lt 005 was considered significant
12 Oxidative Medicine and Cellular Longevity
decrease CHOL and TG Therefore H2S may modulate liver
and renal lipidmetabolism through an enhanced SIRT1 signaland contribute to the prevention of renal disease progression
Nrf2 is an important antioxidative molecule that mod-ulates the expression of antioxidant genes [30] and thusprevents age-related oxidative stress [31 32] Here we showedthat the kidney Nrf2 expression decreased during age-ing and that chronic NaHS treatment could rescue Nrf2expression (10 and 50 120583molkgday) and enhance its nucleartranslocation (50120583molkgday) The precise mechanism ofexcessive oxidative stress in ageing is not clear but thedecreased expression of Nrf2antioxidant stress element-linked antioxidant genes is thought to play a role [32ndash34]We found that chronic NaHS treatment could activate Nrf2downstream genes such as HO-1 SOD1 and SOD2 whichconsequently enhanced resistance to oxidative stress in theageing kidney Taken together our data support the idea thatH2S plays a crucial role in protecting the ageing kidney from
antifibrosis and antiapoptosis through the regulation of redoxhomeostasis
5 Conclusion
In summary we demonstrated that 3-MST was expressed inthe ageing kidney and that endogenous H
2S levels decreased
because of the impaired expression ofH2S-producing enzymes
Chronic exogenous H2S treatment could protect the ageing
kidney by reducing oxidative stress decreasing collagendeposition and enhancing Nrf2 nuclear translocation aswell as increasing endogenous H
2S production
Competing Interests
The authors declare no conflict of interests regarding thepublication of this paper
Authorsrsquo ContributionsCui-Lan Hou and Ming-Jie Wang contributed equally to thiswork
AcknowledgmentsThe authors thank Bo Tan for his excellent technical assis-tance in H
2S measurements This present study was sup-
ported by the National Natural Science Foundation of China(NSFC) (no 81230003 to Yi-Chun Zhu no 81670248 toMing-Jie Wang and no 81402917 to Bo Tan) the Ministryof Education (20110071130009 to Yi-Chun Zhu) the Shang-hai Pujiang Program (15PJ1400700 to Ming-Jie Wang) theResearch Center on Ageing and Medicine Fudan University(13dz2260700 to Ming-Jie Wang) and Key Laboratory Pro-gramof the EducationCommission of ShanghaiMunicipality(ZDSYS14005 to Yi-Chun Zhu)
References
[1] J Volkert H SchulzMHarter OWlodarczyk and S AndreasldquoThe prevalence of mental disorders in older people inWesterncountriesmdasha meta-analysisrdquo Ageing Research Reviews vol 12no 1 pp 339ndash353 2013
[2] A Denic R J Glassock and A D Rule ldquoStructural andfunctional changes with the aging kidneyrdquo Advances in ChronicKidney Disease vol 23 no 1 pp 19ndash28 2016
[3] G Yang L Wu B Jiang et al ldquoH2S as a physiologic vasore-
laxant hypertension in mice with deletion of cystathionine 120574-lyaserdquo Science vol 322 no 5901 pp 587ndash590 2008
[4] Y Sun Y Huang R Zhang et al ldquoHydrogen sulfide upreg-ulates KATP channel expression in vascular smooth musclecells of spontaneously hypertensive ratsrdquo Journal of MolecularMedicine vol 93 no 4 pp 439ndash455 2015
[5] C Guo F Liang W Shah Masood and X Yan ldquoHydrogen sul-fide protected gastric epithelial cell from ischemiareperfusioninjury by Keap1 s-sulfhydration MAPK dependent anti-apoptosis and NF-120581B dependent anti-inflammation pathwayrdquoEuropean Journal of Pharmacology vol 725 no 1 pp 70ndash782014
[6] J Du Y Huang H Yan et al ldquoHydrogen sulfide suppresses oxi-dized low-density lipoprotein (Ox-LDL)-stimulated monocytechemoattractant protein 1 generation frommacrophages via thenuclear factor 120581b (NF-120581B) pathwayrdquo The Journal of BiologicalChemistry vol 289 no 14 pp 9741ndash9753 2014
[7] J Zheng T Zhao Y Yuan N Hu and X Tang ldquoHydrogensulfide (H
through oxidative stress and inflammatory response via Nrf2-NF-120581B pathwaysrdquo Chemico-Biological Interactions vol 242 no1 pp 353ndash362 2015
[8] D L Miller and M B Roth ldquoHydrogen sulfide increasesthermotolerance and lifespan in Caenorhabditis elegansrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 104 no 51 pp 20618ndash20622 2007
[9] Y Wei and C Kenyon ldquoRoles for ROS and hydrogen sulfidein the longevity response to germline loss in Caenorhabditiselegansrdquo Proceedings of the National Academy of Sciences vol113 no 20 pp e2832ndashe2841 2016
[10] Y Zhang Z-H Tang Z Ren et al ldquoHydrogen sulfide thenext potent preventive and therapeutic agent in aging and age-associated diseaserdquo Molecular and Cellular Biology vol 33 no6 pp 1104ndash1113 2013
[11] C Wei Y Zhao L Wang et al ldquoH2S restores the cardiopro-
tection from ischemic post-conditioning in isolated aged ratheartsrdquo Cell Biology International vol 39 no 10 pp 1173ndash11762015
[12] R E Sandu A Buga A T Balseanu M Moldovan and APopa-Wagner ldquoTwenty-four hours hypothermia has temporaryefficacy in reducing brain infarction and inflammation in agedratsrdquo Neurobiology of Aging vol 38 no 1 pp 127ndash140 2016
[13] S Jin S X Pu C L Hou et al ldquoCardiac H2S generation
is reduced in ageing diabetic micerdquo Oxidative Medicine andCellular Longevity vol 2015 Article ID 758358 14 pages 2015
[14] K L Quick and L L Dugan ldquoSuperoxide stress identifiesneurons at risk in a model of ataxia-telangiectasiardquo Annals ofNeurology vol 49 no 5 pp 627ndash635 2001
[15] X Shen C B Pattillo S Pardue S C Bir R Wang and C GKevil ldquoMeasurement of plasma hydrogen sulfide in vivo and invitrordquo Free Radical Biology andMedicine vol 50 no 9 pp 1021ndash1031 2011
[16] B-B Tao S-Y Liu C-C Zhang et al ldquoVEGFR2 functionsas an H
2S-targeting receptor protein kinase with its novel
Cys1045-Cys1024 disulfide bond serving as a specific molecularswitch for hydrogen sulfide actions in vascular endothelialcellsrdquo Antioxidants amp Redox Signaling vol 19 no 5 pp 448ndash464 2013
Oxidative Medicine and Cellular Longevity 13
[17] K J Bitterman R M Anderson H Y Cohen M Latorre-Esteves and D A Sinclair ldquoInhibition of silencing and accel-erated aging by nicotinamide a putative negative regulatorof yeast Sir2 and human SIRT1rdquo The Journal of BiologicalChemistry vol 277 no 47 pp 45099ndash45107 2002
[18] X Zhou Y Feng Z Zhan and J Chen ldquoHydrogen sulfidealleviates diabetic nephropathy in a streptozotocin-induceddiabetic ratmodelrdquoThe Journal of Biological Chemistry vol 289no 42 pp 28827ndash28834 2014
[19] P Wang C K Isaak Y L Siow and K O ldquoDownregulationof cystathionine 120573-synthase and cystathionine 120574-lyase expres-sion stimulates inflammation in kidney ischemiamdashreperfusioninjuryrdquo Physiological Reports vol 24 no 12 article e12251 2014
[20] F Yi and P-L Li ldquoMechanisms of homocysteine-inducedglomerular injury and sclerosisrdquo American Journal of Nephrol-ogy vol 28 no 2 pp 254ndash264 2008
[21] R Xue D-D Hao J-P Sun et al ldquoHydrogen sulfide treat-ment promotes glucose uptake by increasing insulin receptorsensitivity and ameliorates kidney lesions in type 2 diabetesrdquoAntioxidants amp Redox Signaling vol 19 no 1 pp 5ndash23 2013
[22] N Li M-J Wang S Jin et al ldquoThe H2S donor NaHS changes
the expression pattern ofH2S-producing enzymes aftermyocar-
dial infarctionrdquo Oxidative Medicine and Cellular Longevity vol2016 Article ID 6492469 11 pages 2016
[23] U Sen W E Rodriguez N Tyagi M Kumar S Kundu andS C Tyagi ldquoCiglitazone a PPAR120574 agonist ameliorates diabeticnephropathy in part through homocysteine clearancerdquo Ameri-can Journal of PhysiologymdashEndocrinology and Metabolism vol295 no 5 pp E1205ndashE1212 2008
[24] W E Rodriguez U Sen N Tyagi et al ldquoPPAR gammaagonist normalizes glomerular filtration rate tissue levels ofhomocysteine and attenuates endothelial-myocyte uncouplingin alloxan induced diabetic micerdquo International Journal ofBiological Sciences vol 4 no 4 pp 236ndash244 2008
[25] C Hine E Harputlugil Y Zhang et al ldquoEndogenous hydrogensulfide production is essential for dietary restriction benefitsrdquoCell vol 160 no 1-2 pp 132ndash144 2015
[26] S-I Imai ldquoDissecting systemic control ofmetabolism and agingin the NAD World the importance of SIRT1 and NAMPT-mediated NAD biosynthesisrdquo FEBS Letters vol 585 no 11 pp1657ndash1662 2011
[27] N Braidy G J Guillemin H Mansour T Chan-Ling APoljak and R Grant ldquoAge related changes inNAD+metabolismoxidative stress and sirt1 activity in wistar ratsrdquo PLoS ONE vol6 no 4 Article ID e19194 pp 1ndash18 2011
[28] A PurushothamT T SchugQXu S Surapureddi XGuo andX Li ldquoHepatocyte-specific deletion of SIRT1 alters fatty acidmetabolism and results in hepatic steatosis and inflammationrdquoCell Metabolism vol 9 no 4 pp 327ndash338 2009
[29] A K Walker F Yang K Jiang et al ldquoConserved roleof SIRT1 orthologs in fasting-dependent inhibition of thelipidcholesterol regulator SREBPrdquoGenes and Development vol24 no 13 pp 1403ndash1417 2010
[30] X Li S Zhang G Blander J G Tse M Krieger and L Guar-ente ldquoSIRT1 deacetylates and positively regulates the nuclearreceptor LXRrdquoMolecular Cell vol 28 no 1 pp 91ndash106 2007
[31] M M Sachdeva M Cano and J T Handa ldquoNrf2 signaling isimpaired in the aging RPE given an oxidative insultrdquo Experi-mental Eye Research vol 119 no 1 pp 111ndash114 2014
[32] A Loboda M Damulewicz E Pyza A Jozkowicz and JDulak ldquoRole of Nrf2HO-1 system in development oxidative
stress response and diseases an evolutionarily conservedmech-anismrdquo Cellular and Molecular Life Sciences vol 73 no 17 pp3221ndash3247 2016
[33] G Yang K Zhao Y Ju et al ldquoHydrogen sulfide protects againstcellular senescence via S-sulfhydration of Keap1 and activationof Nrf2rdquo Antioxidants amp Redox Signaling vol 18 no 15 pp1906ndash1919 2013
[34] A J Done M J Gage N C Nieto and T TraustadottirldquoExercise-induced Nrf2-signaling is impaired in agingrdquo FreeRadical Biology and Medicine vol 96 no 1 pp 130ndash138 2016
Table 1 Results are means plusmn SE Old groups were treated with a variety of NaHS (0 10 50 and 100120583molkgday) treatments for 10 weekslowast119875 lt 005 and lowastlowast119875 lt 001 compared with old control (young group119873 = 14 old groups119873 = 18)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
P = 0039
0
2
4
6
8
10
Wat
er in
take
(mL
day)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(b)
P = 0002P = 0040
00
02
04
06
08
Urin
e vol
ume (
mL
day)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(c)
P = 0034P = 0041
0
500
1000
1500
2000
2500
Urin
e pro
tein
leve
l (m
gL)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(d)
Figure 1 Twenty-four-hour metabolic characteristics of young and old mice The old groups were treated with different doses of NaHS orsaline as the control for 10 weeks (a) Food intake (b) Water intake (c) Urine volume (d)The level of urinary protein Values are mean plusmn SE119875 lt 005 was considered significant (young groups119873 = 12 old groups119873 = 14)
version 210 (SPSS Inc Chicago IL USA) Comparisonsamong groups were performed by one-way ANOVA Paireddata were evaluated by two-tailed Studentrsquos 119905-test Statisticalsignificance was considered when 119875 lt 005
3 Results
31 Hydrogen Sulfide Donor NaHS Has a Protective Effect inAgeing Mice Metabolism There was no significant differencein fasting blood-glucose between the young and old groupsand NaHS (10 50 and 100 120583molkgday) treatment changedneither the fasting blood-glucose nor the body weight among
old groups (Table 1) Compared with young mice foodand water intake were decreased whereas the urine volumeincreased in old control mice Chronic NaHS treatmentfor 10 weeks did not change the food and water intake(Figures 1(a) and 1(b)) but could decrease the 24-hour urinevolume and the contents of urine protein (Figures 1(c) and1(d)) The maximum effect of NaHS in decreased urinevolumewas achieved in the 100 120583molkgdayNaHS treatmentgroup whereas the 50 120583molkgday NaHS treatment groupdecreased urine protein contents the most (Figures 1(c) and1(d)) The blood level of Crea and LDL-C did not change(Figures 2(a) and 2(e)) whereas the contents of BUN CHOL
4 Oxidative Medicine and Cellular Longevity
P = 0022
0
5
10
15Cr
ea le
vel (
mm
olL
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
P = 0033 P = 0045
0
5
10
15
BUN
leve
l (m
mol
L)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(b)
P = 0045P = 0015
00
05
10
15
20
CHO
L le
vel (
mm
olL
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(c)
P = 0042 P = 0002
00
02
04
06
08
10
TG le
vel (
mm
olL
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(d)
000
005
010
015
020
025
LDL-
C le
vel (
mm
olL
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(e)
P = 0047P = 0026
00
02
04
06
08
10
HD
L-C
leve
l (m
mol
L)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(f)
Figure 2 Blood biochemical results for young and old mice (a) Crea creatinine (b) BUN blood urea nitrogen (c) CHOL total cholesterol(d) TG triglycerides (e) LDL-C low-density lipoprotein (f)HDL-C high-density lipoprotein Values aremeanplusmn SE119875 lt 005was consideredsignificant (young groups119873 = 12 old groups119873 = 14)
TG and HDL-C increased dramatically in old control micecomparedwith youngmice (Figures 2(b) 2(c) 2(d) and 2(f))Ten weeks of 50120583molkgday NaHS treatment significantlyalleviated the increase of Crea BUN CHOL and HDL-C (Figures 2(a) 2(b) 2(c) and 2(f)) and 100 120583molkgdayNaHS therapy decreased the TG contents (Figure 2(d)) Ourresults indicate that there is impaired kidney function duringageing and exogenous H
2S treatment could partially reverse
such impairment
32 NaHS Alleviates the Level of Oxidative Stress in the AgeingKidney ROS levels in the kidney were examined after 10weeks of NaHS therapy DHE fluorescence intensity andmal-ondialdehyde (MDA) levels were elevated significantly in theold control group compared with young mice and chronicNaHS treatment could mitigate these changes (Figures 3(a)3(b) and 3(c)) Accordingly SOD activity and glutathioneperoxidase (GPx) levels were decreased in old mice and50 120583molkgday NaHS treatment could partially rescue these
Oxidative Medicine and Cellular Longevity 5
Young Old
200120583m200120583m200120583m
Old-50120583m NaHS
(a)
00
05
10
15
DH
E flu
ores
cenc
e (fo
lds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
P = 0042P = 0025
(b)
P lt 0001
P = 0030
P = 0004
00
05
10
15
MD
A le
vel (
fold
s of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(c)
P lt 0001
P = 0001
P = 0016
00
02
04
06
08
SOD
in k
idne
y (U
mg
prot
ein)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(d)
P = 0010 P = 0018
00
05
10
15
20
GPx
leve
l (fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(e)
Figure 3 H2S donor NaHS protected the ageing kidney from oxidative stress ((a) and (b)) DHE staining and the DHE fluorescence in the
kidney tissue (c) Malondialdehyde (MDA) levels in the kidney (d) Total SOD activity renal tissue (e) Glutathione peroxidase (GPx) levelsin the kidney Values are mean plusmn SE 119875 lt 005 was considered significant (young groups119873 = 6 old groups119873 = 8)
changes (Figures 3(d) and 3(e)) Our results indicated that theH2S donor could protect the ageing kidney from oxidative
stress
33 Ageing Mice Exhibit Kidney Remodeling and Apopto-sis and Chronic NaHS Treatment Mitigates These ProcessesMasson staining showed significantly increased interstitialfibrosis compared with young groups and 10 weeks of
50 120583molkgday NaHS treatment could partially reduce col-lagen deposition (Figures 4(a) and 4(b)) There was higherexpression of Col III and FN in the ageing kidney and thistrend became less significant with chronic NaHS treatment(Figures 4(d) and 4(e)) Col I expressionwas also increased inthe ageing kidney but with no significant difference betweenthe NaHS-treated and nontreated groups (Figure 4(c)) InadditionmRNA levels of Col I Col III and FN also increased
6 Oxidative Medicine and Cellular Longevity
Young
100120583m100120583m100120583m
Old-50120583m NaHSOld
(a)
Old Old-50YoungNaHS (120583molkgday)
00
05
10
15
Col
lage
n co
nten
t (fo
lds o
f old
cont
rol)
P = 0034P = 0024
(b)
GAPDH
Col I
Young Old-100Old-50Old-10OldNaHS (120583molkgday)
00
05
10
15
Col
IG
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P = 0025
(c)
GAPDH
Col III
00
05
10
15
Col
IIIG
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P = 0037
P = 0001 P = 0025
Young Old-100Old-50Old-10OldNaHS (120583molkgday)
(d)
GAPDH
FN
00
05
10
15
FNG
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P lt 0001P lt 0001
Young Old-100Old-50Old-10OldNaHS (120583molkgday)
(e)
Figure 4 Renal pathological changes of ageing mice ((a) and (b)) Masson staining of the kidney tissue (c) Col I and (d) Col III expressionin ageing mice (119873 = 8) (e) FN expression in ageing mice (119873 = 10) 119875 lt 005 was considered significant
Oxidative Medicine and Cellular Longevity 7
during ageing (Figure S1 in SupplementaryMaterial availableonline at httpdxdoiorg10115520167570489)
The ageing kidney appeared to have a higher apoptosislevel compared with that of the young group evidencedby increased expression of proapoptotic Bax and decreasedexpression of antiapoptotic Bcl-2 (Figure 5(b)) TUNELstaining revealed the same changes (Figure 5(a)) HE stainingshowed tubular dilation and inflammatory infiltration amongageing mice whereas chronic NaHS therapy could mitigatethis damage (Figure 5(c)) Our results indicated that kidneyremodeling and apoptosis had occurred in ageing mice andchronic NaHS treatment could mitigate these processes
34 Endogenous Hydrogen Sulfide Production Is Decreasedin Ageing Mice NaHS Treatment Alleviates the Reduction byIncreasing the Expression and Activity of Hydrogen Sulfide-Producing Enzymes Compared with young groups ageingmice showed lower CSE and CBS expression although3-MST expression remained unchanged Ten weeks ofNaHS treatments (50 120583molkgday) significantly increasedthe expression of CSE and CBS but not the 3-MST (Figures6(a1) 6(a2) and 6(a3)) Plasma urine and kidney H
2S levels
decreased significantly during ageing (Figures 6(b1) 6(b2)and 6(b3)) whereas 10 weeks of NaHS (50 120583molkgday)treatment alleviated the plasma and urine H
2S levels (Figures
6(b2) and 6(b3)) H2S levels in ageing kidney tissues were also
increased to some extent after NaHS treatment but withoutstatistical significance (Figure 6(b1)) CSECBS activity inthe ageing kidney decreased and 100 120583molkgday NaHStreatment diminished this reduction (Figure 6(c))
35 NaHSActivates SIRT1 in the Ageing Kidney To determinewhether H
2S protects the ageing kidney through the SIRT1-
mediated pathway the protein and transcriptional levelsof SIRT1 were examined Both the protein expression andmRNA transcription of SIRT1 were decreased in ageing micecompared with young ones and NaHS (50120583molkgday)treatment could improve SIRT1 protein expression but notthe mRNA levels (Figure 7(a)) SIRT1 is a deacetylase and10 weeks of NaHS treatment did not affect total deacetylaseactivity in the ageing kidney (Figure 7(b)) but 25120583molLNaHS could directly increase the activity of recombinantSIRT1 protein in vitro (Figure 7(c)) Measurement of SIRT1activity in the ageing kidney will help to make clear whetherchronic NaHS treatment selectively influences SIRT1 activitywhile total deacetylase activity remains the same
36 Effects of Chronic NaHS Treatment on the Expressionof Antioxidant-Related Proteins in the Ageing Kidney Theexpression of antioxidant proteins in kidney tissue wasexamined by western blot analysis at the end of chronicNaHS treatment Ten weeks of NaHS (50 120583molkgday) treat-ment could increase the Nrf2 expression and improve itsdownstream antioxidative proteins such as HO-1 SOD1 andSOD2 (Figure 8(a)) Moreover compared with young groupsboth the nuclear and cytosol Nrf2 levels were decreased andNaHS (50120583molkgday) treatment could selectively increasenuclear Nrf2 (Figure 8(b)) As shown in Figure S2A theNrf2 nuclear translation in the kidney tissue was insufficient
As shown in Figure S2B Nrf2 translocation in the NRK-52E cells treated with NaHS (50 120583molL) was significantlyinduced from cytosol to nucleus and peaked at 60min Thetranslocation ofNrf2 from cytoplasm into nuclearmay be oneof the protective mechanisms of H
2S against ageing-related
oxidative stress
4 Discussion
In this study we employed an ageing mouse model toinvestigate chronic NaHS treatment in the process of kidneysenescence Our work reveals two important findings (1)lower plasma urine and kidney H
2S levels and reduction of
kidneyCSE andCBS expression and activity are accompaniedwith ageing (2) exogenous administration of H
2S donor
NaHS mitigates ageing-related kidney dysfunction and theprotective effect of NaHS may at least partially relate toimproved endogenous H
2S production and its antioxidative
natureDuring ageing an elevated amount of ROS generated
from glycolysis specifically caused by the defects in thepolyol pathway uncoupling of nitric oxide synthase xanthineoxidase and advanced glycation results in the progressivedeterioration of renal function [18] Emerging data suggestthat ROS are related to the pathophysiology of glomerulardysfunction interstitial fibrosis and glomerulosclerosis [1920] In streptozotocin-induced diabetic rats H
2S therapy
(14 120583molkgday) improved renal function and decreasedglomerular basement thickening mesangial expansion andinterstitial fibrosis [18] Our previous study showed thatchronic NaHS treatment (30 60 and 120 120583molkgday) sig-nificantly reduced ROS levels in the kidney of GK rats [21]Consistently we found that chronic NaHS treatment couldreduce ROS levels (50 120583molkgday) and MDA contents (50and 100 120583molkgday) and increase GPx levels and SODactivity in the kidney of ageingmice H
2S takes part in a great
variety of physiological and pathophysiological processesbecause of its antiapoptotic antioxidative anti-inflammatoryand proangiogenic activities in mammals and the reduc-tion of endogenous H
2S levels has been related to various
diseases Zhou et al reported that endogenous H2S gen-
eration and CSE protein expression decreased significantlyin the streptozotocin-induced diabetic rat model and thatexogenous H
2S (14 120583molkgday) protected against diabetic
nephropathy [18] Our previous study also showed thatchronic NaHS (30 120583molkgday) treatment might amelioratediabetic complications of the kidney [21]
In this study we confirmed intensified interstitial fibrosisin the ageing kidney and observed decreased accumulation ofCol III (50 and 100 120583molkgday) and FN (at 50120583molkgday)with exogenous NaHSWe also observed increased apoptosisin the ageing kidney and chronic NaHS treatment could par-tially reverse this deterioration Age-related kidney damagemight reversely correlate with endogenous H
2S production
We found that both H2S levels in the plasma urine and
kidney and the kidney expression of CSE and CBS decreasedsignificantly with ageing As part of our bodyrsquos antioxidativedefense an insufficient H
2S system may decrease the overall
ability of ROS scavenging and render the kidney to be
8 Oxidative Medicine and Cellular Longevity
Young Old Old-50120583m NaHS
100120583m 100120583m 100120583m
(a)
GAPDH GAPDH
BAX Bcl-2
Young Old-50Old
00
05
10
15
BAX
GA
PDH
(fol
ds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
NaHS (120583molkgday)Young Old-50Old
NaHS (120583molkgday)
Old Old-50YoungNaHS (120583molkgday)
00
05
10
15
20
25
Bcl-2
GA
PDH
(fol
ds o
f old
cont
rol) P = 0001 P = 0029
P = 0034P = 0012
(b)
Young Old Old-50120583m NaHS
100120583m100120583m100120583m
(c)
Figure 5 Renal pathological changes of ageing mice (a) TUNEL staining of the kidney tissue (b) Bax (119873 = 6) and Bcl-2 (119873 = 7) expressionin ageing mice (c) HE staining of the kidney tissue Values are means plusmn SE 119875 lt 005 was considered significant
Oxidative Medicine and Cellular Longevity 9
GAPDH
GAPDH
CBS
CSE
Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old
GAPDH
MST
Young Old-100Old-50Old-10Old
NaHS (120583molkgday)P = 0008
P = 0029 P = 0028
P = 0020
P = 0048
P = 0007
P = 0029
P = 0001 P = 0005
P = 0043
P = 0014Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
(a1)
(a2)
(a3)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
Kidn
ey H
2S
(120583m
olg
)(fo
lds o
f old
cont
rol)
(b1)
(b2)
(b3)
(c)
00
05
10
15
Plas
ma H
2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
5
Urin
e H2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
(fold
s of o
ld co
ntro
l)CS
ECB
S ac
tivity
(120583m
olg
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
CSE
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
20
25
CBS
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
MST
GA
PDH
(fold
s of o
ld co
ntro
l)
Figure 6 Exogenous H2S alleviated ageing-related decreasing of H
2S production (a1)ndash(a3) Kidney expression of CSE (119873 = 11) and CBS
decreased significantly whereas 3-MST kept constant during ageing (119873 = 10) (b1)ndash(b3) Plasma urine and kidney tissueH2S levels decreased
significantly during ageing and rose again after chronic NaHS treatment (c) The activity of CSECBS in the ageing kidney declined whereas100 120583molkgday NaHS treatment could mitigate the reduction Values are means plusmn SE 119875 lt 005 was considered significant (119873 = 12)
10 Oxidative Medicine and Cellular Longevity
GAPDH
SIRT1
Young Old-100Old-50Old-10Old
P = 0007 P = 0015 P = 0023
NaHS (120583molkgday)
00
05
10
15
20
SIRT
1G
APD
H (f
olds
of o
ld co
ntro
l)
0
1
2
3
SIRT
1 m
RNA
(fol
ds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
P = 0049
00
05
10
15
Tota
l hist
one d
eace
tyla
se ac
tivity
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
P = 0001
P lt 0001
00
05
10
15
20
25
SIRT
1 ac
tivat
ion
in v
itro
(fold
s of c
ontro
l)
125 25 50 100ControlNaHS (120583molL)
(c)
Figure 7 SIRT1 was involved in NaHS-mediated ageing kidney protection (a) SIRT1 protein and mRNA expressions in the kidney afterchronic NaHS treatment for 10 weeks (119873 = 8) (b) Total deacetylase activity exchangeable in the kidney (119873 = 10) (c) NaHS-activatedrecombinant protein SIRT1 activity in vitro (119873 = 10) Values are mean plusmn SE 119875 lt 005 was considered significant
under oxidative stress Reasonably chronic NaHS treatmentprotects the ageing kidney by ROS scavenging and enhancingendogenous H
2S production in kidney tissue as reported
previously inmyocardium [22] DecreasedCBS activity in theageing kidney may also lead to homocysteine accumulationBecause the latter plays an important role in chronic renalfailure [23 24] further study is needed to explore whetherhomocysteine metabolism disorder is involved in ageing-related kidney damage and whether exogenous NaHS couldhelp restore the normal metabolic pathway
Christopher Hine reported that sulfur amino acid restric-tion could alter H
2S production and protect the liver from
ischemia-reperfusion injury [25] indicating the beneficialrole of endogenous H
2S SIRT1 which emerges as a major
life span regulator has been widely explored in the car-diovascular system and nervous system but it is rare in
the urinary system SIRT1 has robust biological effects andcan affect metabolic homeostasis and ageing Consistentwith previous studies [26 27] we found that both theexpression and activity of SIRT1 decreased rapidly in theageing kidney The reduction of NAD+ biosynthesis maybe responsible for reduced SIRT1 activity [26] Here wereport an interesting finding that a low concentration ofNaHS (25120583molL) could directly induce SIRT1 activationin a cell-free system whereas chronic treatment of NaHS(50 120583molkgday) selectively improves the expression but notthe activity of SIRT1 in the ageing kidney SIRT1 also regulateslipid metabolism by manipulating PPAR-120572 LXR FXR andSREBP signals [28 29] For example SIRT1 positively regu-lates LXR proteins which act as cholesterol sensor and adjustwhole body cholesterol and lipid homeostasis [30] Our bloodbiochemical tests showed that chronic NaHS treatment could
Oxidative Medicine and Cellular Longevity 11
Nrf2
GAPDH
GAPDH
SOD2
GAPDH
SOD1
GAPDH
HO-1
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday) NaHS (120583molkgday)
00
05
10
15
Nrf2
GA
PDH
(fol
ds o
f old
cont
rol)
00
05
10
15
20
HO
-1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
2G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
NS
GAPDH
Cyto Nrf2
Young Old-50OldNaHS (120583molkgday)
P lt 0001
00
05
10
15
20
Cyto
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
Nuclear Nrf2
Lamin B
Young Old-50Old
P = 0010 P = 0044
NaHS (120583molkgday)
00
05
10
15
20
Nuc
lear
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
Figure 8 The protective effects of the H2S donor on the expression of antioxidant-related proteins in the ageing kidney tissue (a) The
expression change of Nrf2 (119873 = 11) HO-1 (119873 = 10) SOD1 (119873 = 11) and SOD2 (119873 = 10) after 10 weeks of exogenous H2S donor (b) Nrf2
was translocated from cytosol to nucleus Lamin B was used as nuclear control and GAPDH was used as cytosol control (119873 = 6) Values aremean plusmn SE 119875 lt 005 was considered significant
12 Oxidative Medicine and Cellular Longevity
decrease CHOL and TG Therefore H2S may modulate liver
and renal lipidmetabolism through an enhanced SIRT1 signaland contribute to the prevention of renal disease progression
Nrf2 is an important antioxidative molecule that mod-ulates the expression of antioxidant genes [30] and thusprevents age-related oxidative stress [31 32] Here we showedthat the kidney Nrf2 expression decreased during age-ing and that chronic NaHS treatment could rescue Nrf2expression (10 and 50 120583molkgday) and enhance its nucleartranslocation (50120583molkgday) The precise mechanism ofexcessive oxidative stress in ageing is not clear but thedecreased expression of Nrf2antioxidant stress element-linked antioxidant genes is thought to play a role [32ndash34]We found that chronic NaHS treatment could activate Nrf2downstream genes such as HO-1 SOD1 and SOD2 whichconsequently enhanced resistance to oxidative stress in theageing kidney Taken together our data support the idea thatH2S plays a crucial role in protecting the ageing kidney from
antifibrosis and antiapoptosis through the regulation of redoxhomeostasis
5 Conclusion
In summary we demonstrated that 3-MST was expressed inthe ageing kidney and that endogenous H
2S levels decreased
because of the impaired expression ofH2S-producing enzymes
Chronic exogenous H2S treatment could protect the ageing
kidney by reducing oxidative stress decreasing collagendeposition and enhancing Nrf2 nuclear translocation aswell as increasing endogenous H
2S production
Competing Interests
The authors declare no conflict of interests regarding thepublication of this paper
Authorsrsquo ContributionsCui-Lan Hou and Ming-Jie Wang contributed equally to thiswork
AcknowledgmentsThe authors thank Bo Tan for his excellent technical assis-tance in H
2S measurements This present study was sup-
ported by the National Natural Science Foundation of China(NSFC) (no 81230003 to Yi-Chun Zhu no 81670248 toMing-Jie Wang and no 81402917 to Bo Tan) the Ministryof Education (20110071130009 to Yi-Chun Zhu) the Shang-hai Pujiang Program (15PJ1400700 to Ming-Jie Wang) theResearch Center on Ageing and Medicine Fudan University(13dz2260700 to Ming-Jie Wang) and Key Laboratory Pro-gramof the EducationCommission of ShanghaiMunicipality(ZDSYS14005 to Yi-Chun Zhu)
References
[1] J Volkert H SchulzMHarter OWlodarczyk and S AndreasldquoThe prevalence of mental disorders in older people inWesterncountriesmdasha meta-analysisrdquo Ageing Research Reviews vol 12no 1 pp 339ndash353 2013
[2] A Denic R J Glassock and A D Rule ldquoStructural andfunctional changes with the aging kidneyrdquo Advances in ChronicKidney Disease vol 23 no 1 pp 19ndash28 2016
[3] G Yang L Wu B Jiang et al ldquoH2S as a physiologic vasore-
laxant hypertension in mice with deletion of cystathionine 120574-lyaserdquo Science vol 322 no 5901 pp 587ndash590 2008
[4] Y Sun Y Huang R Zhang et al ldquoHydrogen sulfide upreg-ulates KATP channel expression in vascular smooth musclecells of spontaneously hypertensive ratsrdquo Journal of MolecularMedicine vol 93 no 4 pp 439ndash455 2015
[5] C Guo F Liang W Shah Masood and X Yan ldquoHydrogen sul-fide protected gastric epithelial cell from ischemiareperfusioninjury by Keap1 s-sulfhydration MAPK dependent anti-apoptosis and NF-120581B dependent anti-inflammation pathwayrdquoEuropean Journal of Pharmacology vol 725 no 1 pp 70ndash782014
[6] J Du Y Huang H Yan et al ldquoHydrogen sulfide suppresses oxi-dized low-density lipoprotein (Ox-LDL)-stimulated monocytechemoattractant protein 1 generation frommacrophages via thenuclear factor 120581b (NF-120581B) pathwayrdquo The Journal of BiologicalChemistry vol 289 no 14 pp 9741ndash9753 2014
[7] J Zheng T Zhao Y Yuan N Hu and X Tang ldquoHydrogensulfide (H
through oxidative stress and inflammatory response via Nrf2-NF-120581B pathwaysrdquo Chemico-Biological Interactions vol 242 no1 pp 353ndash362 2015
[8] D L Miller and M B Roth ldquoHydrogen sulfide increasesthermotolerance and lifespan in Caenorhabditis elegansrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 104 no 51 pp 20618ndash20622 2007
[9] Y Wei and C Kenyon ldquoRoles for ROS and hydrogen sulfidein the longevity response to germline loss in Caenorhabditiselegansrdquo Proceedings of the National Academy of Sciences vol113 no 20 pp e2832ndashe2841 2016
[10] Y Zhang Z-H Tang Z Ren et al ldquoHydrogen sulfide thenext potent preventive and therapeutic agent in aging and age-associated diseaserdquo Molecular and Cellular Biology vol 33 no6 pp 1104ndash1113 2013
[11] C Wei Y Zhao L Wang et al ldquoH2S restores the cardiopro-
tection from ischemic post-conditioning in isolated aged ratheartsrdquo Cell Biology International vol 39 no 10 pp 1173ndash11762015
[12] R E Sandu A Buga A T Balseanu M Moldovan and APopa-Wagner ldquoTwenty-four hours hypothermia has temporaryefficacy in reducing brain infarction and inflammation in agedratsrdquo Neurobiology of Aging vol 38 no 1 pp 127ndash140 2016
[13] S Jin S X Pu C L Hou et al ldquoCardiac H2S generation
is reduced in ageing diabetic micerdquo Oxidative Medicine andCellular Longevity vol 2015 Article ID 758358 14 pages 2015
[14] K L Quick and L L Dugan ldquoSuperoxide stress identifiesneurons at risk in a model of ataxia-telangiectasiardquo Annals ofNeurology vol 49 no 5 pp 627ndash635 2001
[15] X Shen C B Pattillo S Pardue S C Bir R Wang and C GKevil ldquoMeasurement of plasma hydrogen sulfide in vivo and invitrordquo Free Radical Biology andMedicine vol 50 no 9 pp 1021ndash1031 2011
[16] B-B Tao S-Y Liu C-C Zhang et al ldquoVEGFR2 functionsas an H
2S-targeting receptor protein kinase with its novel
Cys1045-Cys1024 disulfide bond serving as a specific molecularswitch for hydrogen sulfide actions in vascular endothelialcellsrdquo Antioxidants amp Redox Signaling vol 19 no 5 pp 448ndash464 2013
Oxidative Medicine and Cellular Longevity 13
[17] K J Bitterman R M Anderson H Y Cohen M Latorre-Esteves and D A Sinclair ldquoInhibition of silencing and accel-erated aging by nicotinamide a putative negative regulatorof yeast Sir2 and human SIRT1rdquo The Journal of BiologicalChemistry vol 277 no 47 pp 45099ndash45107 2002
[18] X Zhou Y Feng Z Zhan and J Chen ldquoHydrogen sulfidealleviates diabetic nephropathy in a streptozotocin-induceddiabetic ratmodelrdquoThe Journal of Biological Chemistry vol 289no 42 pp 28827ndash28834 2014
[19] P Wang C K Isaak Y L Siow and K O ldquoDownregulationof cystathionine 120573-synthase and cystathionine 120574-lyase expres-sion stimulates inflammation in kidney ischemiamdashreperfusioninjuryrdquo Physiological Reports vol 24 no 12 article e12251 2014
[20] F Yi and P-L Li ldquoMechanisms of homocysteine-inducedglomerular injury and sclerosisrdquo American Journal of Nephrol-ogy vol 28 no 2 pp 254ndash264 2008
[21] R Xue D-D Hao J-P Sun et al ldquoHydrogen sulfide treat-ment promotes glucose uptake by increasing insulin receptorsensitivity and ameliorates kidney lesions in type 2 diabetesrdquoAntioxidants amp Redox Signaling vol 19 no 1 pp 5ndash23 2013
[22] N Li M-J Wang S Jin et al ldquoThe H2S donor NaHS changes
the expression pattern ofH2S-producing enzymes aftermyocar-
dial infarctionrdquo Oxidative Medicine and Cellular Longevity vol2016 Article ID 6492469 11 pages 2016
[23] U Sen W E Rodriguez N Tyagi M Kumar S Kundu andS C Tyagi ldquoCiglitazone a PPAR120574 agonist ameliorates diabeticnephropathy in part through homocysteine clearancerdquo Ameri-can Journal of PhysiologymdashEndocrinology and Metabolism vol295 no 5 pp E1205ndashE1212 2008
[24] W E Rodriguez U Sen N Tyagi et al ldquoPPAR gammaagonist normalizes glomerular filtration rate tissue levels ofhomocysteine and attenuates endothelial-myocyte uncouplingin alloxan induced diabetic micerdquo International Journal ofBiological Sciences vol 4 no 4 pp 236ndash244 2008
[25] C Hine E Harputlugil Y Zhang et al ldquoEndogenous hydrogensulfide production is essential for dietary restriction benefitsrdquoCell vol 160 no 1-2 pp 132ndash144 2015
[26] S-I Imai ldquoDissecting systemic control ofmetabolism and agingin the NAD World the importance of SIRT1 and NAMPT-mediated NAD biosynthesisrdquo FEBS Letters vol 585 no 11 pp1657ndash1662 2011
[27] N Braidy G J Guillemin H Mansour T Chan-Ling APoljak and R Grant ldquoAge related changes inNAD+metabolismoxidative stress and sirt1 activity in wistar ratsrdquo PLoS ONE vol6 no 4 Article ID e19194 pp 1ndash18 2011
[28] A PurushothamT T SchugQXu S Surapureddi XGuo andX Li ldquoHepatocyte-specific deletion of SIRT1 alters fatty acidmetabolism and results in hepatic steatosis and inflammationrdquoCell Metabolism vol 9 no 4 pp 327ndash338 2009
[29] A K Walker F Yang K Jiang et al ldquoConserved roleof SIRT1 orthologs in fasting-dependent inhibition of thelipidcholesterol regulator SREBPrdquoGenes and Development vol24 no 13 pp 1403ndash1417 2010
[30] X Li S Zhang G Blander J G Tse M Krieger and L Guar-ente ldquoSIRT1 deacetylates and positively regulates the nuclearreceptor LXRrdquoMolecular Cell vol 28 no 1 pp 91ndash106 2007
[31] M M Sachdeva M Cano and J T Handa ldquoNrf2 signaling isimpaired in the aging RPE given an oxidative insultrdquo Experi-mental Eye Research vol 119 no 1 pp 111ndash114 2014
[32] A Loboda M Damulewicz E Pyza A Jozkowicz and JDulak ldquoRole of Nrf2HO-1 system in development oxidative
stress response and diseases an evolutionarily conservedmech-anismrdquo Cellular and Molecular Life Sciences vol 73 no 17 pp3221ndash3247 2016
[33] G Yang K Zhao Y Ju et al ldquoHydrogen sulfide protects againstcellular senescence via S-sulfhydration of Keap1 and activationof Nrf2rdquo Antioxidants amp Redox Signaling vol 18 no 15 pp1906ndash1919 2013
[34] A J Done M J Gage N C Nieto and T TraustadottirldquoExercise-induced Nrf2-signaling is impaired in agingrdquo FreeRadical Biology and Medicine vol 96 no 1 pp 130ndash138 2016
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
P = 0033 P = 0045
0
5
10
15
BUN
leve
l (m
mol
L)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(b)
P = 0045P = 0015
00
05
10
15
20
CHO
L le
vel (
mm
olL
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(c)
P = 0042 P = 0002
00
02
04
06
08
10
TG le
vel (
mm
olL
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(d)
000
005
010
015
020
025
LDL-
C le
vel (
mm
olL
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(e)
P = 0047P = 0026
00
02
04
06
08
10
HD
L-C
leve
l (m
mol
L)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(f)
Figure 2 Blood biochemical results for young and old mice (a) Crea creatinine (b) BUN blood urea nitrogen (c) CHOL total cholesterol(d) TG triglycerides (e) LDL-C low-density lipoprotein (f)HDL-C high-density lipoprotein Values aremeanplusmn SE119875 lt 005was consideredsignificant (young groups119873 = 12 old groups119873 = 14)
TG and HDL-C increased dramatically in old control micecomparedwith youngmice (Figures 2(b) 2(c) 2(d) and 2(f))Ten weeks of 50120583molkgday NaHS treatment significantlyalleviated the increase of Crea BUN CHOL and HDL-C (Figures 2(a) 2(b) 2(c) and 2(f)) and 100 120583molkgdayNaHS therapy decreased the TG contents (Figure 2(d)) Ourresults indicate that there is impaired kidney function duringageing and exogenous H
2S treatment could partially reverse
such impairment
32 NaHS Alleviates the Level of Oxidative Stress in the AgeingKidney ROS levels in the kidney were examined after 10weeks of NaHS therapy DHE fluorescence intensity andmal-ondialdehyde (MDA) levels were elevated significantly in theold control group compared with young mice and chronicNaHS treatment could mitigate these changes (Figures 3(a)3(b) and 3(c)) Accordingly SOD activity and glutathioneperoxidase (GPx) levels were decreased in old mice and50 120583molkgday NaHS treatment could partially rescue these
Oxidative Medicine and Cellular Longevity 5
Young Old
200120583m200120583m200120583m
Old-50120583m NaHS
(a)
00
05
10
15
DH
E flu
ores
cenc
e (fo
lds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
P = 0042P = 0025
(b)
P lt 0001
P = 0030
P = 0004
00
05
10
15
MD
A le
vel (
fold
s of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(c)
P lt 0001
P = 0001
P = 0016
00
02
04
06
08
SOD
in k
idne
y (U
mg
prot
ein)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(d)
P = 0010 P = 0018
00
05
10
15
20
GPx
leve
l (fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(e)
Figure 3 H2S donor NaHS protected the ageing kidney from oxidative stress ((a) and (b)) DHE staining and the DHE fluorescence in the
kidney tissue (c) Malondialdehyde (MDA) levels in the kidney (d) Total SOD activity renal tissue (e) Glutathione peroxidase (GPx) levelsin the kidney Values are mean plusmn SE 119875 lt 005 was considered significant (young groups119873 = 6 old groups119873 = 8)
changes (Figures 3(d) and 3(e)) Our results indicated that theH2S donor could protect the ageing kidney from oxidative
stress
33 Ageing Mice Exhibit Kidney Remodeling and Apopto-sis and Chronic NaHS Treatment Mitigates These ProcessesMasson staining showed significantly increased interstitialfibrosis compared with young groups and 10 weeks of
50 120583molkgday NaHS treatment could partially reduce col-lagen deposition (Figures 4(a) and 4(b)) There was higherexpression of Col III and FN in the ageing kidney and thistrend became less significant with chronic NaHS treatment(Figures 4(d) and 4(e)) Col I expressionwas also increased inthe ageing kidney but with no significant difference betweenthe NaHS-treated and nontreated groups (Figure 4(c)) InadditionmRNA levels of Col I Col III and FN also increased
6 Oxidative Medicine and Cellular Longevity
Young
100120583m100120583m100120583m
Old-50120583m NaHSOld
(a)
Old Old-50YoungNaHS (120583molkgday)
00
05
10
15
Col
lage
n co
nten
t (fo
lds o
f old
cont
rol)
P = 0034P = 0024
(b)
GAPDH
Col I
Young Old-100Old-50Old-10OldNaHS (120583molkgday)
00
05
10
15
Col
IG
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P = 0025
(c)
GAPDH
Col III
00
05
10
15
Col
IIIG
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P = 0037
P = 0001 P = 0025
Young Old-100Old-50Old-10OldNaHS (120583molkgday)
(d)
GAPDH
FN
00
05
10
15
FNG
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P lt 0001P lt 0001
Young Old-100Old-50Old-10OldNaHS (120583molkgday)
(e)
Figure 4 Renal pathological changes of ageing mice ((a) and (b)) Masson staining of the kidney tissue (c) Col I and (d) Col III expressionin ageing mice (119873 = 8) (e) FN expression in ageing mice (119873 = 10) 119875 lt 005 was considered significant
Oxidative Medicine and Cellular Longevity 7
during ageing (Figure S1 in SupplementaryMaterial availableonline at httpdxdoiorg10115520167570489)
The ageing kidney appeared to have a higher apoptosislevel compared with that of the young group evidencedby increased expression of proapoptotic Bax and decreasedexpression of antiapoptotic Bcl-2 (Figure 5(b)) TUNELstaining revealed the same changes (Figure 5(a)) HE stainingshowed tubular dilation and inflammatory infiltration amongageing mice whereas chronic NaHS therapy could mitigatethis damage (Figure 5(c)) Our results indicated that kidneyremodeling and apoptosis had occurred in ageing mice andchronic NaHS treatment could mitigate these processes
34 Endogenous Hydrogen Sulfide Production Is Decreasedin Ageing Mice NaHS Treatment Alleviates the Reduction byIncreasing the Expression and Activity of Hydrogen Sulfide-Producing Enzymes Compared with young groups ageingmice showed lower CSE and CBS expression although3-MST expression remained unchanged Ten weeks ofNaHS treatments (50 120583molkgday) significantly increasedthe expression of CSE and CBS but not the 3-MST (Figures6(a1) 6(a2) and 6(a3)) Plasma urine and kidney H
2S levels
decreased significantly during ageing (Figures 6(b1) 6(b2)and 6(b3)) whereas 10 weeks of NaHS (50 120583molkgday)treatment alleviated the plasma and urine H
2S levels (Figures
6(b2) and 6(b3)) H2S levels in ageing kidney tissues were also
increased to some extent after NaHS treatment but withoutstatistical significance (Figure 6(b1)) CSECBS activity inthe ageing kidney decreased and 100 120583molkgday NaHStreatment diminished this reduction (Figure 6(c))
35 NaHSActivates SIRT1 in the Ageing Kidney To determinewhether H
2S protects the ageing kidney through the SIRT1-
mediated pathway the protein and transcriptional levelsof SIRT1 were examined Both the protein expression andmRNA transcription of SIRT1 were decreased in ageing micecompared with young ones and NaHS (50120583molkgday)treatment could improve SIRT1 protein expression but notthe mRNA levels (Figure 7(a)) SIRT1 is a deacetylase and10 weeks of NaHS treatment did not affect total deacetylaseactivity in the ageing kidney (Figure 7(b)) but 25120583molLNaHS could directly increase the activity of recombinantSIRT1 protein in vitro (Figure 7(c)) Measurement of SIRT1activity in the ageing kidney will help to make clear whetherchronic NaHS treatment selectively influences SIRT1 activitywhile total deacetylase activity remains the same
36 Effects of Chronic NaHS Treatment on the Expressionof Antioxidant-Related Proteins in the Ageing Kidney Theexpression of antioxidant proteins in kidney tissue wasexamined by western blot analysis at the end of chronicNaHS treatment Ten weeks of NaHS (50 120583molkgday) treat-ment could increase the Nrf2 expression and improve itsdownstream antioxidative proteins such as HO-1 SOD1 andSOD2 (Figure 8(a)) Moreover compared with young groupsboth the nuclear and cytosol Nrf2 levels were decreased andNaHS (50120583molkgday) treatment could selectively increasenuclear Nrf2 (Figure 8(b)) As shown in Figure S2A theNrf2 nuclear translation in the kidney tissue was insufficient
As shown in Figure S2B Nrf2 translocation in the NRK-52E cells treated with NaHS (50 120583molL) was significantlyinduced from cytosol to nucleus and peaked at 60min Thetranslocation ofNrf2 from cytoplasm into nuclearmay be oneof the protective mechanisms of H
2S against ageing-related
oxidative stress
4 Discussion
In this study we employed an ageing mouse model toinvestigate chronic NaHS treatment in the process of kidneysenescence Our work reveals two important findings (1)lower plasma urine and kidney H
2S levels and reduction of
kidneyCSE andCBS expression and activity are accompaniedwith ageing (2) exogenous administration of H
2S donor
NaHS mitigates ageing-related kidney dysfunction and theprotective effect of NaHS may at least partially relate toimproved endogenous H
2S production and its antioxidative
natureDuring ageing an elevated amount of ROS generated
from glycolysis specifically caused by the defects in thepolyol pathway uncoupling of nitric oxide synthase xanthineoxidase and advanced glycation results in the progressivedeterioration of renal function [18] Emerging data suggestthat ROS are related to the pathophysiology of glomerulardysfunction interstitial fibrosis and glomerulosclerosis [1920] In streptozotocin-induced diabetic rats H
2S therapy
(14 120583molkgday) improved renal function and decreasedglomerular basement thickening mesangial expansion andinterstitial fibrosis [18] Our previous study showed thatchronic NaHS treatment (30 60 and 120 120583molkgday) sig-nificantly reduced ROS levels in the kidney of GK rats [21]Consistently we found that chronic NaHS treatment couldreduce ROS levels (50 120583molkgday) and MDA contents (50and 100 120583molkgday) and increase GPx levels and SODactivity in the kidney of ageingmice H
2S takes part in a great
variety of physiological and pathophysiological processesbecause of its antiapoptotic antioxidative anti-inflammatoryand proangiogenic activities in mammals and the reduc-tion of endogenous H
2S levels has been related to various
diseases Zhou et al reported that endogenous H2S gen-
eration and CSE protein expression decreased significantlyin the streptozotocin-induced diabetic rat model and thatexogenous H
2S (14 120583molkgday) protected against diabetic
nephropathy [18] Our previous study also showed thatchronic NaHS (30 120583molkgday) treatment might amelioratediabetic complications of the kidney [21]
In this study we confirmed intensified interstitial fibrosisin the ageing kidney and observed decreased accumulation ofCol III (50 and 100 120583molkgday) and FN (at 50120583molkgday)with exogenous NaHSWe also observed increased apoptosisin the ageing kidney and chronic NaHS treatment could par-tially reverse this deterioration Age-related kidney damagemight reversely correlate with endogenous H
2S production
We found that both H2S levels in the plasma urine and
kidney and the kidney expression of CSE and CBS decreasedsignificantly with ageing As part of our bodyrsquos antioxidativedefense an insufficient H
2S system may decrease the overall
ability of ROS scavenging and render the kidney to be
8 Oxidative Medicine and Cellular Longevity
Young Old Old-50120583m NaHS
100120583m 100120583m 100120583m
(a)
GAPDH GAPDH
BAX Bcl-2
Young Old-50Old
00
05
10
15
BAX
GA
PDH
(fol
ds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
NaHS (120583molkgday)Young Old-50Old
NaHS (120583molkgday)
Old Old-50YoungNaHS (120583molkgday)
00
05
10
15
20
25
Bcl-2
GA
PDH
(fol
ds o
f old
cont
rol) P = 0001 P = 0029
P = 0034P = 0012
(b)
Young Old Old-50120583m NaHS
100120583m100120583m100120583m
(c)
Figure 5 Renal pathological changes of ageing mice (a) TUNEL staining of the kidney tissue (b) Bax (119873 = 6) and Bcl-2 (119873 = 7) expressionin ageing mice (c) HE staining of the kidney tissue Values are means plusmn SE 119875 lt 005 was considered significant
Oxidative Medicine and Cellular Longevity 9
GAPDH
GAPDH
CBS
CSE
Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old
GAPDH
MST
Young Old-100Old-50Old-10Old
NaHS (120583molkgday)P = 0008
P = 0029 P = 0028
P = 0020
P = 0048
P = 0007
P = 0029
P = 0001 P = 0005
P = 0043
P = 0014Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
(a1)
(a2)
(a3)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
Kidn
ey H
2S
(120583m
olg
)(fo
lds o
f old
cont
rol)
(b1)
(b2)
(b3)
(c)
00
05
10
15
Plas
ma H
2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
5
Urin
e H2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
(fold
s of o
ld co
ntro
l)CS
ECB
S ac
tivity
(120583m
olg
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
CSE
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
20
25
CBS
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
MST
GA
PDH
(fold
s of o
ld co
ntro
l)
Figure 6 Exogenous H2S alleviated ageing-related decreasing of H
2S production (a1)ndash(a3) Kidney expression of CSE (119873 = 11) and CBS
decreased significantly whereas 3-MST kept constant during ageing (119873 = 10) (b1)ndash(b3) Plasma urine and kidney tissueH2S levels decreased
significantly during ageing and rose again after chronic NaHS treatment (c) The activity of CSECBS in the ageing kidney declined whereas100 120583molkgday NaHS treatment could mitigate the reduction Values are means plusmn SE 119875 lt 005 was considered significant (119873 = 12)
10 Oxidative Medicine and Cellular Longevity
GAPDH
SIRT1
Young Old-100Old-50Old-10Old
P = 0007 P = 0015 P = 0023
NaHS (120583molkgday)
00
05
10
15
20
SIRT
1G
APD
H (f
olds
of o
ld co
ntro
l)
0
1
2
3
SIRT
1 m
RNA
(fol
ds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
P = 0049
00
05
10
15
Tota
l hist
one d
eace
tyla
se ac
tivity
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
P = 0001
P lt 0001
00
05
10
15
20
25
SIRT
1 ac
tivat
ion
in v
itro
(fold
s of c
ontro
l)
125 25 50 100ControlNaHS (120583molL)
(c)
Figure 7 SIRT1 was involved in NaHS-mediated ageing kidney protection (a) SIRT1 protein and mRNA expressions in the kidney afterchronic NaHS treatment for 10 weeks (119873 = 8) (b) Total deacetylase activity exchangeable in the kidney (119873 = 10) (c) NaHS-activatedrecombinant protein SIRT1 activity in vitro (119873 = 10) Values are mean plusmn SE 119875 lt 005 was considered significant
under oxidative stress Reasonably chronic NaHS treatmentprotects the ageing kidney by ROS scavenging and enhancingendogenous H
2S production in kidney tissue as reported
previously inmyocardium [22] DecreasedCBS activity in theageing kidney may also lead to homocysteine accumulationBecause the latter plays an important role in chronic renalfailure [23 24] further study is needed to explore whetherhomocysteine metabolism disorder is involved in ageing-related kidney damage and whether exogenous NaHS couldhelp restore the normal metabolic pathway
Christopher Hine reported that sulfur amino acid restric-tion could alter H
2S production and protect the liver from
ischemia-reperfusion injury [25] indicating the beneficialrole of endogenous H
2S SIRT1 which emerges as a major
life span regulator has been widely explored in the car-diovascular system and nervous system but it is rare in
the urinary system SIRT1 has robust biological effects andcan affect metabolic homeostasis and ageing Consistentwith previous studies [26 27] we found that both theexpression and activity of SIRT1 decreased rapidly in theageing kidney The reduction of NAD+ biosynthesis maybe responsible for reduced SIRT1 activity [26] Here wereport an interesting finding that a low concentration ofNaHS (25120583molL) could directly induce SIRT1 activationin a cell-free system whereas chronic treatment of NaHS(50 120583molkgday) selectively improves the expression but notthe activity of SIRT1 in the ageing kidney SIRT1 also regulateslipid metabolism by manipulating PPAR-120572 LXR FXR andSREBP signals [28 29] For example SIRT1 positively regu-lates LXR proteins which act as cholesterol sensor and adjustwhole body cholesterol and lipid homeostasis [30] Our bloodbiochemical tests showed that chronic NaHS treatment could
Oxidative Medicine and Cellular Longevity 11
Nrf2
GAPDH
GAPDH
SOD2
GAPDH
SOD1
GAPDH
HO-1
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday) NaHS (120583molkgday)
00
05
10
15
Nrf2
GA
PDH
(fol
ds o
f old
cont
rol)
00
05
10
15
20
HO
-1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
2G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
NS
GAPDH
Cyto Nrf2
Young Old-50OldNaHS (120583molkgday)
P lt 0001
00
05
10
15
20
Cyto
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
Nuclear Nrf2
Lamin B
Young Old-50Old
P = 0010 P = 0044
NaHS (120583molkgday)
00
05
10
15
20
Nuc
lear
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
Figure 8 The protective effects of the H2S donor on the expression of antioxidant-related proteins in the ageing kidney tissue (a) The
expression change of Nrf2 (119873 = 11) HO-1 (119873 = 10) SOD1 (119873 = 11) and SOD2 (119873 = 10) after 10 weeks of exogenous H2S donor (b) Nrf2
was translocated from cytosol to nucleus Lamin B was used as nuclear control and GAPDH was used as cytosol control (119873 = 6) Values aremean plusmn SE 119875 lt 005 was considered significant
12 Oxidative Medicine and Cellular Longevity
decrease CHOL and TG Therefore H2S may modulate liver
and renal lipidmetabolism through an enhanced SIRT1 signaland contribute to the prevention of renal disease progression
Nrf2 is an important antioxidative molecule that mod-ulates the expression of antioxidant genes [30] and thusprevents age-related oxidative stress [31 32] Here we showedthat the kidney Nrf2 expression decreased during age-ing and that chronic NaHS treatment could rescue Nrf2expression (10 and 50 120583molkgday) and enhance its nucleartranslocation (50120583molkgday) The precise mechanism ofexcessive oxidative stress in ageing is not clear but thedecreased expression of Nrf2antioxidant stress element-linked antioxidant genes is thought to play a role [32ndash34]We found that chronic NaHS treatment could activate Nrf2downstream genes such as HO-1 SOD1 and SOD2 whichconsequently enhanced resistance to oxidative stress in theageing kidney Taken together our data support the idea thatH2S plays a crucial role in protecting the ageing kidney from
antifibrosis and antiapoptosis through the regulation of redoxhomeostasis
5 Conclusion
In summary we demonstrated that 3-MST was expressed inthe ageing kidney and that endogenous H
2S levels decreased
because of the impaired expression ofH2S-producing enzymes
Chronic exogenous H2S treatment could protect the ageing
kidney by reducing oxidative stress decreasing collagendeposition and enhancing Nrf2 nuclear translocation aswell as increasing endogenous H
2S production
Competing Interests
The authors declare no conflict of interests regarding thepublication of this paper
Authorsrsquo ContributionsCui-Lan Hou and Ming-Jie Wang contributed equally to thiswork
AcknowledgmentsThe authors thank Bo Tan for his excellent technical assis-tance in H
2S measurements This present study was sup-
ported by the National Natural Science Foundation of China(NSFC) (no 81230003 to Yi-Chun Zhu no 81670248 toMing-Jie Wang and no 81402917 to Bo Tan) the Ministryof Education (20110071130009 to Yi-Chun Zhu) the Shang-hai Pujiang Program (15PJ1400700 to Ming-Jie Wang) theResearch Center on Ageing and Medicine Fudan University(13dz2260700 to Ming-Jie Wang) and Key Laboratory Pro-gramof the EducationCommission of ShanghaiMunicipality(ZDSYS14005 to Yi-Chun Zhu)
References
[1] J Volkert H SchulzMHarter OWlodarczyk and S AndreasldquoThe prevalence of mental disorders in older people inWesterncountriesmdasha meta-analysisrdquo Ageing Research Reviews vol 12no 1 pp 339ndash353 2013
[2] A Denic R J Glassock and A D Rule ldquoStructural andfunctional changes with the aging kidneyrdquo Advances in ChronicKidney Disease vol 23 no 1 pp 19ndash28 2016
[3] G Yang L Wu B Jiang et al ldquoH2S as a physiologic vasore-
laxant hypertension in mice with deletion of cystathionine 120574-lyaserdquo Science vol 322 no 5901 pp 587ndash590 2008
[4] Y Sun Y Huang R Zhang et al ldquoHydrogen sulfide upreg-ulates KATP channel expression in vascular smooth musclecells of spontaneously hypertensive ratsrdquo Journal of MolecularMedicine vol 93 no 4 pp 439ndash455 2015
[5] C Guo F Liang W Shah Masood and X Yan ldquoHydrogen sul-fide protected gastric epithelial cell from ischemiareperfusioninjury by Keap1 s-sulfhydration MAPK dependent anti-apoptosis and NF-120581B dependent anti-inflammation pathwayrdquoEuropean Journal of Pharmacology vol 725 no 1 pp 70ndash782014
[6] J Du Y Huang H Yan et al ldquoHydrogen sulfide suppresses oxi-dized low-density lipoprotein (Ox-LDL)-stimulated monocytechemoattractant protein 1 generation frommacrophages via thenuclear factor 120581b (NF-120581B) pathwayrdquo The Journal of BiologicalChemistry vol 289 no 14 pp 9741ndash9753 2014
[7] J Zheng T Zhao Y Yuan N Hu and X Tang ldquoHydrogensulfide (H
through oxidative stress and inflammatory response via Nrf2-NF-120581B pathwaysrdquo Chemico-Biological Interactions vol 242 no1 pp 353ndash362 2015
[8] D L Miller and M B Roth ldquoHydrogen sulfide increasesthermotolerance and lifespan in Caenorhabditis elegansrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 104 no 51 pp 20618ndash20622 2007
[9] Y Wei and C Kenyon ldquoRoles for ROS and hydrogen sulfidein the longevity response to germline loss in Caenorhabditiselegansrdquo Proceedings of the National Academy of Sciences vol113 no 20 pp e2832ndashe2841 2016
[10] Y Zhang Z-H Tang Z Ren et al ldquoHydrogen sulfide thenext potent preventive and therapeutic agent in aging and age-associated diseaserdquo Molecular and Cellular Biology vol 33 no6 pp 1104ndash1113 2013
[11] C Wei Y Zhao L Wang et al ldquoH2S restores the cardiopro-
tection from ischemic post-conditioning in isolated aged ratheartsrdquo Cell Biology International vol 39 no 10 pp 1173ndash11762015
[12] R E Sandu A Buga A T Balseanu M Moldovan and APopa-Wagner ldquoTwenty-four hours hypothermia has temporaryefficacy in reducing brain infarction and inflammation in agedratsrdquo Neurobiology of Aging vol 38 no 1 pp 127ndash140 2016
[13] S Jin S X Pu C L Hou et al ldquoCardiac H2S generation
is reduced in ageing diabetic micerdquo Oxidative Medicine andCellular Longevity vol 2015 Article ID 758358 14 pages 2015
[14] K L Quick and L L Dugan ldquoSuperoxide stress identifiesneurons at risk in a model of ataxia-telangiectasiardquo Annals ofNeurology vol 49 no 5 pp 627ndash635 2001
[15] X Shen C B Pattillo S Pardue S C Bir R Wang and C GKevil ldquoMeasurement of plasma hydrogen sulfide in vivo and invitrordquo Free Radical Biology andMedicine vol 50 no 9 pp 1021ndash1031 2011
[16] B-B Tao S-Y Liu C-C Zhang et al ldquoVEGFR2 functionsas an H
2S-targeting receptor protein kinase with its novel
Cys1045-Cys1024 disulfide bond serving as a specific molecularswitch for hydrogen sulfide actions in vascular endothelialcellsrdquo Antioxidants amp Redox Signaling vol 19 no 5 pp 448ndash464 2013
Oxidative Medicine and Cellular Longevity 13
[17] K J Bitterman R M Anderson H Y Cohen M Latorre-Esteves and D A Sinclair ldquoInhibition of silencing and accel-erated aging by nicotinamide a putative negative regulatorof yeast Sir2 and human SIRT1rdquo The Journal of BiologicalChemistry vol 277 no 47 pp 45099ndash45107 2002
[18] X Zhou Y Feng Z Zhan and J Chen ldquoHydrogen sulfidealleviates diabetic nephropathy in a streptozotocin-induceddiabetic ratmodelrdquoThe Journal of Biological Chemistry vol 289no 42 pp 28827ndash28834 2014
[19] P Wang C K Isaak Y L Siow and K O ldquoDownregulationof cystathionine 120573-synthase and cystathionine 120574-lyase expres-sion stimulates inflammation in kidney ischemiamdashreperfusioninjuryrdquo Physiological Reports vol 24 no 12 article e12251 2014
[20] F Yi and P-L Li ldquoMechanisms of homocysteine-inducedglomerular injury and sclerosisrdquo American Journal of Nephrol-ogy vol 28 no 2 pp 254ndash264 2008
[21] R Xue D-D Hao J-P Sun et al ldquoHydrogen sulfide treat-ment promotes glucose uptake by increasing insulin receptorsensitivity and ameliorates kidney lesions in type 2 diabetesrdquoAntioxidants amp Redox Signaling vol 19 no 1 pp 5ndash23 2013
[22] N Li M-J Wang S Jin et al ldquoThe H2S donor NaHS changes
the expression pattern ofH2S-producing enzymes aftermyocar-
dial infarctionrdquo Oxidative Medicine and Cellular Longevity vol2016 Article ID 6492469 11 pages 2016
[23] U Sen W E Rodriguez N Tyagi M Kumar S Kundu andS C Tyagi ldquoCiglitazone a PPAR120574 agonist ameliorates diabeticnephropathy in part through homocysteine clearancerdquo Ameri-can Journal of PhysiologymdashEndocrinology and Metabolism vol295 no 5 pp E1205ndashE1212 2008
[24] W E Rodriguez U Sen N Tyagi et al ldquoPPAR gammaagonist normalizes glomerular filtration rate tissue levels ofhomocysteine and attenuates endothelial-myocyte uncouplingin alloxan induced diabetic micerdquo International Journal ofBiological Sciences vol 4 no 4 pp 236ndash244 2008
[25] C Hine E Harputlugil Y Zhang et al ldquoEndogenous hydrogensulfide production is essential for dietary restriction benefitsrdquoCell vol 160 no 1-2 pp 132ndash144 2015
[26] S-I Imai ldquoDissecting systemic control ofmetabolism and agingin the NAD World the importance of SIRT1 and NAMPT-mediated NAD biosynthesisrdquo FEBS Letters vol 585 no 11 pp1657ndash1662 2011
[27] N Braidy G J Guillemin H Mansour T Chan-Ling APoljak and R Grant ldquoAge related changes inNAD+metabolismoxidative stress and sirt1 activity in wistar ratsrdquo PLoS ONE vol6 no 4 Article ID e19194 pp 1ndash18 2011
[28] A PurushothamT T SchugQXu S Surapureddi XGuo andX Li ldquoHepatocyte-specific deletion of SIRT1 alters fatty acidmetabolism and results in hepatic steatosis and inflammationrdquoCell Metabolism vol 9 no 4 pp 327ndash338 2009
[29] A K Walker F Yang K Jiang et al ldquoConserved roleof SIRT1 orthologs in fasting-dependent inhibition of thelipidcholesterol regulator SREBPrdquoGenes and Development vol24 no 13 pp 1403ndash1417 2010
[30] X Li S Zhang G Blander J G Tse M Krieger and L Guar-ente ldquoSIRT1 deacetylates and positively regulates the nuclearreceptor LXRrdquoMolecular Cell vol 28 no 1 pp 91ndash106 2007
[31] M M Sachdeva M Cano and J T Handa ldquoNrf2 signaling isimpaired in the aging RPE given an oxidative insultrdquo Experi-mental Eye Research vol 119 no 1 pp 111ndash114 2014
[32] A Loboda M Damulewicz E Pyza A Jozkowicz and JDulak ldquoRole of Nrf2HO-1 system in development oxidative
stress response and diseases an evolutionarily conservedmech-anismrdquo Cellular and Molecular Life Sciences vol 73 no 17 pp3221ndash3247 2016
[33] G Yang K Zhao Y Ju et al ldquoHydrogen sulfide protects againstcellular senescence via S-sulfhydration of Keap1 and activationof Nrf2rdquo Antioxidants amp Redox Signaling vol 18 no 15 pp1906ndash1919 2013
[34] A J Done M J Gage N C Nieto and T TraustadottirldquoExercise-induced Nrf2-signaling is impaired in agingrdquo FreeRadical Biology and Medicine vol 96 no 1 pp 130ndash138 2016
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(c)
P lt 0001
P = 0001
P = 0016
00
02
04
06
08
SOD
in k
idne
y (U
mg
prot
ein)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(d)
P = 0010 P = 0018
00
05
10
15
20
GPx
leve
l (fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(e)
Figure 3 H2S donor NaHS protected the ageing kidney from oxidative stress ((a) and (b)) DHE staining and the DHE fluorescence in the
kidney tissue (c) Malondialdehyde (MDA) levels in the kidney (d) Total SOD activity renal tissue (e) Glutathione peroxidase (GPx) levelsin the kidney Values are mean plusmn SE 119875 lt 005 was considered significant (young groups119873 = 6 old groups119873 = 8)
changes (Figures 3(d) and 3(e)) Our results indicated that theH2S donor could protect the ageing kidney from oxidative
stress
33 Ageing Mice Exhibit Kidney Remodeling and Apopto-sis and Chronic NaHS Treatment Mitigates These ProcessesMasson staining showed significantly increased interstitialfibrosis compared with young groups and 10 weeks of
50 120583molkgday NaHS treatment could partially reduce col-lagen deposition (Figures 4(a) and 4(b)) There was higherexpression of Col III and FN in the ageing kidney and thistrend became less significant with chronic NaHS treatment(Figures 4(d) and 4(e)) Col I expressionwas also increased inthe ageing kidney but with no significant difference betweenthe NaHS-treated and nontreated groups (Figure 4(c)) InadditionmRNA levels of Col I Col III and FN also increased
6 Oxidative Medicine and Cellular Longevity
Young
100120583m100120583m100120583m
Old-50120583m NaHSOld
(a)
Old Old-50YoungNaHS (120583molkgday)
00
05
10
15
Col
lage
n co
nten
t (fo
lds o
f old
cont
rol)
P = 0034P = 0024
(b)
GAPDH
Col I
Young Old-100Old-50Old-10OldNaHS (120583molkgday)
00
05
10
15
Col
IG
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P = 0025
(c)
GAPDH
Col III
00
05
10
15
Col
IIIG
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P = 0037
P = 0001 P = 0025
Young Old-100Old-50Old-10OldNaHS (120583molkgday)
(d)
GAPDH
FN
00
05
10
15
FNG
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P lt 0001P lt 0001
Young Old-100Old-50Old-10OldNaHS (120583molkgday)
(e)
Figure 4 Renal pathological changes of ageing mice ((a) and (b)) Masson staining of the kidney tissue (c) Col I and (d) Col III expressionin ageing mice (119873 = 8) (e) FN expression in ageing mice (119873 = 10) 119875 lt 005 was considered significant
Oxidative Medicine and Cellular Longevity 7
during ageing (Figure S1 in SupplementaryMaterial availableonline at httpdxdoiorg10115520167570489)
The ageing kidney appeared to have a higher apoptosislevel compared with that of the young group evidencedby increased expression of proapoptotic Bax and decreasedexpression of antiapoptotic Bcl-2 (Figure 5(b)) TUNELstaining revealed the same changes (Figure 5(a)) HE stainingshowed tubular dilation and inflammatory infiltration amongageing mice whereas chronic NaHS therapy could mitigatethis damage (Figure 5(c)) Our results indicated that kidneyremodeling and apoptosis had occurred in ageing mice andchronic NaHS treatment could mitigate these processes
34 Endogenous Hydrogen Sulfide Production Is Decreasedin Ageing Mice NaHS Treatment Alleviates the Reduction byIncreasing the Expression and Activity of Hydrogen Sulfide-Producing Enzymes Compared with young groups ageingmice showed lower CSE and CBS expression although3-MST expression remained unchanged Ten weeks ofNaHS treatments (50 120583molkgday) significantly increasedthe expression of CSE and CBS but not the 3-MST (Figures6(a1) 6(a2) and 6(a3)) Plasma urine and kidney H
2S levels
decreased significantly during ageing (Figures 6(b1) 6(b2)and 6(b3)) whereas 10 weeks of NaHS (50 120583molkgday)treatment alleviated the plasma and urine H
2S levels (Figures
6(b2) and 6(b3)) H2S levels in ageing kidney tissues were also
increased to some extent after NaHS treatment but withoutstatistical significance (Figure 6(b1)) CSECBS activity inthe ageing kidney decreased and 100 120583molkgday NaHStreatment diminished this reduction (Figure 6(c))
35 NaHSActivates SIRT1 in the Ageing Kidney To determinewhether H
2S protects the ageing kidney through the SIRT1-
mediated pathway the protein and transcriptional levelsof SIRT1 were examined Both the protein expression andmRNA transcription of SIRT1 were decreased in ageing micecompared with young ones and NaHS (50120583molkgday)treatment could improve SIRT1 protein expression but notthe mRNA levels (Figure 7(a)) SIRT1 is a deacetylase and10 weeks of NaHS treatment did not affect total deacetylaseactivity in the ageing kidney (Figure 7(b)) but 25120583molLNaHS could directly increase the activity of recombinantSIRT1 protein in vitro (Figure 7(c)) Measurement of SIRT1activity in the ageing kidney will help to make clear whetherchronic NaHS treatment selectively influences SIRT1 activitywhile total deacetylase activity remains the same
36 Effects of Chronic NaHS Treatment on the Expressionof Antioxidant-Related Proteins in the Ageing Kidney Theexpression of antioxidant proteins in kidney tissue wasexamined by western blot analysis at the end of chronicNaHS treatment Ten weeks of NaHS (50 120583molkgday) treat-ment could increase the Nrf2 expression and improve itsdownstream antioxidative proteins such as HO-1 SOD1 andSOD2 (Figure 8(a)) Moreover compared with young groupsboth the nuclear and cytosol Nrf2 levels were decreased andNaHS (50120583molkgday) treatment could selectively increasenuclear Nrf2 (Figure 8(b)) As shown in Figure S2A theNrf2 nuclear translation in the kidney tissue was insufficient
As shown in Figure S2B Nrf2 translocation in the NRK-52E cells treated with NaHS (50 120583molL) was significantlyinduced from cytosol to nucleus and peaked at 60min Thetranslocation ofNrf2 from cytoplasm into nuclearmay be oneof the protective mechanisms of H
2S against ageing-related
oxidative stress
4 Discussion
In this study we employed an ageing mouse model toinvestigate chronic NaHS treatment in the process of kidneysenescence Our work reveals two important findings (1)lower plasma urine and kidney H
2S levels and reduction of
kidneyCSE andCBS expression and activity are accompaniedwith ageing (2) exogenous administration of H
2S donor
NaHS mitigates ageing-related kidney dysfunction and theprotective effect of NaHS may at least partially relate toimproved endogenous H
2S production and its antioxidative
natureDuring ageing an elevated amount of ROS generated
from glycolysis specifically caused by the defects in thepolyol pathway uncoupling of nitric oxide synthase xanthineoxidase and advanced glycation results in the progressivedeterioration of renal function [18] Emerging data suggestthat ROS are related to the pathophysiology of glomerulardysfunction interstitial fibrosis and glomerulosclerosis [1920] In streptozotocin-induced diabetic rats H
2S therapy
(14 120583molkgday) improved renal function and decreasedglomerular basement thickening mesangial expansion andinterstitial fibrosis [18] Our previous study showed thatchronic NaHS treatment (30 60 and 120 120583molkgday) sig-nificantly reduced ROS levels in the kidney of GK rats [21]Consistently we found that chronic NaHS treatment couldreduce ROS levels (50 120583molkgday) and MDA contents (50and 100 120583molkgday) and increase GPx levels and SODactivity in the kidney of ageingmice H
2S takes part in a great
variety of physiological and pathophysiological processesbecause of its antiapoptotic antioxidative anti-inflammatoryand proangiogenic activities in mammals and the reduc-tion of endogenous H
2S levels has been related to various
diseases Zhou et al reported that endogenous H2S gen-
eration and CSE protein expression decreased significantlyin the streptozotocin-induced diabetic rat model and thatexogenous H
2S (14 120583molkgday) protected against diabetic
nephropathy [18] Our previous study also showed thatchronic NaHS (30 120583molkgday) treatment might amelioratediabetic complications of the kidney [21]
In this study we confirmed intensified interstitial fibrosisin the ageing kidney and observed decreased accumulation ofCol III (50 and 100 120583molkgday) and FN (at 50120583molkgday)with exogenous NaHSWe also observed increased apoptosisin the ageing kidney and chronic NaHS treatment could par-tially reverse this deterioration Age-related kidney damagemight reversely correlate with endogenous H
2S production
We found that both H2S levels in the plasma urine and
kidney and the kidney expression of CSE and CBS decreasedsignificantly with ageing As part of our bodyrsquos antioxidativedefense an insufficient H
2S system may decrease the overall
ability of ROS scavenging and render the kidney to be
8 Oxidative Medicine and Cellular Longevity
Young Old Old-50120583m NaHS
100120583m 100120583m 100120583m
(a)
GAPDH GAPDH
BAX Bcl-2
Young Old-50Old
00
05
10
15
BAX
GA
PDH
(fol
ds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
NaHS (120583molkgday)Young Old-50Old
NaHS (120583molkgday)
Old Old-50YoungNaHS (120583molkgday)
00
05
10
15
20
25
Bcl-2
GA
PDH
(fol
ds o
f old
cont
rol) P = 0001 P = 0029
P = 0034P = 0012
(b)
Young Old Old-50120583m NaHS
100120583m100120583m100120583m
(c)
Figure 5 Renal pathological changes of ageing mice (a) TUNEL staining of the kidney tissue (b) Bax (119873 = 6) and Bcl-2 (119873 = 7) expressionin ageing mice (c) HE staining of the kidney tissue Values are means plusmn SE 119875 lt 005 was considered significant
Oxidative Medicine and Cellular Longevity 9
GAPDH
GAPDH
CBS
CSE
Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old
GAPDH
MST
Young Old-100Old-50Old-10Old
NaHS (120583molkgday)P = 0008
P = 0029 P = 0028
P = 0020
P = 0048
P = 0007
P = 0029
P = 0001 P = 0005
P = 0043
P = 0014Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
(a1)
(a2)
(a3)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
Kidn
ey H
2S
(120583m
olg
)(fo
lds o
f old
cont
rol)
(b1)
(b2)
(b3)
(c)
00
05
10
15
Plas
ma H
2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
5
Urin
e H2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
(fold
s of o
ld co
ntro
l)CS
ECB
S ac
tivity
(120583m
olg
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
CSE
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
20
25
CBS
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
MST
GA
PDH
(fold
s of o
ld co
ntro
l)
Figure 6 Exogenous H2S alleviated ageing-related decreasing of H
2S production (a1)ndash(a3) Kidney expression of CSE (119873 = 11) and CBS
decreased significantly whereas 3-MST kept constant during ageing (119873 = 10) (b1)ndash(b3) Plasma urine and kidney tissueH2S levels decreased
significantly during ageing and rose again after chronic NaHS treatment (c) The activity of CSECBS in the ageing kidney declined whereas100 120583molkgday NaHS treatment could mitigate the reduction Values are means plusmn SE 119875 lt 005 was considered significant (119873 = 12)
10 Oxidative Medicine and Cellular Longevity
GAPDH
SIRT1
Young Old-100Old-50Old-10Old
P = 0007 P = 0015 P = 0023
NaHS (120583molkgday)
00
05
10
15
20
SIRT
1G
APD
H (f
olds
of o
ld co
ntro
l)
0
1
2
3
SIRT
1 m
RNA
(fol
ds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
P = 0049
00
05
10
15
Tota
l hist
one d
eace
tyla
se ac
tivity
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
P = 0001
P lt 0001
00
05
10
15
20
25
SIRT
1 ac
tivat
ion
in v
itro
(fold
s of c
ontro
l)
125 25 50 100ControlNaHS (120583molL)
(c)
Figure 7 SIRT1 was involved in NaHS-mediated ageing kidney protection (a) SIRT1 protein and mRNA expressions in the kidney afterchronic NaHS treatment for 10 weeks (119873 = 8) (b) Total deacetylase activity exchangeable in the kidney (119873 = 10) (c) NaHS-activatedrecombinant protein SIRT1 activity in vitro (119873 = 10) Values are mean plusmn SE 119875 lt 005 was considered significant
under oxidative stress Reasonably chronic NaHS treatmentprotects the ageing kidney by ROS scavenging and enhancingendogenous H
2S production in kidney tissue as reported
previously inmyocardium [22] DecreasedCBS activity in theageing kidney may also lead to homocysteine accumulationBecause the latter plays an important role in chronic renalfailure [23 24] further study is needed to explore whetherhomocysteine metabolism disorder is involved in ageing-related kidney damage and whether exogenous NaHS couldhelp restore the normal metabolic pathway
Christopher Hine reported that sulfur amino acid restric-tion could alter H
2S production and protect the liver from
ischemia-reperfusion injury [25] indicating the beneficialrole of endogenous H
2S SIRT1 which emerges as a major
life span regulator has been widely explored in the car-diovascular system and nervous system but it is rare in
the urinary system SIRT1 has robust biological effects andcan affect metabolic homeostasis and ageing Consistentwith previous studies [26 27] we found that both theexpression and activity of SIRT1 decreased rapidly in theageing kidney The reduction of NAD+ biosynthesis maybe responsible for reduced SIRT1 activity [26] Here wereport an interesting finding that a low concentration ofNaHS (25120583molL) could directly induce SIRT1 activationin a cell-free system whereas chronic treatment of NaHS(50 120583molkgday) selectively improves the expression but notthe activity of SIRT1 in the ageing kidney SIRT1 also regulateslipid metabolism by manipulating PPAR-120572 LXR FXR andSREBP signals [28 29] For example SIRT1 positively regu-lates LXR proteins which act as cholesterol sensor and adjustwhole body cholesterol and lipid homeostasis [30] Our bloodbiochemical tests showed that chronic NaHS treatment could
Oxidative Medicine and Cellular Longevity 11
Nrf2
GAPDH
GAPDH
SOD2
GAPDH
SOD1
GAPDH
HO-1
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday) NaHS (120583molkgday)
00
05
10
15
Nrf2
GA
PDH
(fol
ds o
f old
cont
rol)
00
05
10
15
20
HO
-1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
2G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
NS
GAPDH
Cyto Nrf2
Young Old-50OldNaHS (120583molkgday)
P lt 0001
00
05
10
15
20
Cyto
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
Nuclear Nrf2
Lamin B
Young Old-50Old
P = 0010 P = 0044
NaHS (120583molkgday)
00
05
10
15
20
Nuc
lear
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
Figure 8 The protective effects of the H2S donor on the expression of antioxidant-related proteins in the ageing kidney tissue (a) The
expression change of Nrf2 (119873 = 11) HO-1 (119873 = 10) SOD1 (119873 = 11) and SOD2 (119873 = 10) after 10 weeks of exogenous H2S donor (b) Nrf2
was translocated from cytosol to nucleus Lamin B was used as nuclear control and GAPDH was used as cytosol control (119873 = 6) Values aremean plusmn SE 119875 lt 005 was considered significant
12 Oxidative Medicine and Cellular Longevity
decrease CHOL and TG Therefore H2S may modulate liver
and renal lipidmetabolism through an enhanced SIRT1 signaland contribute to the prevention of renal disease progression
Nrf2 is an important antioxidative molecule that mod-ulates the expression of antioxidant genes [30] and thusprevents age-related oxidative stress [31 32] Here we showedthat the kidney Nrf2 expression decreased during age-ing and that chronic NaHS treatment could rescue Nrf2expression (10 and 50 120583molkgday) and enhance its nucleartranslocation (50120583molkgday) The precise mechanism ofexcessive oxidative stress in ageing is not clear but thedecreased expression of Nrf2antioxidant stress element-linked antioxidant genes is thought to play a role [32ndash34]We found that chronic NaHS treatment could activate Nrf2downstream genes such as HO-1 SOD1 and SOD2 whichconsequently enhanced resistance to oxidative stress in theageing kidney Taken together our data support the idea thatH2S plays a crucial role in protecting the ageing kidney from
antifibrosis and antiapoptosis through the regulation of redoxhomeostasis
5 Conclusion
In summary we demonstrated that 3-MST was expressed inthe ageing kidney and that endogenous H
2S levels decreased
because of the impaired expression ofH2S-producing enzymes
Chronic exogenous H2S treatment could protect the ageing
kidney by reducing oxidative stress decreasing collagendeposition and enhancing Nrf2 nuclear translocation aswell as increasing endogenous H
2S production
Competing Interests
The authors declare no conflict of interests regarding thepublication of this paper
Authorsrsquo ContributionsCui-Lan Hou and Ming-Jie Wang contributed equally to thiswork
AcknowledgmentsThe authors thank Bo Tan for his excellent technical assis-tance in H
2S measurements This present study was sup-
ported by the National Natural Science Foundation of China(NSFC) (no 81230003 to Yi-Chun Zhu no 81670248 toMing-Jie Wang and no 81402917 to Bo Tan) the Ministryof Education (20110071130009 to Yi-Chun Zhu) the Shang-hai Pujiang Program (15PJ1400700 to Ming-Jie Wang) theResearch Center on Ageing and Medicine Fudan University(13dz2260700 to Ming-Jie Wang) and Key Laboratory Pro-gramof the EducationCommission of ShanghaiMunicipality(ZDSYS14005 to Yi-Chun Zhu)
References
[1] J Volkert H SchulzMHarter OWlodarczyk and S AndreasldquoThe prevalence of mental disorders in older people inWesterncountriesmdasha meta-analysisrdquo Ageing Research Reviews vol 12no 1 pp 339ndash353 2013
[2] A Denic R J Glassock and A D Rule ldquoStructural andfunctional changes with the aging kidneyrdquo Advances in ChronicKidney Disease vol 23 no 1 pp 19ndash28 2016
[3] G Yang L Wu B Jiang et al ldquoH2S as a physiologic vasore-
laxant hypertension in mice with deletion of cystathionine 120574-lyaserdquo Science vol 322 no 5901 pp 587ndash590 2008
[4] Y Sun Y Huang R Zhang et al ldquoHydrogen sulfide upreg-ulates KATP channel expression in vascular smooth musclecells of spontaneously hypertensive ratsrdquo Journal of MolecularMedicine vol 93 no 4 pp 439ndash455 2015
[5] C Guo F Liang W Shah Masood and X Yan ldquoHydrogen sul-fide protected gastric epithelial cell from ischemiareperfusioninjury by Keap1 s-sulfhydration MAPK dependent anti-apoptosis and NF-120581B dependent anti-inflammation pathwayrdquoEuropean Journal of Pharmacology vol 725 no 1 pp 70ndash782014
[6] J Du Y Huang H Yan et al ldquoHydrogen sulfide suppresses oxi-dized low-density lipoprotein (Ox-LDL)-stimulated monocytechemoattractant protein 1 generation frommacrophages via thenuclear factor 120581b (NF-120581B) pathwayrdquo The Journal of BiologicalChemistry vol 289 no 14 pp 9741ndash9753 2014
[7] J Zheng T Zhao Y Yuan N Hu and X Tang ldquoHydrogensulfide (H
through oxidative stress and inflammatory response via Nrf2-NF-120581B pathwaysrdquo Chemico-Biological Interactions vol 242 no1 pp 353ndash362 2015
[8] D L Miller and M B Roth ldquoHydrogen sulfide increasesthermotolerance and lifespan in Caenorhabditis elegansrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 104 no 51 pp 20618ndash20622 2007
[9] Y Wei and C Kenyon ldquoRoles for ROS and hydrogen sulfidein the longevity response to germline loss in Caenorhabditiselegansrdquo Proceedings of the National Academy of Sciences vol113 no 20 pp e2832ndashe2841 2016
[10] Y Zhang Z-H Tang Z Ren et al ldquoHydrogen sulfide thenext potent preventive and therapeutic agent in aging and age-associated diseaserdquo Molecular and Cellular Biology vol 33 no6 pp 1104ndash1113 2013
[11] C Wei Y Zhao L Wang et al ldquoH2S restores the cardiopro-
tection from ischemic post-conditioning in isolated aged ratheartsrdquo Cell Biology International vol 39 no 10 pp 1173ndash11762015
[12] R E Sandu A Buga A T Balseanu M Moldovan and APopa-Wagner ldquoTwenty-four hours hypothermia has temporaryefficacy in reducing brain infarction and inflammation in agedratsrdquo Neurobiology of Aging vol 38 no 1 pp 127ndash140 2016
[13] S Jin S X Pu C L Hou et al ldquoCardiac H2S generation
is reduced in ageing diabetic micerdquo Oxidative Medicine andCellular Longevity vol 2015 Article ID 758358 14 pages 2015
[14] K L Quick and L L Dugan ldquoSuperoxide stress identifiesneurons at risk in a model of ataxia-telangiectasiardquo Annals ofNeurology vol 49 no 5 pp 627ndash635 2001
[15] X Shen C B Pattillo S Pardue S C Bir R Wang and C GKevil ldquoMeasurement of plasma hydrogen sulfide in vivo and invitrordquo Free Radical Biology andMedicine vol 50 no 9 pp 1021ndash1031 2011
[16] B-B Tao S-Y Liu C-C Zhang et al ldquoVEGFR2 functionsas an H
2S-targeting receptor protein kinase with its novel
Cys1045-Cys1024 disulfide bond serving as a specific molecularswitch for hydrogen sulfide actions in vascular endothelialcellsrdquo Antioxidants amp Redox Signaling vol 19 no 5 pp 448ndash464 2013
Oxidative Medicine and Cellular Longevity 13
[17] K J Bitterman R M Anderson H Y Cohen M Latorre-Esteves and D A Sinclair ldquoInhibition of silencing and accel-erated aging by nicotinamide a putative negative regulatorof yeast Sir2 and human SIRT1rdquo The Journal of BiologicalChemistry vol 277 no 47 pp 45099ndash45107 2002
[18] X Zhou Y Feng Z Zhan and J Chen ldquoHydrogen sulfidealleviates diabetic nephropathy in a streptozotocin-induceddiabetic ratmodelrdquoThe Journal of Biological Chemistry vol 289no 42 pp 28827ndash28834 2014
[19] P Wang C K Isaak Y L Siow and K O ldquoDownregulationof cystathionine 120573-synthase and cystathionine 120574-lyase expres-sion stimulates inflammation in kidney ischemiamdashreperfusioninjuryrdquo Physiological Reports vol 24 no 12 article e12251 2014
[20] F Yi and P-L Li ldquoMechanisms of homocysteine-inducedglomerular injury and sclerosisrdquo American Journal of Nephrol-ogy vol 28 no 2 pp 254ndash264 2008
[21] R Xue D-D Hao J-P Sun et al ldquoHydrogen sulfide treat-ment promotes glucose uptake by increasing insulin receptorsensitivity and ameliorates kidney lesions in type 2 diabetesrdquoAntioxidants amp Redox Signaling vol 19 no 1 pp 5ndash23 2013
[22] N Li M-J Wang S Jin et al ldquoThe H2S donor NaHS changes
the expression pattern ofH2S-producing enzymes aftermyocar-
dial infarctionrdquo Oxidative Medicine and Cellular Longevity vol2016 Article ID 6492469 11 pages 2016
[23] U Sen W E Rodriguez N Tyagi M Kumar S Kundu andS C Tyagi ldquoCiglitazone a PPAR120574 agonist ameliorates diabeticnephropathy in part through homocysteine clearancerdquo Ameri-can Journal of PhysiologymdashEndocrinology and Metabolism vol295 no 5 pp E1205ndashE1212 2008
[24] W E Rodriguez U Sen N Tyagi et al ldquoPPAR gammaagonist normalizes glomerular filtration rate tissue levels ofhomocysteine and attenuates endothelial-myocyte uncouplingin alloxan induced diabetic micerdquo International Journal ofBiological Sciences vol 4 no 4 pp 236ndash244 2008
[25] C Hine E Harputlugil Y Zhang et al ldquoEndogenous hydrogensulfide production is essential for dietary restriction benefitsrdquoCell vol 160 no 1-2 pp 132ndash144 2015
[26] S-I Imai ldquoDissecting systemic control ofmetabolism and agingin the NAD World the importance of SIRT1 and NAMPT-mediated NAD biosynthesisrdquo FEBS Letters vol 585 no 11 pp1657ndash1662 2011
[27] N Braidy G J Guillemin H Mansour T Chan-Ling APoljak and R Grant ldquoAge related changes inNAD+metabolismoxidative stress and sirt1 activity in wistar ratsrdquo PLoS ONE vol6 no 4 Article ID e19194 pp 1ndash18 2011
[28] A PurushothamT T SchugQXu S Surapureddi XGuo andX Li ldquoHepatocyte-specific deletion of SIRT1 alters fatty acidmetabolism and results in hepatic steatosis and inflammationrdquoCell Metabolism vol 9 no 4 pp 327ndash338 2009
[29] A K Walker F Yang K Jiang et al ldquoConserved roleof SIRT1 orthologs in fasting-dependent inhibition of thelipidcholesterol regulator SREBPrdquoGenes and Development vol24 no 13 pp 1403ndash1417 2010
[30] X Li S Zhang G Blander J G Tse M Krieger and L Guar-ente ldquoSIRT1 deacetylates and positively regulates the nuclearreceptor LXRrdquoMolecular Cell vol 28 no 1 pp 91ndash106 2007
[31] M M Sachdeva M Cano and J T Handa ldquoNrf2 signaling isimpaired in the aging RPE given an oxidative insultrdquo Experi-mental Eye Research vol 119 no 1 pp 111ndash114 2014
[32] A Loboda M Damulewicz E Pyza A Jozkowicz and JDulak ldquoRole of Nrf2HO-1 system in development oxidative
stress response and diseases an evolutionarily conservedmech-anismrdquo Cellular and Molecular Life Sciences vol 73 no 17 pp3221ndash3247 2016
[33] G Yang K Zhao Y Ju et al ldquoHydrogen sulfide protects againstcellular senescence via S-sulfhydration of Keap1 and activationof Nrf2rdquo Antioxidants amp Redox Signaling vol 18 no 15 pp1906ndash1919 2013
[34] A J Done M J Gage N C Nieto and T TraustadottirldquoExercise-induced Nrf2-signaling is impaired in agingrdquo FreeRadical Biology and Medicine vol 96 no 1 pp 130ndash138 2016
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P = 0025
(c)
GAPDH
Col III
00
05
10
15
Col
IIIG
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P = 0037
P = 0001 P = 0025
Young Old-100Old-50Old-10OldNaHS (120583molkgday)
(d)
GAPDH
FN
00
05
10
15
FNG
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
P lt 0001P lt 0001
Young Old-100Old-50Old-10OldNaHS (120583molkgday)
(e)
Figure 4 Renal pathological changes of ageing mice ((a) and (b)) Masson staining of the kidney tissue (c) Col I and (d) Col III expressionin ageing mice (119873 = 8) (e) FN expression in ageing mice (119873 = 10) 119875 lt 005 was considered significant
Oxidative Medicine and Cellular Longevity 7
during ageing (Figure S1 in SupplementaryMaterial availableonline at httpdxdoiorg10115520167570489)
The ageing kidney appeared to have a higher apoptosislevel compared with that of the young group evidencedby increased expression of proapoptotic Bax and decreasedexpression of antiapoptotic Bcl-2 (Figure 5(b)) TUNELstaining revealed the same changes (Figure 5(a)) HE stainingshowed tubular dilation and inflammatory infiltration amongageing mice whereas chronic NaHS therapy could mitigatethis damage (Figure 5(c)) Our results indicated that kidneyremodeling and apoptosis had occurred in ageing mice andchronic NaHS treatment could mitigate these processes
34 Endogenous Hydrogen Sulfide Production Is Decreasedin Ageing Mice NaHS Treatment Alleviates the Reduction byIncreasing the Expression and Activity of Hydrogen Sulfide-Producing Enzymes Compared with young groups ageingmice showed lower CSE and CBS expression although3-MST expression remained unchanged Ten weeks ofNaHS treatments (50 120583molkgday) significantly increasedthe expression of CSE and CBS but not the 3-MST (Figures6(a1) 6(a2) and 6(a3)) Plasma urine and kidney H
2S levels
decreased significantly during ageing (Figures 6(b1) 6(b2)and 6(b3)) whereas 10 weeks of NaHS (50 120583molkgday)treatment alleviated the plasma and urine H
2S levels (Figures
6(b2) and 6(b3)) H2S levels in ageing kidney tissues were also
increased to some extent after NaHS treatment but withoutstatistical significance (Figure 6(b1)) CSECBS activity inthe ageing kidney decreased and 100 120583molkgday NaHStreatment diminished this reduction (Figure 6(c))
35 NaHSActivates SIRT1 in the Ageing Kidney To determinewhether H
2S protects the ageing kidney through the SIRT1-
mediated pathway the protein and transcriptional levelsof SIRT1 were examined Both the protein expression andmRNA transcription of SIRT1 were decreased in ageing micecompared with young ones and NaHS (50120583molkgday)treatment could improve SIRT1 protein expression but notthe mRNA levels (Figure 7(a)) SIRT1 is a deacetylase and10 weeks of NaHS treatment did not affect total deacetylaseactivity in the ageing kidney (Figure 7(b)) but 25120583molLNaHS could directly increase the activity of recombinantSIRT1 protein in vitro (Figure 7(c)) Measurement of SIRT1activity in the ageing kidney will help to make clear whetherchronic NaHS treatment selectively influences SIRT1 activitywhile total deacetylase activity remains the same
36 Effects of Chronic NaHS Treatment on the Expressionof Antioxidant-Related Proteins in the Ageing Kidney Theexpression of antioxidant proteins in kidney tissue wasexamined by western blot analysis at the end of chronicNaHS treatment Ten weeks of NaHS (50 120583molkgday) treat-ment could increase the Nrf2 expression and improve itsdownstream antioxidative proteins such as HO-1 SOD1 andSOD2 (Figure 8(a)) Moreover compared with young groupsboth the nuclear and cytosol Nrf2 levels were decreased andNaHS (50120583molkgday) treatment could selectively increasenuclear Nrf2 (Figure 8(b)) As shown in Figure S2A theNrf2 nuclear translation in the kidney tissue was insufficient
As shown in Figure S2B Nrf2 translocation in the NRK-52E cells treated with NaHS (50 120583molL) was significantlyinduced from cytosol to nucleus and peaked at 60min Thetranslocation ofNrf2 from cytoplasm into nuclearmay be oneof the protective mechanisms of H
2S against ageing-related
oxidative stress
4 Discussion
In this study we employed an ageing mouse model toinvestigate chronic NaHS treatment in the process of kidneysenescence Our work reveals two important findings (1)lower plasma urine and kidney H
2S levels and reduction of
kidneyCSE andCBS expression and activity are accompaniedwith ageing (2) exogenous administration of H
2S donor
NaHS mitigates ageing-related kidney dysfunction and theprotective effect of NaHS may at least partially relate toimproved endogenous H
2S production and its antioxidative
natureDuring ageing an elevated amount of ROS generated
from glycolysis specifically caused by the defects in thepolyol pathway uncoupling of nitric oxide synthase xanthineoxidase and advanced glycation results in the progressivedeterioration of renal function [18] Emerging data suggestthat ROS are related to the pathophysiology of glomerulardysfunction interstitial fibrosis and glomerulosclerosis [1920] In streptozotocin-induced diabetic rats H
2S therapy
(14 120583molkgday) improved renal function and decreasedglomerular basement thickening mesangial expansion andinterstitial fibrosis [18] Our previous study showed thatchronic NaHS treatment (30 60 and 120 120583molkgday) sig-nificantly reduced ROS levels in the kidney of GK rats [21]Consistently we found that chronic NaHS treatment couldreduce ROS levels (50 120583molkgday) and MDA contents (50and 100 120583molkgday) and increase GPx levels and SODactivity in the kidney of ageingmice H
2S takes part in a great
variety of physiological and pathophysiological processesbecause of its antiapoptotic antioxidative anti-inflammatoryand proangiogenic activities in mammals and the reduc-tion of endogenous H
2S levels has been related to various
diseases Zhou et al reported that endogenous H2S gen-
eration and CSE protein expression decreased significantlyin the streptozotocin-induced diabetic rat model and thatexogenous H
2S (14 120583molkgday) protected against diabetic
nephropathy [18] Our previous study also showed thatchronic NaHS (30 120583molkgday) treatment might amelioratediabetic complications of the kidney [21]
In this study we confirmed intensified interstitial fibrosisin the ageing kidney and observed decreased accumulation ofCol III (50 and 100 120583molkgday) and FN (at 50120583molkgday)with exogenous NaHSWe also observed increased apoptosisin the ageing kidney and chronic NaHS treatment could par-tially reverse this deterioration Age-related kidney damagemight reversely correlate with endogenous H
2S production
We found that both H2S levels in the plasma urine and
kidney and the kidney expression of CSE and CBS decreasedsignificantly with ageing As part of our bodyrsquos antioxidativedefense an insufficient H
2S system may decrease the overall
ability of ROS scavenging and render the kidney to be
8 Oxidative Medicine and Cellular Longevity
Young Old Old-50120583m NaHS
100120583m 100120583m 100120583m
(a)
GAPDH GAPDH
BAX Bcl-2
Young Old-50Old
00
05
10
15
BAX
GA
PDH
(fol
ds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
NaHS (120583molkgday)Young Old-50Old
NaHS (120583molkgday)
Old Old-50YoungNaHS (120583molkgday)
00
05
10
15
20
25
Bcl-2
GA
PDH
(fol
ds o
f old
cont
rol) P = 0001 P = 0029
P = 0034P = 0012
(b)
Young Old Old-50120583m NaHS
100120583m100120583m100120583m
(c)
Figure 5 Renal pathological changes of ageing mice (a) TUNEL staining of the kidney tissue (b) Bax (119873 = 6) and Bcl-2 (119873 = 7) expressionin ageing mice (c) HE staining of the kidney tissue Values are means plusmn SE 119875 lt 005 was considered significant
Oxidative Medicine and Cellular Longevity 9
GAPDH
GAPDH
CBS
CSE
Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old
GAPDH
MST
Young Old-100Old-50Old-10Old
NaHS (120583molkgday)P = 0008
P = 0029 P = 0028
P = 0020
P = 0048
P = 0007
P = 0029
P = 0001 P = 0005
P = 0043
P = 0014Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
(a1)
(a2)
(a3)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
Kidn
ey H
2S
(120583m
olg
)(fo
lds o
f old
cont
rol)
(b1)
(b2)
(b3)
(c)
00
05
10
15
Plas
ma H
2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
5
Urin
e H2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
(fold
s of o
ld co
ntro
l)CS
ECB
S ac
tivity
(120583m
olg
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
CSE
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
20
25
CBS
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
MST
GA
PDH
(fold
s of o
ld co
ntro
l)
Figure 6 Exogenous H2S alleviated ageing-related decreasing of H
2S production (a1)ndash(a3) Kidney expression of CSE (119873 = 11) and CBS
decreased significantly whereas 3-MST kept constant during ageing (119873 = 10) (b1)ndash(b3) Plasma urine and kidney tissueH2S levels decreased
significantly during ageing and rose again after chronic NaHS treatment (c) The activity of CSECBS in the ageing kidney declined whereas100 120583molkgday NaHS treatment could mitigate the reduction Values are means plusmn SE 119875 lt 005 was considered significant (119873 = 12)
10 Oxidative Medicine and Cellular Longevity
GAPDH
SIRT1
Young Old-100Old-50Old-10Old
P = 0007 P = 0015 P = 0023
NaHS (120583molkgday)
00
05
10
15
20
SIRT
1G
APD
H (f
olds
of o
ld co
ntro
l)
0
1
2
3
SIRT
1 m
RNA
(fol
ds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
P = 0049
00
05
10
15
Tota
l hist
one d
eace
tyla
se ac
tivity
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
P = 0001
P lt 0001
00
05
10
15
20
25
SIRT
1 ac
tivat
ion
in v
itro
(fold
s of c
ontro
l)
125 25 50 100ControlNaHS (120583molL)
(c)
Figure 7 SIRT1 was involved in NaHS-mediated ageing kidney protection (a) SIRT1 protein and mRNA expressions in the kidney afterchronic NaHS treatment for 10 weeks (119873 = 8) (b) Total deacetylase activity exchangeable in the kidney (119873 = 10) (c) NaHS-activatedrecombinant protein SIRT1 activity in vitro (119873 = 10) Values are mean plusmn SE 119875 lt 005 was considered significant
under oxidative stress Reasonably chronic NaHS treatmentprotects the ageing kidney by ROS scavenging and enhancingendogenous H
2S production in kidney tissue as reported
previously inmyocardium [22] DecreasedCBS activity in theageing kidney may also lead to homocysteine accumulationBecause the latter plays an important role in chronic renalfailure [23 24] further study is needed to explore whetherhomocysteine metabolism disorder is involved in ageing-related kidney damage and whether exogenous NaHS couldhelp restore the normal metabolic pathway
Christopher Hine reported that sulfur amino acid restric-tion could alter H
2S production and protect the liver from
ischemia-reperfusion injury [25] indicating the beneficialrole of endogenous H
2S SIRT1 which emerges as a major
life span regulator has been widely explored in the car-diovascular system and nervous system but it is rare in
the urinary system SIRT1 has robust biological effects andcan affect metabolic homeostasis and ageing Consistentwith previous studies [26 27] we found that both theexpression and activity of SIRT1 decreased rapidly in theageing kidney The reduction of NAD+ biosynthesis maybe responsible for reduced SIRT1 activity [26] Here wereport an interesting finding that a low concentration ofNaHS (25120583molL) could directly induce SIRT1 activationin a cell-free system whereas chronic treatment of NaHS(50 120583molkgday) selectively improves the expression but notthe activity of SIRT1 in the ageing kidney SIRT1 also regulateslipid metabolism by manipulating PPAR-120572 LXR FXR andSREBP signals [28 29] For example SIRT1 positively regu-lates LXR proteins which act as cholesterol sensor and adjustwhole body cholesterol and lipid homeostasis [30] Our bloodbiochemical tests showed that chronic NaHS treatment could
Oxidative Medicine and Cellular Longevity 11
Nrf2
GAPDH
GAPDH
SOD2
GAPDH
SOD1
GAPDH
HO-1
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday) NaHS (120583molkgday)
00
05
10
15
Nrf2
GA
PDH
(fol
ds o
f old
cont
rol)
00
05
10
15
20
HO
-1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
2G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
NS
GAPDH
Cyto Nrf2
Young Old-50OldNaHS (120583molkgday)
P lt 0001
00
05
10
15
20
Cyto
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
Nuclear Nrf2
Lamin B
Young Old-50Old
P = 0010 P = 0044
NaHS (120583molkgday)
00
05
10
15
20
Nuc
lear
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
Figure 8 The protective effects of the H2S donor on the expression of antioxidant-related proteins in the ageing kidney tissue (a) The
expression change of Nrf2 (119873 = 11) HO-1 (119873 = 10) SOD1 (119873 = 11) and SOD2 (119873 = 10) after 10 weeks of exogenous H2S donor (b) Nrf2
was translocated from cytosol to nucleus Lamin B was used as nuclear control and GAPDH was used as cytosol control (119873 = 6) Values aremean plusmn SE 119875 lt 005 was considered significant
12 Oxidative Medicine and Cellular Longevity
decrease CHOL and TG Therefore H2S may modulate liver
and renal lipidmetabolism through an enhanced SIRT1 signaland contribute to the prevention of renal disease progression
Nrf2 is an important antioxidative molecule that mod-ulates the expression of antioxidant genes [30] and thusprevents age-related oxidative stress [31 32] Here we showedthat the kidney Nrf2 expression decreased during age-ing and that chronic NaHS treatment could rescue Nrf2expression (10 and 50 120583molkgday) and enhance its nucleartranslocation (50120583molkgday) The precise mechanism ofexcessive oxidative stress in ageing is not clear but thedecreased expression of Nrf2antioxidant stress element-linked antioxidant genes is thought to play a role [32ndash34]We found that chronic NaHS treatment could activate Nrf2downstream genes such as HO-1 SOD1 and SOD2 whichconsequently enhanced resistance to oxidative stress in theageing kidney Taken together our data support the idea thatH2S plays a crucial role in protecting the ageing kidney from
antifibrosis and antiapoptosis through the regulation of redoxhomeostasis
5 Conclusion
In summary we demonstrated that 3-MST was expressed inthe ageing kidney and that endogenous H
2S levels decreased
because of the impaired expression ofH2S-producing enzymes
Chronic exogenous H2S treatment could protect the ageing
kidney by reducing oxidative stress decreasing collagendeposition and enhancing Nrf2 nuclear translocation aswell as increasing endogenous H
2S production
Competing Interests
The authors declare no conflict of interests regarding thepublication of this paper
Authorsrsquo ContributionsCui-Lan Hou and Ming-Jie Wang contributed equally to thiswork
AcknowledgmentsThe authors thank Bo Tan for his excellent technical assis-tance in H
2S measurements This present study was sup-
ported by the National Natural Science Foundation of China(NSFC) (no 81230003 to Yi-Chun Zhu no 81670248 toMing-Jie Wang and no 81402917 to Bo Tan) the Ministryof Education (20110071130009 to Yi-Chun Zhu) the Shang-hai Pujiang Program (15PJ1400700 to Ming-Jie Wang) theResearch Center on Ageing and Medicine Fudan University(13dz2260700 to Ming-Jie Wang) and Key Laboratory Pro-gramof the EducationCommission of ShanghaiMunicipality(ZDSYS14005 to Yi-Chun Zhu)
References
[1] J Volkert H SchulzMHarter OWlodarczyk and S AndreasldquoThe prevalence of mental disorders in older people inWesterncountriesmdasha meta-analysisrdquo Ageing Research Reviews vol 12no 1 pp 339ndash353 2013
[2] A Denic R J Glassock and A D Rule ldquoStructural andfunctional changes with the aging kidneyrdquo Advances in ChronicKidney Disease vol 23 no 1 pp 19ndash28 2016
[3] G Yang L Wu B Jiang et al ldquoH2S as a physiologic vasore-
laxant hypertension in mice with deletion of cystathionine 120574-lyaserdquo Science vol 322 no 5901 pp 587ndash590 2008
[4] Y Sun Y Huang R Zhang et al ldquoHydrogen sulfide upreg-ulates KATP channel expression in vascular smooth musclecells of spontaneously hypertensive ratsrdquo Journal of MolecularMedicine vol 93 no 4 pp 439ndash455 2015
[5] C Guo F Liang W Shah Masood and X Yan ldquoHydrogen sul-fide protected gastric epithelial cell from ischemiareperfusioninjury by Keap1 s-sulfhydration MAPK dependent anti-apoptosis and NF-120581B dependent anti-inflammation pathwayrdquoEuropean Journal of Pharmacology vol 725 no 1 pp 70ndash782014
[6] J Du Y Huang H Yan et al ldquoHydrogen sulfide suppresses oxi-dized low-density lipoprotein (Ox-LDL)-stimulated monocytechemoattractant protein 1 generation frommacrophages via thenuclear factor 120581b (NF-120581B) pathwayrdquo The Journal of BiologicalChemistry vol 289 no 14 pp 9741ndash9753 2014
[7] J Zheng T Zhao Y Yuan N Hu and X Tang ldquoHydrogensulfide (H
through oxidative stress and inflammatory response via Nrf2-NF-120581B pathwaysrdquo Chemico-Biological Interactions vol 242 no1 pp 353ndash362 2015
[8] D L Miller and M B Roth ldquoHydrogen sulfide increasesthermotolerance and lifespan in Caenorhabditis elegansrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 104 no 51 pp 20618ndash20622 2007
[9] Y Wei and C Kenyon ldquoRoles for ROS and hydrogen sulfidein the longevity response to germline loss in Caenorhabditiselegansrdquo Proceedings of the National Academy of Sciences vol113 no 20 pp e2832ndashe2841 2016
[10] Y Zhang Z-H Tang Z Ren et al ldquoHydrogen sulfide thenext potent preventive and therapeutic agent in aging and age-associated diseaserdquo Molecular and Cellular Biology vol 33 no6 pp 1104ndash1113 2013
[11] C Wei Y Zhao L Wang et al ldquoH2S restores the cardiopro-
tection from ischemic post-conditioning in isolated aged ratheartsrdquo Cell Biology International vol 39 no 10 pp 1173ndash11762015
[12] R E Sandu A Buga A T Balseanu M Moldovan and APopa-Wagner ldquoTwenty-four hours hypothermia has temporaryefficacy in reducing brain infarction and inflammation in agedratsrdquo Neurobiology of Aging vol 38 no 1 pp 127ndash140 2016
[13] S Jin S X Pu C L Hou et al ldquoCardiac H2S generation
is reduced in ageing diabetic micerdquo Oxidative Medicine andCellular Longevity vol 2015 Article ID 758358 14 pages 2015
[14] K L Quick and L L Dugan ldquoSuperoxide stress identifiesneurons at risk in a model of ataxia-telangiectasiardquo Annals ofNeurology vol 49 no 5 pp 627ndash635 2001
[15] X Shen C B Pattillo S Pardue S C Bir R Wang and C GKevil ldquoMeasurement of plasma hydrogen sulfide in vivo and invitrordquo Free Radical Biology andMedicine vol 50 no 9 pp 1021ndash1031 2011
[16] B-B Tao S-Y Liu C-C Zhang et al ldquoVEGFR2 functionsas an H
2S-targeting receptor protein kinase with its novel
Cys1045-Cys1024 disulfide bond serving as a specific molecularswitch for hydrogen sulfide actions in vascular endothelialcellsrdquo Antioxidants amp Redox Signaling vol 19 no 5 pp 448ndash464 2013
Oxidative Medicine and Cellular Longevity 13
[17] K J Bitterman R M Anderson H Y Cohen M Latorre-Esteves and D A Sinclair ldquoInhibition of silencing and accel-erated aging by nicotinamide a putative negative regulatorof yeast Sir2 and human SIRT1rdquo The Journal of BiologicalChemistry vol 277 no 47 pp 45099ndash45107 2002
[18] X Zhou Y Feng Z Zhan and J Chen ldquoHydrogen sulfidealleviates diabetic nephropathy in a streptozotocin-induceddiabetic ratmodelrdquoThe Journal of Biological Chemistry vol 289no 42 pp 28827ndash28834 2014
[19] P Wang C K Isaak Y L Siow and K O ldquoDownregulationof cystathionine 120573-synthase and cystathionine 120574-lyase expres-sion stimulates inflammation in kidney ischemiamdashreperfusioninjuryrdquo Physiological Reports vol 24 no 12 article e12251 2014
[20] F Yi and P-L Li ldquoMechanisms of homocysteine-inducedglomerular injury and sclerosisrdquo American Journal of Nephrol-ogy vol 28 no 2 pp 254ndash264 2008
[21] R Xue D-D Hao J-P Sun et al ldquoHydrogen sulfide treat-ment promotes glucose uptake by increasing insulin receptorsensitivity and ameliorates kidney lesions in type 2 diabetesrdquoAntioxidants amp Redox Signaling vol 19 no 1 pp 5ndash23 2013
[22] N Li M-J Wang S Jin et al ldquoThe H2S donor NaHS changes
the expression pattern ofH2S-producing enzymes aftermyocar-
dial infarctionrdquo Oxidative Medicine and Cellular Longevity vol2016 Article ID 6492469 11 pages 2016
[23] U Sen W E Rodriguez N Tyagi M Kumar S Kundu andS C Tyagi ldquoCiglitazone a PPAR120574 agonist ameliorates diabeticnephropathy in part through homocysteine clearancerdquo Ameri-can Journal of PhysiologymdashEndocrinology and Metabolism vol295 no 5 pp E1205ndashE1212 2008
[24] W E Rodriguez U Sen N Tyagi et al ldquoPPAR gammaagonist normalizes glomerular filtration rate tissue levels ofhomocysteine and attenuates endothelial-myocyte uncouplingin alloxan induced diabetic micerdquo International Journal ofBiological Sciences vol 4 no 4 pp 236ndash244 2008
[25] C Hine E Harputlugil Y Zhang et al ldquoEndogenous hydrogensulfide production is essential for dietary restriction benefitsrdquoCell vol 160 no 1-2 pp 132ndash144 2015
[26] S-I Imai ldquoDissecting systemic control ofmetabolism and agingin the NAD World the importance of SIRT1 and NAMPT-mediated NAD biosynthesisrdquo FEBS Letters vol 585 no 11 pp1657ndash1662 2011
[27] N Braidy G J Guillemin H Mansour T Chan-Ling APoljak and R Grant ldquoAge related changes inNAD+metabolismoxidative stress and sirt1 activity in wistar ratsrdquo PLoS ONE vol6 no 4 Article ID e19194 pp 1ndash18 2011
[28] A PurushothamT T SchugQXu S Surapureddi XGuo andX Li ldquoHepatocyte-specific deletion of SIRT1 alters fatty acidmetabolism and results in hepatic steatosis and inflammationrdquoCell Metabolism vol 9 no 4 pp 327ndash338 2009
[29] A K Walker F Yang K Jiang et al ldquoConserved roleof SIRT1 orthologs in fasting-dependent inhibition of thelipidcholesterol regulator SREBPrdquoGenes and Development vol24 no 13 pp 1403ndash1417 2010
[30] X Li S Zhang G Blander J G Tse M Krieger and L Guar-ente ldquoSIRT1 deacetylates and positively regulates the nuclearreceptor LXRrdquoMolecular Cell vol 28 no 1 pp 91ndash106 2007
[31] M M Sachdeva M Cano and J T Handa ldquoNrf2 signaling isimpaired in the aging RPE given an oxidative insultrdquo Experi-mental Eye Research vol 119 no 1 pp 111ndash114 2014
[32] A Loboda M Damulewicz E Pyza A Jozkowicz and JDulak ldquoRole of Nrf2HO-1 system in development oxidative
stress response and diseases an evolutionarily conservedmech-anismrdquo Cellular and Molecular Life Sciences vol 73 no 17 pp3221ndash3247 2016
[33] G Yang K Zhao Y Ju et al ldquoHydrogen sulfide protects againstcellular senescence via S-sulfhydration of Keap1 and activationof Nrf2rdquo Antioxidants amp Redox Signaling vol 18 no 15 pp1906ndash1919 2013
[34] A J Done M J Gage N C Nieto and T TraustadottirldquoExercise-induced Nrf2-signaling is impaired in agingrdquo FreeRadical Biology and Medicine vol 96 no 1 pp 130ndash138 2016
during ageing (Figure S1 in SupplementaryMaterial availableonline at httpdxdoiorg10115520167570489)
The ageing kidney appeared to have a higher apoptosislevel compared with that of the young group evidencedby increased expression of proapoptotic Bax and decreasedexpression of antiapoptotic Bcl-2 (Figure 5(b)) TUNELstaining revealed the same changes (Figure 5(a)) HE stainingshowed tubular dilation and inflammatory infiltration amongageing mice whereas chronic NaHS therapy could mitigatethis damage (Figure 5(c)) Our results indicated that kidneyremodeling and apoptosis had occurred in ageing mice andchronic NaHS treatment could mitigate these processes
34 Endogenous Hydrogen Sulfide Production Is Decreasedin Ageing Mice NaHS Treatment Alleviates the Reduction byIncreasing the Expression and Activity of Hydrogen Sulfide-Producing Enzymes Compared with young groups ageingmice showed lower CSE and CBS expression although3-MST expression remained unchanged Ten weeks ofNaHS treatments (50 120583molkgday) significantly increasedthe expression of CSE and CBS but not the 3-MST (Figures6(a1) 6(a2) and 6(a3)) Plasma urine and kidney H
2S levels
decreased significantly during ageing (Figures 6(b1) 6(b2)and 6(b3)) whereas 10 weeks of NaHS (50 120583molkgday)treatment alleviated the plasma and urine H
2S levels (Figures
6(b2) and 6(b3)) H2S levels in ageing kidney tissues were also
increased to some extent after NaHS treatment but withoutstatistical significance (Figure 6(b1)) CSECBS activity inthe ageing kidney decreased and 100 120583molkgday NaHStreatment diminished this reduction (Figure 6(c))
35 NaHSActivates SIRT1 in the Ageing Kidney To determinewhether H
2S protects the ageing kidney through the SIRT1-
mediated pathway the protein and transcriptional levelsof SIRT1 were examined Both the protein expression andmRNA transcription of SIRT1 were decreased in ageing micecompared with young ones and NaHS (50120583molkgday)treatment could improve SIRT1 protein expression but notthe mRNA levels (Figure 7(a)) SIRT1 is a deacetylase and10 weeks of NaHS treatment did not affect total deacetylaseactivity in the ageing kidney (Figure 7(b)) but 25120583molLNaHS could directly increase the activity of recombinantSIRT1 protein in vitro (Figure 7(c)) Measurement of SIRT1activity in the ageing kidney will help to make clear whetherchronic NaHS treatment selectively influences SIRT1 activitywhile total deacetylase activity remains the same
36 Effects of Chronic NaHS Treatment on the Expressionof Antioxidant-Related Proteins in the Ageing Kidney Theexpression of antioxidant proteins in kidney tissue wasexamined by western blot analysis at the end of chronicNaHS treatment Ten weeks of NaHS (50 120583molkgday) treat-ment could increase the Nrf2 expression and improve itsdownstream antioxidative proteins such as HO-1 SOD1 andSOD2 (Figure 8(a)) Moreover compared with young groupsboth the nuclear and cytosol Nrf2 levels were decreased andNaHS (50120583molkgday) treatment could selectively increasenuclear Nrf2 (Figure 8(b)) As shown in Figure S2A theNrf2 nuclear translation in the kidney tissue was insufficient
As shown in Figure S2B Nrf2 translocation in the NRK-52E cells treated with NaHS (50 120583molL) was significantlyinduced from cytosol to nucleus and peaked at 60min Thetranslocation ofNrf2 from cytoplasm into nuclearmay be oneof the protective mechanisms of H
2S against ageing-related
oxidative stress
4 Discussion
In this study we employed an ageing mouse model toinvestigate chronic NaHS treatment in the process of kidneysenescence Our work reveals two important findings (1)lower plasma urine and kidney H
2S levels and reduction of
kidneyCSE andCBS expression and activity are accompaniedwith ageing (2) exogenous administration of H
2S donor
NaHS mitigates ageing-related kidney dysfunction and theprotective effect of NaHS may at least partially relate toimproved endogenous H
2S production and its antioxidative
natureDuring ageing an elevated amount of ROS generated
from glycolysis specifically caused by the defects in thepolyol pathway uncoupling of nitric oxide synthase xanthineoxidase and advanced glycation results in the progressivedeterioration of renal function [18] Emerging data suggestthat ROS are related to the pathophysiology of glomerulardysfunction interstitial fibrosis and glomerulosclerosis [1920] In streptozotocin-induced diabetic rats H
2S therapy
(14 120583molkgday) improved renal function and decreasedglomerular basement thickening mesangial expansion andinterstitial fibrosis [18] Our previous study showed thatchronic NaHS treatment (30 60 and 120 120583molkgday) sig-nificantly reduced ROS levels in the kidney of GK rats [21]Consistently we found that chronic NaHS treatment couldreduce ROS levels (50 120583molkgday) and MDA contents (50and 100 120583molkgday) and increase GPx levels and SODactivity in the kidney of ageingmice H
2S takes part in a great
variety of physiological and pathophysiological processesbecause of its antiapoptotic antioxidative anti-inflammatoryand proangiogenic activities in mammals and the reduc-tion of endogenous H
2S levels has been related to various
diseases Zhou et al reported that endogenous H2S gen-
eration and CSE protein expression decreased significantlyin the streptozotocin-induced diabetic rat model and thatexogenous H
2S (14 120583molkgday) protected against diabetic
nephropathy [18] Our previous study also showed thatchronic NaHS (30 120583molkgday) treatment might amelioratediabetic complications of the kidney [21]
In this study we confirmed intensified interstitial fibrosisin the ageing kidney and observed decreased accumulation ofCol III (50 and 100 120583molkgday) and FN (at 50120583molkgday)with exogenous NaHSWe also observed increased apoptosisin the ageing kidney and chronic NaHS treatment could par-tially reverse this deterioration Age-related kidney damagemight reversely correlate with endogenous H
2S production
We found that both H2S levels in the plasma urine and
kidney and the kidney expression of CSE and CBS decreasedsignificantly with ageing As part of our bodyrsquos antioxidativedefense an insufficient H
2S system may decrease the overall
ability of ROS scavenging and render the kidney to be
8 Oxidative Medicine and Cellular Longevity
Young Old Old-50120583m NaHS
100120583m 100120583m 100120583m
(a)
GAPDH GAPDH
BAX Bcl-2
Young Old-50Old
00
05
10
15
BAX
GA
PDH
(fol
ds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
NaHS (120583molkgday)Young Old-50Old
NaHS (120583molkgday)
Old Old-50YoungNaHS (120583molkgday)
00
05
10
15
20
25
Bcl-2
GA
PDH
(fol
ds o
f old
cont
rol) P = 0001 P = 0029
P = 0034P = 0012
(b)
Young Old Old-50120583m NaHS
100120583m100120583m100120583m
(c)
Figure 5 Renal pathological changes of ageing mice (a) TUNEL staining of the kidney tissue (b) Bax (119873 = 6) and Bcl-2 (119873 = 7) expressionin ageing mice (c) HE staining of the kidney tissue Values are means plusmn SE 119875 lt 005 was considered significant
Oxidative Medicine and Cellular Longevity 9
GAPDH
GAPDH
CBS
CSE
Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old
GAPDH
MST
Young Old-100Old-50Old-10Old
NaHS (120583molkgday)P = 0008
P = 0029 P = 0028
P = 0020
P = 0048
P = 0007
P = 0029
P = 0001 P = 0005
P = 0043
P = 0014Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
(a1)
(a2)
(a3)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
Kidn
ey H
2S
(120583m
olg
)(fo
lds o
f old
cont
rol)
(b1)
(b2)
(b3)
(c)
00
05
10
15
Plas
ma H
2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
5
Urin
e H2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
(fold
s of o
ld co
ntro
l)CS
ECB
S ac
tivity
(120583m
olg
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
CSE
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
20
25
CBS
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
MST
GA
PDH
(fold
s of o
ld co
ntro
l)
Figure 6 Exogenous H2S alleviated ageing-related decreasing of H
2S production (a1)ndash(a3) Kidney expression of CSE (119873 = 11) and CBS
decreased significantly whereas 3-MST kept constant during ageing (119873 = 10) (b1)ndash(b3) Plasma urine and kidney tissueH2S levels decreased
significantly during ageing and rose again after chronic NaHS treatment (c) The activity of CSECBS in the ageing kidney declined whereas100 120583molkgday NaHS treatment could mitigate the reduction Values are means plusmn SE 119875 lt 005 was considered significant (119873 = 12)
10 Oxidative Medicine and Cellular Longevity
GAPDH
SIRT1
Young Old-100Old-50Old-10Old
P = 0007 P = 0015 P = 0023
NaHS (120583molkgday)
00
05
10
15
20
SIRT
1G
APD
H (f
olds
of o
ld co
ntro
l)
0
1
2
3
SIRT
1 m
RNA
(fol
ds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
P = 0049
00
05
10
15
Tota
l hist
one d
eace
tyla
se ac
tivity
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
P = 0001
P lt 0001
00
05
10
15
20
25
SIRT
1 ac
tivat
ion
in v
itro
(fold
s of c
ontro
l)
125 25 50 100ControlNaHS (120583molL)
(c)
Figure 7 SIRT1 was involved in NaHS-mediated ageing kidney protection (a) SIRT1 protein and mRNA expressions in the kidney afterchronic NaHS treatment for 10 weeks (119873 = 8) (b) Total deacetylase activity exchangeable in the kidney (119873 = 10) (c) NaHS-activatedrecombinant protein SIRT1 activity in vitro (119873 = 10) Values are mean plusmn SE 119875 lt 005 was considered significant
under oxidative stress Reasonably chronic NaHS treatmentprotects the ageing kidney by ROS scavenging and enhancingendogenous H
2S production in kidney tissue as reported
previously inmyocardium [22] DecreasedCBS activity in theageing kidney may also lead to homocysteine accumulationBecause the latter plays an important role in chronic renalfailure [23 24] further study is needed to explore whetherhomocysteine metabolism disorder is involved in ageing-related kidney damage and whether exogenous NaHS couldhelp restore the normal metabolic pathway
Christopher Hine reported that sulfur amino acid restric-tion could alter H
2S production and protect the liver from
ischemia-reperfusion injury [25] indicating the beneficialrole of endogenous H
2S SIRT1 which emerges as a major
life span regulator has been widely explored in the car-diovascular system and nervous system but it is rare in
the urinary system SIRT1 has robust biological effects andcan affect metabolic homeostasis and ageing Consistentwith previous studies [26 27] we found that both theexpression and activity of SIRT1 decreased rapidly in theageing kidney The reduction of NAD+ biosynthesis maybe responsible for reduced SIRT1 activity [26] Here wereport an interesting finding that a low concentration ofNaHS (25120583molL) could directly induce SIRT1 activationin a cell-free system whereas chronic treatment of NaHS(50 120583molkgday) selectively improves the expression but notthe activity of SIRT1 in the ageing kidney SIRT1 also regulateslipid metabolism by manipulating PPAR-120572 LXR FXR andSREBP signals [28 29] For example SIRT1 positively regu-lates LXR proteins which act as cholesterol sensor and adjustwhole body cholesterol and lipid homeostasis [30] Our bloodbiochemical tests showed that chronic NaHS treatment could
Oxidative Medicine and Cellular Longevity 11
Nrf2
GAPDH
GAPDH
SOD2
GAPDH
SOD1
GAPDH
HO-1
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday) NaHS (120583molkgday)
00
05
10
15
Nrf2
GA
PDH
(fol
ds o
f old
cont
rol)
00
05
10
15
20
HO
-1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
2G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
NS
GAPDH
Cyto Nrf2
Young Old-50OldNaHS (120583molkgday)
P lt 0001
00
05
10
15
20
Cyto
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
Nuclear Nrf2
Lamin B
Young Old-50Old
P = 0010 P = 0044
NaHS (120583molkgday)
00
05
10
15
20
Nuc
lear
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
Figure 8 The protective effects of the H2S donor on the expression of antioxidant-related proteins in the ageing kidney tissue (a) The
expression change of Nrf2 (119873 = 11) HO-1 (119873 = 10) SOD1 (119873 = 11) and SOD2 (119873 = 10) after 10 weeks of exogenous H2S donor (b) Nrf2
was translocated from cytosol to nucleus Lamin B was used as nuclear control and GAPDH was used as cytosol control (119873 = 6) Values aremean plusmn SE 119875 lt 005 was considered significant
12 Oxidative Medicine and Cellular Longevity
decrease CHOL and TG Therefore H2S may modulate liver
and renal lipidmetabolism through an enhanced SIRT1 signaland contribute to the prevention of renal disease progression
Nrf2 is an important antioxidative molecule that mod-ulates the expression of antioxidant genes [30] and thusprevents age-related oxidative stress [31 32] Here we showedthat the kidney Nrf2 expression decreased during age-ing and that chronic NaHS treatment could rescue Nrf2expression (10 and 50 120583molkgday) and enhance its nucleartranslocation (50120583molkgday) The precise mechanism ofexcessive oxidative stress in ageing is not clear but thedecreased expression of Nrf2antioxidant stress element-linked antioxidant genes is thought to play a role [32ndash34]We found that chronic NaHS treatment could activate Nrf2downstream genes such as HO-1 SOD1 and SOD2 whichconsequently enhanced resistance to oxidative stress in theageing kidney Taken together our data support the idea thatH2S plays a crucial role in protecting the ageing kidney from
antifibrosis and antiapoptosis through the regulation of redoxhomeostasis
5 Conclusion
In summary we demonstrated that 3-MST was expressed inthe ageing kidney and that endogenous H
2S levels decreased
because of the impaired expression ofH2S-producing enzymes
Chronic exogenous H2S treatment could protect the ageing
kidney by reducing oxidative stress decreasing collagendeposition and enhancing Nrf2 nuclear translocation aswell as increasing endogenous H
2S production
Competing Interests
The authors declare no conflict of interests regarding thepublication of this paper
Authorsrsquo ContributionsCui-Lan Hou and Ming-Jie Wang contributed equally to thiswork
AcknowledgmentsThe authors thank Bo Tan for his excellent technical assis-tance in H
2S measurements This present study was sup-
ported by the National Natural Science Foundation of China(NSFC) (no 81230003 to Yi-Chun Zhu no 81670248 toMing-Jie Wang and no 81402917 to Bo Tan) the Ministryof Education (20110071130009 to Yi-Chun Zhu) the Shang-hai Pujiang Program (15PJ1400700 to Ming-Jie Wang) theResearch Center on Ageing and Medicine Fudan University(13dz2260700 to Ming-Jie Wang) and Key Laboratory Pro-gramof the EducationCommission of ShanghaiMunicipality(ZDSYS14005 to Yi-Chun Zhu)
References
[1] J Volkert H SchulzMHarter OWlodarczyk and S AndreasldquoThe prevalence of mental disorders in older people inWesterncountriesmdasha meta-analysisrdquo Ageing Research Reviews vol 12no 1 pp 339ndash353 2013
[2] A Denic R J Glassock and A D Rule ldquoStructural andfunctional changes with the aging kidneyrdquo Advances in ChronicKidney Disease vol 23 no 1 pp 19ndash28 2016
[3] G Yang L Wu B Jiang et al ldquoH2S as a physiologic vasore-
laxant hypertension in mice with deletion of cystathionine 120574-lyaserdquo Science vol 322 no 5901 pp 587ndash590 2008
[4] Y Sun Y Huang R Zhang et al ldquoHydrogen sulfide upreg-ulates KATP channel expression in vascular smooth musclecells of spontaneously hypertensive ratsrdquo Journal of MolecularMedicine vol 93 no 4 pp 439ndash455 2015
[5] C Guo F Liang W Shah Masood and X Yan ldquoHydrogen sul-fide protected gastric epithelial cell from ischemiareperfusioninjury by Keap1 s-sulfhydration MAPK dependent anti-apoptosis and NF-120581B dependent anti-inflammation pathwayrdquoEuropean Journal of Pharmacology vol 725 no 1 pp 70ndash782014
[6] J Du Y Huang H Yan et al ldquoHydrogen sulfide suppresses oxi-dized low-density lipoprotein (Ox-LDL)-stimulated monocytechemoattractant protein 1 generation frommacrophages via thenuclear factor 120581b (NF-120581B) pathwayrdquo The Journal of BiologicalChemistry vol 289 no 14 pp 9741ndash9753 2014
[7] J Zheng T Zhao Y Yuan N Hu and X Tang ldquoHydrogensulfide (H
through oxidative stress and inflammatory response via Nrf2-NF-120581B pathwaysrdquo Chemico-Biological Interactions vol 242 no1 pp 353ndash362 2015
[8] D L Miller and M B Roth ldquoHydrogen sulfide increasesthermotolerance and lifespan in Caenorhabditis elegansrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 104 no 51 pp 20618ndash20622 2007
[9] Y Wei and C Kenyon ldquoRoles for ROS and hydrogen sulfidein the longevity response to germline loss in Caenorhabditiselegansrdquo Proceedings of the National Academy of Sciences vol113 no 20 pp e2832ndashe2841 2016
[10] Y Zhang Z-H Tang Z Ren et al ldquoHydrogen sulfide thenext potent preventive and therapeutic agent in aging and age-associated diseaserdquo Molecular and Cellular Biology vol 33 no6 pp 1104ndash1113 2013
[11] C Wei Y Zhao L Wang et al ldquoH2S restores the cardiopro-
tection from ischemic post-conditioning in isolated aged ratheartsrdquo Cell Biology International vol 39 no 10 pp 1173ndash11762015
[12] R E Sandu A Buga A T Balseanu M Moldovan and APopa-Wagner ldquoTwenty-four hours hypothermia has temporaryefficacy in reducing brain infarction and inflammation in agedratsrdquo Neurobiology of Aging vol 38 no 1 pp 127ndash140 2016
[13] S Jin S X Pu C L Hou et al ldquoCardiac H2S generation
is reduced in ageing diabetic micerdquo Oxidative Medicine andCellular Longevity vol 2015 Article ID 758358 14 pages 2015
[14] K L Quick and L L Dugan ldquoSuperoxide stress identifiesneurons at risk in a model of ataxia-telangiectasiardquo Annals ofNeurology vol 49 no 5 pp 627ndash635 2001
[15] X Shen C B Pattillo S Pardue S C Bir R Wang and C GKevil ldquoMeasurement of plasma hydrogen sulfide in vivo and invitrordquo Free Radical Biology andMedicine vol 50 no 9 pp 1021ndash1031 2011
[16] B-B Tao S-Y Liu C-C Zhang et al ldquoVEGFR2 functionsas an H
2S-targeting receptor protein kinase with its novel
Cys1045-Cys1024 disulfide bond serving as a specific molecularswitch for hydrogen sulfide actions in vascular endothelialcellsrdquo Antioxidants amp Redox Signaling vol 19 no 5 pp 448ndash464 2013
Oxidative Medicine and Cellular Longevity 13
[17] K J Bitterman R M Anderson H Y Cohen M Latorre-Esteves and D A Sinclair ldquoInhibition of silencing and accel-erated aging by nicotinamide a putative negative regulatorof yeast Sir2 and human SIRT1rdquo The Journal of BiologicalChemistry vol 277 no 47 pp 45099ndash45107 2002
[18] X Zhou Y Feng Z Zhan and J Chen ldquoHydrogen sulfidealleviates diabetic nephropathy in a streptozotocin-induceddiabetic ratmodelrdquoThe Journal of Biological Chemistry vol 289no 42 pp 28827ndash28834 2014
[19] P Wang C K Isaak Y L Siow and K O ldquoDownregulationof cystathionine 120573-synthase and cystathionine 120574-lyase expres-sion stimulates inflammation in kidney ischemiamdashreperfusioninjuryrdquo Physiological Reports vol 24 no 12 article e12251 2014
[20] F Yi and P-L Li ldquoMechanisms of homocysteine-inducedglomerular injury and sclerosisrdquo American Journal of Nephrol-ogy vol 28 no 2 pp 254ndash264 2008
[21] R Xue D-D Hao J-P Sun et al ldquoHydrogen sulfide treat-ment promotes glucose uptake by increasing insulin receptorsensitivity and ameliorates kidney lesions in type 2 diabetesrdquoAntioxidants amp Redox Signaling vol 19 no 1 pp 5ndash23 2013
[22] N Li M-J Wang S Jin et al ldquoThe H2S donor NaHS changes
the expression pattern ofH2S-producing enzymes aftermyocar-
dial infarctionrdquo Oxidative Medicine and Cellular Longevity vol2016 Article ID 6492469 11 pages 2016
[23] U Sen W E Rodriguez N Tyagi M Kumar S Kundu andS C Tyagi ldquoCiglitazone a PPAR120574 agonist ameliorates diabeticnephropathy in part through homocysteine clearancerdquo Ameri-can Journal of PhysiologymdashEndocrinology and Metabolism vol295 no 5 pp E1205ndashE1212 2008
[24] W E Rodriguez U Sen N Tyagi et al ldquoPPAR gammaagonist normalizes glomerular filtration rate tissue levels ofhomocysteine and attenuates endothelial-myocyte uncouplingin alloxan induced diabetic micerdquo International Journal ofBiological Sciences vol 4 no 4 pp 236ndash244 2008
[25] C Hine E Harputlugil Y Zhang et al ldquoEndogenous hydrogensulfide production is essential for dietary restriction benefitsrdquoCell vol 160 no 1-2 pp 132ndash144 2015
[26] S-I Imai ldquoDissecting systemic control ofmetabolism and agingin the NAD World the importance of SIRT1 and NAMPT-mediated NAD biosynthesisrdquo FEBS Letters vol 585 no 11 pp1657ndash1662 2011
[27] N Braidy G J Guillemin H Mansour T Chan-Ling APoljak and R Grant ldquoAge related changes inNAD+metabolismoxidative stress and sirt1 activity in wistar ratsrdquo PLoS ONE vol6 no 4 Article ID e19194 pp 1ndash18 2011
[28] A PurushothamT T SchugQXu S Surapureddi XGuo andX Li ldquoHepatocyte-specific deletion of SIRT1 alters fatty acidmetabolism and results in hepatic steatosis and inflammationrdquoCell Metabolism vol 9 no 4 pp 327ndash338 2009
[29] A K Walker F Yang K Jiang et al ldquoConserved roleof SIRT1 orthologs in fasting-dependent inhibition of thelipidcholesterol regulator SREBPrdquoGenes and Development vol24 no 13 pp 1403ndash1417 2010
[30] X Li S Zhang G Blander J G Tse M Krieger and L Guar-ente ldquoSIRT1 deacetylates and positively regulates the nuclearreceptor LXRrdquoMolecular Cell vol 28 no 1 pp 91ndash106 2007
[31] M M Sachdeva M Cano and J T Handa ldquoNrf2 signaling isimpaired in the aging RPE given an oxidative insultrdquo Experi-mental Eye Research vol 119 no 1 pp 111ndash114 2014
[32] A Loboda M Damulewicz E Pyza A Jozkowicz and JDulak ldquoRole of Nrf2HO-1 system in development oxidative
stress response and diseases an evolutionarily conservedmech-anismrdquo Cellular and Molecular Life Sciences vol 73 no 17 pp3221ndash3247 2016
[33] G Yang K Zhao Y Ju et al ldquoHydrogen sulfide protects againstcellular senescence via S-sulfhydration of Keap1 and activationof Nrf2rdquo Antioxidants amp Redox Signaling vol 18 no 15 pp1906ndash1919 2013
[34] A J Done M J Gage N C Nieto and T TraustadottirldquoExercise-induced Nrf2-signaling is impaired in agingrdquo FreeRadical Biology and Medicine vol 96 no 1 pp 130ndash138 2016
Figure 5 Renal pathological changes of ageing mice (a) TUNEL staining of the kidney tissue (b) Bax (119873 = 6) and Bcl-2 (119873 = 7) expressionin ageing mice (c) HE staining of the kidney tissue Values are means plusmn SE 119875 lt 005 was considered significant
Oxidative Medicine and Cellular Longevity 9
GAPDH
GAPDH
CBS
CSE
Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old
GAPDH
MST
Young Old-100Old-50Old-10Old
NaHS (120583molkgday)P = 0008
P = 0029 P = 0028
P = 0020
P = 0048
P = 0007
P = 0029
P = 0001 P = 0005
P = 0043
P = 0014Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
(a1)
(a2)
(a3)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
Kidn
ey H
2S
(120583m
olg
)(fo
lds o
f old
cont
rol)
(b1)
(b2)
(b3)
(c)
00
05
10
15
Plas
ma H
2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
5
Urin
e H2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
(fold
s of o
ld co
ntro
l)CS
ECB
S ac
tivity
(120583m
olg
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
CSE
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
20
25
CBS
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
MST
GA
PDH
(fold
s of o
ld co
ntro
l)
Figure 6 Exogenous H2S alleviated ageing-related decreasing of H
2S production (a1)ndash(a3) Kidney expression of CSE (119873 = 11) and CBS
decreased significantly whereas 3-MST kept constant during ageing (119873 = 10) (b1)ndash(b3) Plasma urine and kidney tissueH2S levels decreased
significantly during ageing and rose again after chronic NaHS treatment (c) The activity of CSECBS in the ageing kidney declined whereas100 120583molkgday NaHS treatment could mitigate the reduction Values are means plusmn SE 119875 lt 005 was considered significant (119873 = 12)
10 Oxidative Medicine and Cellular Longevity
GAPDH
SIRT1
Young Old-100Old-50Old-10Old
P = 0007 P = 0015 P = 0023
NaHS (120583molkgday)
00
05
10
15
20
SIRT
1G
APD
H (f
olds
of o
ld co
ntro
l)
0
1
2
3
SIRT
1 m
RNA
(fol
ds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
P = 0049
00
05
10
15
Tota
l hist
one d
eace
tyla
se ac
tivity
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
P = 0001
P lt 0001
00
05
10
15
20
25
SIRT
1 ac
tivat
ion
in v
itro
(fold
s of c
ontro
l)
125 25 50 100ControlNaHS (120583molL)
(c)
Figure 7 SIRT1 was involved in NaHS-mediated ageing kidney protection (a) SIRT1 protein and mRNA expressions in the kidney afterchronic NaHS treatment for 10 weeks (119873 = 8) (b) Total deacetylase activity exchangeable in the kidney (119873 = 10) (c) NaHS-activatedrecombinant protein SIRT1 activity in vitro (119873 = 10) Values are mean plusmn SE 119875 lt 005 was considered significant
under oxidative stress Reasonably chronic NaHS treatmentprotects the ageing kidney by ROS scavenging and enhancingendogenous H
2S production in kidney tissue as reported
previously inmyocardium [22] DecreasedCBS activity in theageing kidney may also lead to homocysteine accumulationBecause the latter plays an important role in chronic renalfailure [23 24] further study is needed to explore whetherhomocysteine metabolism disorder is involved in ageing-related kidney damage and whether exogenous NaHS couldhelp restore the normal metabolic pathway
Christopher Hine reported that sulfur amino acid restric-tion could alter H
2S production and protect the liver from
ischemia-reperfusion injury [25] indicating the beneficialrole of endogenous H
2S SIRT1 which emerges as a major
life span regulator has been widely explored in the car-diovascular system and nervous system but it is rare in
the urinary system SIRT1 has robust biological effects andcan affect metabolic homeostasis and ageing Consistentwith previous studies [26 27] we found that both theexpression and activity of SIRT1 decreased rapidly in theageing kidney The reduction of NAD+ biosynthesis maybe responsible for reduced SIRT1 activity [26] Here wereport an interesting finding that a low concentration ofNaHS (25120583molL) could directly induce SIRT1 activationin a cell-free system whereas chronic treatment of NaHS(50 120583molkgday) selectively improves the expression but notthe activity of SIRT1 in the ageing kidney SIRT1 also regulateslipid metabolism by manipulating PPAR-120572 LXR FXR andSREBP signals [28 29] For example SIRT1 positively regu-lates LXR proteins which act as cholesterol sensor and adjustwhole body cholesterol and lipid homeostasis [30] Our bloodbiochemical tests showed that chronic NaHS treatment could
Oxidative Medicine and Cellular Longevity 11
Nrf2
GAPDH
GAPDH
SOD2
GAPDH
SOD1
GAPDH
HO-1
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday) NaHS (120583molkgday)
00
05
10
15
Nrf2
GA
PDH
(fol
ds o
f old
cont
rol)
00
05
10
15
20
HO
-1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
2G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
NS
GAPDH
Cyto Nrf2
Young Old-50OldNaHS (120583molkgday)
P lt 0001
00
05
10
15
20
Cyto
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
Nuclear Nrf2
Lamin B
Young Old-50Old
P = 0010 P = 0044
NaHS (120583molkgday)
00
05
10
15
20
Nuc
lear
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
Figure 8 The protective effects of the H2S donor on the expression of antioxidant-related proteins in the ageing kidney tissue (a) The
expression change of Nrf2 (119873 = 11) HO-1 (119873 = 10) SOD1 (119873 = 11) and SOD2 (119873 = 10) after 10 weeks of exogenous H2S donor (b) Nrf2
was translocated from cytosol to nucleus Lamin B was used as nuclear control and GAPDH was used as cytosol control (119873 = 6) Values aremean plusmn SE 119875 lt 005 was considered significant
12 Oxidative Medicine and Cellular Longevity
decrease CHOL and TG Therefore H2S may modulate liver
and renal lipidmetabolism through an enhanced SIRT1 signaland contribute to the prevention of renal disease progression
Nrf2 is an important antioxidative molecule that mod-ulates the expression of antioxidant genes [30] and thusprevents age-related oxidative stress [31 32] Here we showedthat the kidney Nrf2 expression decreased during age-ing and that chronic NaHS treatment could rescue Nrf2expression (10 and 50 120583molkgday) and enhance its nucleartranslocation (50120583molkgday) The precise mechanism ofexcessive oxidative stress in ageing is not clear but thedecreased expression of Nrf2antioxidant stress element-linked antioxidant genes is thought to play a role [32ndash34]We found that chronic NaHS treatment could activate Nrf2downstream genes such as HO-1 SOD1 and SOD2 whichconsequently enhanced resistance to oxidative stress in theageing kidney Taken together our data support the idea thatH2S plays a crucial role in protecting the ageing kidney from
antifibrosis and antiapoptosis through the regulation of redoxhomeostasis
5 Conclusion
In summary we demonstrated that 3-MST was expressed inthe ageing kidney and that endogenous H
2S levels decreased
because of the impaired expression ofH2S-producing enzymes
Chronic exogenous H2S treatment could protect the ageing
kidney by reducing oxidative stress decreasing collagendeposition and enhancing Nrf2 nuclear translocation aswell as increasing endogenous H
2S production
Competing Interests
The authors declare no conflict of interests regarding thepublication of this paper
Authorsrsquo ContributionsCui-Lan Hou and Ming-Jie Wang contributed equally to thiswork
AcknowledgmentsThe authors thank Bo Tan for his excellent technical assis-tance in H
2S measurements This present study was sup-
ported by the National Natural Science Foundation of China(NSFC) (no 81230003 to Yi-Chun Zhu no 81670248 toMing-Jie Wang and no 81402917 to Bo Tan) the Ministryof Education (20110071130009 to Yi-Chun Zhu) the Shang-hai Pujiang Program (15PJ1400700 to Ming-Jie Wang) theResearch Center on Ageing and Medicine Fudan University(13dz2260700 to Ming-Jie Wang) and Key Laboratory Pro-gramof the EducationCommission of ShanghaiMunicipality(ZDSYS14005 to Yi-Chun Zhu)
References
[1] J Volkert H SchulzMHarter OWlodarczyk and S AndreasldquoThe prevalence of mental disorders in older people inWesterncountriesmdasha meta-analysisrdquo Ageing Research Reviews vol 12no 1 pp 339ndash353 2013
[2] A Denic R J Glassock and A D Rule ldquoStructural andfunctional changes with the aging kidneyrdquo Advances in ChronicKidney Disease vol 23 no 1 pp 19ndash28 2016
[3] G Yang L Wu B Jiang et al ldquoH2S as a physiologic vasore-
laxant hypertension in mice with deletion of cystathionine 120574-lyaserdquo Science vol 322 no 5901 pp 587ndash590 2008
[4] Y Sun Y Huang R Zhang et al ldquoHydrogen sulfide upreg-ulates KATP channel expression in vascular smooth musclecells of spontaneously hypertensive ratsrdquo Journal of MolecularMedicine vol 93 no 4 pp 439ndash455 2015
[5] C Guo F Liang W Shah Masood and X Yan ldquoHydrogen sul-fide protected gastric epithelial cell from ischemiareperfusioninjury by Keap1 s-sulfhydration MAPK dependent anti-apoptosis and NF-120581B dependent anti-inflammation pathwayrdquoEuropean Journal of Pharmacology vol 725 no 1 pp 70ndash782014
[6] J Du Y Huang H Yan et al ldquoHydrogen sulfide suppresses oxi-dized low-density lipoprotein (Ox-LDL)-stimulated monocytechemoattractant protein 1 generation frommacrophages via thenuclear factor 120581b (NF-120581B) pathwayrdquo The Journal of BiologicalChemistry vol 289 no 14 pp 9741ndash9753 2014
[7] J Zheng T Zhao Y Yuan N Hu and X Tang ldquoHydrogensulfide (H
through oxidative stress and inflammatory response via Nrf2-NF-120581B pathwaysrdquo Chemico-Biological Interactions vol 242 no1 pp 353ndash362 2015
[8] D L Miller and M B Roth ldquoHydrogen sulfide increasesthermotolerance and lifespan in Caenorhabditis elegansrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 104 no 51 pp 20618ndash20622 2007
[9] Y Wei and C Kenyon ldquoRoles for ROS and hydrogen sulfidein the longevity response to germline loss in Caenorhabditiselegansrdquo Proceedings of the National Academy of Sciences vol113 no 20 pp e2832ndashe2841 2016
[10] Y Zhang Z-H Tang Z Ren et al ldquoHydrogen sulfide thenext potent preventive and therapeutic agent in aging and age-associated diseaserdquo Molecular and Cellular Biology vol 33 no6 pp 1104ndash1113 2013
[11] C Wei Y Zhao L Wang et al ldquoH2S restores the cardiopro-
tection from ischemic post-conditioning in isolated aged ratheartsrdquo Cell Biology International vol 39 no 10 pp 1173ndash11762015
[12] R E Sandu A Buga A T Balseanu M Moldovan and APopa-Wagner ldquoTwenty-four hours hypothermia has temporaryefficacy in reducing brain infarction and inflammation in agedratsrdquo Neurobiology of Aging vol 38 no 1 pp 127ndash140 2016
[13] S Jin S X Pu C L Hou et al ldquoCardiac H2S generation
is reduced in ageing diabetic micerdquo Oxidative Medicine andCellular Longevity vol 2015 Article ID 758358 14 pages 2015
[14] K L Quick and L L Dugan ldquoSuperoxide stress identifiesneurons at risk in a model of ataxia-telangiectasiardquo Annals ofNeurology vol 49 no 5 pp 627ndash635 2001
[15] X Shen C B Pattillo S Pardue S C Bir R Wang and C GKevil ldquoMeasurement of plasma hydrogen sulfide in vivo and invitrordquo Free Radical Biology andMedicine vol 50 no 9 pp 1021ndash1031 2011
[16] B-B Tao S-Y Liu C-C Zhang et al ldquoVEGFR2 functionsas an H
2S-targeting receptor protein kinase with its novel
Cys1045-Cys1024 disulfide bond serving as a specific molecularswitch for hydrogen sulfide actions in vascular endothelialcellsrdquo Antioxidants amp Redox Signaling vol 19 no 5 pp 448ndash464 2013
Oxidative Medicine and Cellular Longevity 13
[17] K J Bitterman R M Anderson H Y Cohen M Latorre-Esteves and D A Sinclair ldquoInhibition of silencing and accel-erated aging by nicotinamide a putative negative regulatorof yeast Sir2 and human SIRT1rdquo The Journal of BiologicalChemistry vol 277 no 47 pp 45099ndash45107 2002
[18] X Zhou Y Feng Z Zhan and J Chen ldquoHydrogen sulfidealleviates diabetic nephropathy in a streptozotocin-induceddiabetic ratmodelrdquoThe Journal of Biological Chemistry vol 289no 42 pp 28827ndash28834 2014
[19] P Wang C K Isaak Y L Siow and K O ldquoDownregulationof cystathionine 120573-synthase and cystathionine 120574-lyase expres-sion stimulates inflammation in kidney ischemiamdashreperfusioninjuryrdquo Physiological Reports vol 24 no 12 article e12251 2014
[20] F Yi and P-L Li ldquoMechanisms of homocysteine-inducedglomerular injury and sclerosisrdquo American Journal of Nephrol-ogy vol 28 no 2 pp 254ndash264 2008
[21] R Xue D-D Hao J-P Sun et al ldquoHydrogen sulfide treat-ment promotes glucose uptake by increasing insulin receptorsensitivity and ameliorates kidney lesions in type 2 diabetesrdquoAntioxidants amp Redox Signaling vol 19 no 1 pp 5ndash23 2013
[22] N Li M-J Wang S Jin et al ldquoThe H2S donor NaHS changes
the expression pattern ofH2S-producing enzymes aftermyocar-
dial infarctionrdquo Oxidative Medicine and Cellular Longevity vol2016 Article ID 6492469 11 pages 2016
[23] U Sen W E Rodriguez N Tyagi M Kumar S Kundu andS C Tyagi ldquoCiglitazone a PPAR120574 agonist ameliorates diabeticnephropathy in part through homocysteine clearancerdquo Ameri-can Journal of PhysiologymdashEndocrinology and Metabolism vol295 no 5 pp E1205ndashE1212 2008
[24] W E Rodriguez U Sen N Tyagi et al ldquoPPAR gammaagonist normalizes glomerular filtration rate tissue levels ofhomocysteine and attenuates endothelial-myocyte uncouplingin alloxan induced diabetic micerdquo International Journal ofBiological Sciences vol 4 no 4 pp 236ndash244 2008
[25] C Hine E Harputlugil Y Zhang et al ldquoEndogenous hydrogensulfide production is essential for dietary restriction benefitsrdquoCell vol 160 no 1-2 pp 132ndash144 2015
[26] S-I Imai ldquoDissecting systemic control ofmetabolism and agingin the NAD World the importance of SIRT1 and NAMPT-mediated NAD biosynthesisrdquo FEBS Letters vol 585 no 11 pp1657ndash1662 2011
[27] N Braidy G J Guillemin H Mansour T Chan-Ling APoljak and R Grant ldquoAge related changes inNAD+metabolismoxidative stress and sirt1 activity in wistar ratsrdquo PLoS ONE vol6 no 4 Article ID e19194 pp 1ndash18 2011
[28] A PurushothamT T SchugQXu S Surapureddi XGuo andX Li ldquoHepatocyte-specific deletion of SIRT1 alters fatty acidmetabolism and results in hepatic steatosis and inflammationrdquoCell Metabolism vol 9 no 4 pp 327ndash338 2009
[29] A K Walker F Yang K Jiang et al ldquoConserved roleof SIRT1 orthologs in fasting-dependent inhibition of thelipidcholesterol regulator SREBPrdquoGenes and Development vol24 no 13 pp 1403ndash1417 2010
[30] X Li S Zhang G Blander J G Tse M Krieger and L Guar-ente ldquoSIRT1 deacetylates and positively regulates the nuclearreceptor LXRrdquoMolecular Cell vol 28 no 1 pp 91ndash106 2007
[31] M M Sachdeva M Cano and J T Handa ldquoNrf2 signaling isimpaired in the aging RPE given an oxidative insultrdquo Experi-mental Eye Research vol 119 no 1 pp 111ndash114 2014
[32] A Loboda M Damulewicz E Pyza A Jozkowicz and JDulak ldquoRole of Nrf2HO-1 system in development oxidative
stress response and diseases an evolutionarily conservedmech-anismrdquo Cellular and Molecular Life Sciences vol 73 no 17 pp3221ndash3247 2016
[33] G Yang K Zhao Y Ju et al ldquoHydrogen sulfide protects againstcellular senescence via S-sulfhydration of Keap1 and activationof Nrf2rdquo Antioxidants amp Redox Signaling vol 18 no 15 pp1906ndash1919 2013
[34] A J Done M J Gage N C Nieto and T TraustadottirldquoExercise-induced Nrf2-signaling is impaired in agingrdquo FreeRadical Biology and Medicine vol 96 no 1 pp 130ndash138 2016
P = 0014Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
(a1)
(a2)
(a3)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
Kidn
ey H
2S
(120583m
olg
)(fo
lds o
f old
cont
rol)
(b1)
(b2)
(b3)
(c)
00
05
10
15
Plas
ma H
2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
0
1
2
3
4
5
Urin
e H2S
(120583m
olL
)(fo
lds o
f old
cont
rol)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
(fold
s of o
ld co
ntro
l)CS
ECB
S ac
tivity
(120583m
olg
)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
CSE
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
20
25
CBS
GA
PDH
(fold
s of o
ld co
ntro
l)
00
05
10
15
MST
GA
PDH
(fold
s of o
ld co
ntro
l)
Figure 6 Exogenous H2S alleviated ageing-related decreasing of H
2S production (a1)ndash(a3) Kidney expression of CSE (119873 = 11) and CBS
decreased significantly whereas 3-MST kept constant during ageing (119873 = 10) (b1)ndash(b3) Plasma urine and kidney tissueH2S levels decreased
significantly during ageing and rose again after chronic NaHS treatment (c) The activity of CSECBS in the ageing kidney declined whereas100 120583molkgday NaHS treatment could mitigate the reduction Values are means plusmn SE 119875 lt 005 was considered significant (119873 = 12)
10 Oxidative Medicine and Cellular Longevity
GAPDH
SIRT1
Young Old-100Old-50Old-10Old
P = 0007 P = 0015 P = 0023
NaHS (120583molkgday)
00
05
10
15
20
SIRT
1G
APD
H (f
olds
of o
ld co
ntro
l)
0
1
2
3
SIRT
1 m
RNA
(fol
ds o
f old
cont
rol)
Old Old-50YoungNaHS (120583molkgday)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
P = 0049
00
05
10
15
Tota
l hist
one d
eace
tyla
se ac
tivity
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
P = 0001
P lt 0001
00
05
10
15
20
25
SIRT
1 ac
tivat
ion
in v
itro
(fold
s of c
ontro
l)
125 25 50 100ControlNaHS (120583molL)
(c)
Figure 7 SIRT1 was involved in NaHS-mediated ageing kidney protection (a) SIRT1 protein and mRNA expressions in the kidney afterchronic NaHS treatment for 10 weeks (119873 = 8) (b) Total deacetylase activity exchangeable in the kidney (119873 = 10) (c) NaHS-activatedrecombinant protein SIRT1 activity in vitro (119873 = 10) Values are mean plusmn SE 119875 lt 005 was considered significant
under oxidative stress Reasonably chronic NaHS treatmentprotects the ageing kidney by ROS scavenging and enhancingendogenous H
2S production in kidney tissue as reported
previously inmyocardium [22] DecreasedCBS activity in theageing kidney may also lead to homocysteine accumulationBecause the latter plays an important role in chronic renalfailure [23 24] further study is needed to explore whetherhomocysteine metabolism disorder is involved in ageing-related kidney damage and whether exogenous NaHS couldhelp restore the normal metabolic pathway
Christopher Hine reported that sulfur amino acid restric-tion could alter H
2S production and protect the liver from
ischemia-reperfusion injury [25] indicating the beneficialrole of endogenous H
2S SIRT1 which emerges as a major
life span regulator has been widely explored in the car-diovascular system and nervous system but it is rare in
the urinary system SIRT1 has robust biological effects andcan affect metabolic homeostasis and ageing Consistentwith previous studies [26 27] we found that both theexpression and activity of SIRT1 decreased rapidly in theageing kidney The reduction of NAD+ biosynthesis maybe responsible for reduced SIRT1 activity [26] Here wereport an interesting finding that a low concentration ofNaHS (25120583molL) could directly induce SIRT1 activationin a cell-free system whereas chronic treatment of NaHS(50 120583molkgday) selectively improves the expression but notthe activity of SIRT1 in the ageing kidney SIRT1 also regulateslipid metabolism by manipulating PPAR-120572 LXR FXR andSREBP signals [28 29] For example SIRT1 positively regu-lates LXR proteins which act as cholesterol sensor and adjustwhole body cholesterol and lipid homeostasis [30] Our bloodbiochemical tests showed that chronic NaHS treatment could
Oxidative Medicine and Cellular Longevity 11
Nrf2
GAPDH
GAPDH
SOD2
GAPDH
SOD1
GAPDH
HO-1
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday) NaHS (120583molkgday)
00
05
10
15
Nrf2
GA
PDH
(fol
ds o
f old
cont
rol)
00
05
10
15
20
HO
-1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
2G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
NS
GAPDH
Cyto Nrf2
Young Old-50OldNaHS (120583molkgday)
P lt 0001
00
05
10
15
20
Cyto
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
Nuclear Nrf2
Lamin B
Young Old-50Old
P = 0010 P = 0044
NaHS (120583molkgday)
00
05
10
15
20
Nuc
lear
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
Figure 8 The protective effects of the H2S donor on the expression of antioxidant-related proteins in the ageing kidney tissue (a) The
expression change of Nrf2 (119873 = 11) HO-1 (119873 = 10) SOD1 (119873 = 11) and SOD2 (119873 = 10) after 10 weeks of exogenous H2S donor (b) Nrf2
was translocated from cytosol to nucleus Lamin B was used as nuclear control and GAPDH was used as cytosol control (119873 = 6) Values aremean plusmn SE 119875 lt 005 was considered significant
12 Oxidative Medicine and Cellular Longevity
decrease CHOL and TG Therefore H2S may modulate liver
and renal lipidmetabolism through an enhanced SIRT1 signaland contribute to the prevention of renal disease progression
Nrf2 is an important antioxidative molecule that mod-ulates the expression of antioxidant genes [30] and thusprevents age-related oxidative stress [31 32] Here we showedthat the kidney Nrf2 expression decreased during age-ing and that chronic NaHS treatment could rescue Nrf2expression (10 and 50 120583molkgday) and enhance its nucleartranslocation (50120583molkgday) The precise mechanism ofexcessive oxidative stress in ageing is not clear but thedecreased expression of Nrf2antioxidant stress element-linked antioxidant genes is thought to play a role [32ndash34]We found that chronic NaHS treatment could activate Nrf2downstream genes such as HO-1 SOD1 and SOD2 whichconsequently enhanced resistance to oxidative stress in theageing kidney Taken together our data support the idea thatH2S plays a crucial role in protecting the ageing kidney from
antifibrosis and antiapoptosis through the regulation of redoxhomeostasis
5 Conclusion
In summary we demonstrated that 3-MST was expressed inthe ageing kidney and that endogenous H
2S levels decreased
because of the impaired expression ofH2S-producing enzymes
Chronic exogenous H2S treatment could protect the ageing
kidney by reducing oxidative stress decreasing collagendeposition and enhancing Nrf2 nuclear translocation aswell as increasing endogenous H
2S production
Competing Interests
The authors declare no conflict of interests regarding thepublication of this paper
Authorsrsquo ContributionsCui-Lan Hou and Ming-Jie Wang contributed equally to thiswork
AcknowledgmentsThe authors thank Bo Tan for his excellent technical assis-tance in H
2S measurements This present study was sup-
ported by the National Natural Science Foundation of China(NSFC) (no 81230003 to Yi-Chun Zhu no 81670248 toMing-Jie Wang and no 81402917 to Bo Tan) the Ministryof Education (20110071130009 to Yi-Chun Zhu) the Shang-hai Pujiang Program (15PJ1400700 to Ming-Jie Wang) theResearch Center on Ageing and Medicine Fudan University(13dz2260700 to Ming-Jie Wang) and Key Laboratory Pro-gramof the EducationCommission of ShanghaiMunicipality(ZDSYS14005 to Yi-Chun Zhu)
References
[1] J Volkert H SchulzMHarter OWlodarczyk and S AndreasldquoThe prevalence of mental disorders in older people inWesterncountriesmdasha meta-analysisrdquo Ageing Research Reviews vol 12no 1 pp 339ndash353 2013
[2] A Denic R J Glassock and A D Rule ldquoStructural andfunctional changes with the aging kidneyrdquo Advances in ChronicKidney Disease vol 23 no 1 pp 19ndash28 2016
[3] G Yang L Wu B Jiang et al ldquoH2S as a physiologic vasore-
laxant hypertension in mice with deletion of cystathionine 120574-lyaserdquo Science vol 322 no 5901 pp 587ndash590 2008
[4] Y Sun Y Huang R Zhang et al ldquoHydrogen sulfide upreg-ulates KATP channel expression in vascular smooth musclecells of spontaneously hypertensive ratsrdquo Journal of MolecularMedicine vol 93 no 4 pp 439ndash455 2015
[5] C Guo F Liang W Shah Masood and X Yan ldquoHydrogen sul-fide protected gastric epithelial cell from ischemiareperfusioninjury by Keap1 s-sulfhydration MAPK dependent anti-apoptosis and NF-120581B dependent anti-inflammation pathwayrdquoEuropean Journal of Pharmacology vol 725 no 1 pp 70ndash782014
[6] J Du Y Huang H Yan et al ldquoHydrogen sulfide suppresses oxi-dized low-density lipoprotein (Ox-LDL)-stimulated monocytechemoattractant protein 1 generation frommacrophages via thenuclear factor 120581b (NF-120581B) pathwayrdquo The Journal of BiologicalChemistry vol 289 no 14 pp 9741ndash9753 2014
[7] J Zheng T Zhao Y Yuan N Hu and X Tang ldquoHydrogensulfide (H
through oxidative stress and inflammatory response via Nrf2-NF-120581B pathwaysrdquo Chemico-Biological Interactions vol 242 no1 pp 353ndash362 2015
[8] D L Miller and M B Roth ldquoHydrogen sulfide increasesthermotolerance and lifespan in Caenorhabditis elegansrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 104 no 51 pp 20618ndash20622 2007
[9] Y Wei and C Kenyon ldquoRoles for ROS and hydrogen sulfidein the longevity response to germline loss in Caenorhabditiselegansrdquo Proceedings of the National Academy of Sciences vol113 no 20 pp e2832ndashe2841 2016
[10] Y Zhang Z-H Tang Z Ren et al ldquoHydrogen sulfide thenext potent preventive and therapeutic agent in aging and age-associated diseaserdquo Molecular and Cellular Biology vol 33 no6 pp 1104ndash1113 2013
[11] C Wei Y Zhao L Wang et al ldquoH2S restores the cardiopro-
tection from ischemic post-conditioning in isolated aged ratheartsrdquo Cell Biology International vol 39 no 10 pp 1173ndash11762015
[12] R E Sandu A Buga A T Balseanu M Moldovan and APopa-Wagner ldquoTwenty-four hours hypothermia has temporaryefficacy in reducing brain infarction and inflammation in agedratsrdquo Neurobiology of Aging vol 38 no 1 pp 127ndash140 2016
[13] S Jin S X Pu C L Hou et al ldquoCardiac H2S generation
is reduced in ageing diabetic micerdquo Oxidative Medicine andCellular Longevity vol 2015 Article ID 758358 14 pages 2015
[14] K L Quick and L L Dugan ldquoSuperoxide stress identifiesneurons at risk in a model of ataxia-telangiectasiardquo Annals ofNeurology vol 49 no 5 pp 627ndash635 2001
[15] X Shen C B Pattillo S Pardue S C Bir R Wang and C GKevil ldquoMeasurement of plasma hydrogen sulfide in vivo and invitrordquo Free Radical Biology andMedicine vol 50 no 9 pp 1021ndash1031 2011
[16] B-B Tao S-Y Liu C-C Zhang et al ldquoVEGFR2 functionsas an H
2S-targeting receptor protein kinase with its novel
Cys1045-Cys1024 disulfide bond serving as a specific molecularswitch for hydrogen sulfide actions in vascular endothelialcellsrdquo Antioxidants amp Redox Signaling vol 19 no 5 pp 448ndash464 2013
Oxidative Medicine and Cellular Longevity 13
[17] K J Bitterman R M Anderson H Y Cohen M Latorre-Esteves and D A Sinclair ldquoInhibition of silencing and accel-erated aging by nicotinamide a putative negative regulatorof yeast Sir2 and human SIRT1rdquo The Journal of BiologicalChemistry vol 277 no 47 pp 45099ndash45107 2002
[18] X Zhou Y Feng Z Zhan and J Chen ldquoHydrogen sulfidealleviates diabetic nephropathy in a streptozotocin-induceddiabetic ratmodelrdquoThe Journal of Biological Chemistry vol 289no 42 pp 28827ndash28834 2014
[19] P Wang C K Isaak Y L Siow and K O ldquoDownregulationof cystathionine 120573-synthase and cystathionine 120574-lyase expres-sion stimulates inflammation in kidney ischemiamdashreperfusioninjuryrdquo Physiological Reports vol 24 no 12 article e12251 2014
[20] F Yi and P-L Li ldquoMechanisms of homocysteine-inducedglomerular injury and sclerosisrdquo American Journal of Nephrol-ogy vol 28 no 2 pp 254ndash264 2008
[21] R Xue D-D Hao J-P Sun et al ldquoHydrogen sulfide treat-ment promotes glucose uptake by increasing insulin receptorsensitivity and ameliorates kidney lesions in type 2 diabetesrdquoAntioxidants amp Redox Signaling vol 19 no 1 pp 5ndash23 2013
[22] N Li M-J Wang S Jin et al ldquoThe H2S donor NaHS changes
the expression pattern ofH2S-producing enzymes aftermyocar-
dial infarctionrdquo Oxidative Medicine and Cellular Longevity vol2016 Article ID 6492469 11 pages 2016
[23] U Sen W E Rodriguez N Tyagi M Kumar S Kundu andS C Tyagi ldquoCiglitazone a PPAR120574 agonist ameliorates diabeticnephropathy in part through homocysteine clearancerdquo Ameri-can Journal of PhysiologymdashEndocrinology and Metabolism vol295 no 5 pp E1205ndashE1212 2008
[24] W E Rodriguez U Sen N Tyagi et al ldquoPPAR gammaagonist normalizes glomerular filtration rate tissue levels ofhomocysteine and attenuates endothelial-myocyte uncouplingin alloxan induced diabetic micerdquo International Journal ofBiological Sciences vol 4 no 4 pp 236ndash244 2008
[25] C Hine E Harputlugil Y Zhang et al ldquoEndogenous hydrogensulfide production is essential for dietary restriction benefitsrdquoCell vol 160 no 1-2 pp 132ndash144 2015
[26] S-I Imai ldquoDissecting systemic control ofmetabolism and agingin the NAD World the importance of SIRT1 and NAMPT-mediated NAD biosynthesisrdquo FEBS Letters vol 585 no 11 pp1657ndash1662 2011
[27] N Braidy G J Guillemin H Mansour T Chan-Ling APoljak and R Grant ldquoAge related changes inNAD+metabolismoxidative stress and sirt1 activity in wistar ratsrdquo PLoS ONE vol6 no 4 Article ID e19194 pp 1ndash18 2011
[28] A PurushothamT T SchugQXu S Surapureddi XGuo andX Li ldquoHepatocyte-specific deletion of SIRT1 alters fatty acidmetabolism and results in hepatic steatosis and inflammationrdquoCell Metabolism vol 9 no 4 pp 327ndash338 2009
[29] A K Walker F Yang K Jiang et al ldquoConserved roleof SIRT1 orthologs in fasting-dependent inhibition of thelipidcholesterol regulator SREBPrdquoGenes and Development vol24 no 13 pp 1403ndash1417 2010
[30] X Li S Zhang G Blander J G Tse M Krieger and L Guar-ente ldquoSIRT1 deacetylates and positively regulates the nuclearreceptor LXRrdquoMolecular Cell vol 28 no 1 pp 91ndash106 2007
[31] M M Sachdeva M Cano and J T Handa ldquoNrf2 signaling isimpaired in the aging RPE given an oxidative insultrdquo Experi-mental Eye Research vol 119 no 1 pp 111ndash114 2014
[32] A Loboda M Damulewicz E Pyza A Jozkowicz and JDulak ldquoRole of Nrf2HO-1 system in development oxidative
stress response and diseases an evolutionarily conservedmech-anismrdquo Cellular and Molecular Life Sciences vol 73 no 17 pp3221ndash3247 2016
[33] G Yang K Zhao Y Ju et al ldquoHydrogen sulfide protects againstcellular senescence via S-sulfhydration of Keap1 and activationof Nrf2rdquo Antioxidants amp Redox Signaling vol 18 no 15 pp1906ndash1919 2013
[34] A J Done M J Gage N C Nieto and T TraustadottirldquoExercise-induced Nrf2-signaling is impaired in agingrdquo FreeRadical Biology and Medicine vol 96 no 1 pp 130ndash138 2016
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
P = 0049
00
05
10
15
Tota
l hist
one d
eace
tyla
se ac
tivity
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
P = 0001
P lt 0001
00
05
10
15
20
25
SIRT
1 ac
tivat
ion
in v
itro
(fold
s of c
ontro
l)
125 25 50 100ControlNaHS (120583molL)
(c)
Figure 7 SIRT1 was involved in NaHS-mediated ageing kidney protection (a) SIRT1 protein and mRNA expressions in the kidney afterchronic NaHS treatment for 10 weeks (119873 = 8) (b) Total deacetylase activity exchangeable in the kidney (119873 = 10) (c) NaHS-activatedrecombinant protein SIRT1 activity in vitro (119873 = 10) Values are mean plusmn SE 119875 lt 005 was considered significant
under oxidative stress Reasonably chronic NaHS treatmentprotects the ageing kidney by ROS scavenging and enhancingendogenous H
2S production in kidney tissue as reported
previously inmyocardium [22] DecreasedCBS activity in theageing kidney may also lead to homocysteine accumulationBecause the latter plays an important role in chronic renalfailure [23 24] further study is needed to explore whetherhomocysteine metabolism disorder is involved in ageing-related kidney damage and whether exogenous NaHS couldhelp restore the normal metabolic pathway
Christopher Hine reported that sulfur amino acid restric-tion could alter H
2S production and protect the liver from
ischemia-reperfusion injury [25] indicating the beneficialrole of endogenous H
2S SIRT1 which emerges as a major
life span regulator has been widely explored in the car-diovascular system and nervous system but it is rare in
the urinary system SIRT1 has robust biological effects andcan affect metabolic homeostasis and ageing Consistentwith previous studies [26 27] we found that both theexpression and activity of SIRT1 decreased rapidly in theageing kidney The reduction of NAD+ biosynthesis maybe responsible for reduced SIRT1 activity [26] Here wereport an interesting finding that a low concentration ofNaHS (25120583molL) could directly induce SIRT1 activationin a cell-free system whereas chronic treatment of NaHS(50 120583molkgday) selectively improves the expression but notthe activity of SIRT1 in the ageing kidney SIRT1 also regulateslipid metabolism by manipulating PPAR-120572 LXR FXR andSREBP signals [28 29] For example SIRT1 positively regu-lates LXR proteins which act as cholesterol sensor and adjustwhole body cholesterol and lipid homeostasis [30] Our bloodbiochemical tests showed that chronic NaHS treatment could
Oxidative Medicine and Cellular Longevity 11
Nrf2
GAPDH
GAPDH
SOD2
GAPDH
SOD1
GAPDH
HO-1
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Young Old-100Old-50Old-10Old Young Old-100Old-50Old-10Old
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday) NaHS (120583molkgday)
00
05
10
15
Nrf2
GA
PDH
(fol
ds o
f old
cont
rol)
00
05
10
15
20
HO
-1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
2G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
NS
GAPDH
Cyto Nrf2
Young Old-50OldNaHS (120583molkgday)
P lt 0001
00
05
10
15
20
Cyto
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
Nuclear Nrf2
Lamin B
Young Old-50Old
P = 0010 P = 0044
NaHS (120583molkgday)
00
05
10
15
20
Nuc
lear
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
Figure 8 The protective effects of the H2S donor on the expression of antioxidant-related proteins in the ageing kidney tissue (a) The
expression change of Nrf2 (119873 = 11) HO-1 (119873 = 10) SOD1 (119873 = 11) and SOD2 (119873 = 10) after 10 weeks of exogenous H2S donor (b) Nrf2
was translocated from cytosol to nucleus Lamin B was used as nuclear control and GAPDH was used as cytosol control (119873 = 6) Values aremean plusmn SE 119875 lt 005 was considered significant
12 Oxidative Medicine and Cellular Longevity
decrease CHOL and TG Therefore H2S may modulate liver
and renal lipidmetabolism through an enhanced SIRT1 signaland contribute to the prevention of renal disease progression
Nrf2 is an important antioxidative molecule that mod-ulates the expression of antioxidant genes [30] and thusprevents age-related oxidative stress [31 32] Here we showedthat the kidney Nrf2 expression decreased during age-ing and that chronic NaHS treatment could rescue Nrf2expression (10 and 50 120583molkgday) and enhance its nucleartranslocation (50120583molkgday) The precise mechanism ofexcessive oxidative stress in ageing is not clear but thedecreased expression of Nrf2antioxidant stress element-linked antioxidant genes is thought to play a role [32ndash34]We found that chronic NaHS treatment could activate Nrf2downstream genes such as HO-1 SOD1 and SOD2 whichconsequently enhanced resistance to oxidative stress in theageing kidney Taken together our data support the idea thatH2S plays a crucial role in protecting the ageing kidney from
antifibrosis and antiapoptosis through the regulation of redoxhomeostasis
5 Conclusion
In summary we demonstrated that 3-MST was expressed inthe ageing kidney and that endogenous H
2S levels decreased
because of the impaired expression ofH2S-producing enzymes
Chronic exogenous H2S treatment could protect the ageing
kidney by reducing oxidative stress decreasing collagendeposition and enhancing Nrf2 nuclear translocation aswell as increasing endogenous H
2S production
Competing Interests
The authors declare no conflict of interests regarding thepublication of this paper
Authorsrsquo ContributionsCui-Lan Hou and Ming-Jie Wang contributed equally to thiswork
AcknowledgmentsThe authors thank Bo Tan for his excellent technical assis-tance in H
2S measurements This present study was sup-
ported by the National Natural Science Foundation of China(NSFC) (no 81230003 to Yi-Chun Zhu no 81670248 toMing-Jie Wang and no 81402917 to Bo Tan) the Ministryof Education (20110071130009 to Yi-Chun Zhu) the Shang-hai Pujiang Program (15PJ1400700 to Ming-Jie Wang) theResearch Center on Ageing and Medicine Fudan University(13dz2260700 to Ming-Jie Wang) and Key Laboratory Pro-gramof the EducationCommission of ShanghaiMunicipality(ZDSYS14005 to Yi-Chun Zhu)
References
[1] J Volkert H SchulzMHarter OWlodarczyk and S AndreasldquoThe prevalence of mental disorders in older people inWesterncountriesmdasha meta-analysisrdquo Ageing Research Reviews vol 12no 1 pp 339ndash353 2013
[2] A Denic R J Glassock and A D Rule ldquoStructural andfunctional changes with the aging kidneyrdquo Advances in ChronicKidney Disease vol 23 no 1 pp 19ndash28 2016
[3] G Yang L Wu B Jiang et al ldquoH2S as a physiologic vasore-
laxant hypertension in mice with deletion of cystathionine 120574-lyaserdquo Science vol 322 no 5901 pp 587ndash590 2008
[4] Y Sun Y Huang R Zhang et al ldquoHydrogen sulfide upreg-ulates KATP channel expression in vascular smooth musclecells of spontaneously hypertensive ratsrdquo Journal of MolecularMedicine vol 93 no 4 pp 439ndash455 2015
[5] C Guo F Liang W Shah Masood and X Yan ldquoHydrogen sul-fide protected gastric epithelial cell from ischemiareperfusioninjury by Keap1 s-sulfhydration MAPK dependent anti-apoptosis and NF-120581B dependent anti-inflammation pathwayrdquoEuropean Journal of Pharmacology vol 725 no 1 pp 70ndash782014
[6] J Du Y Huang H Yan et al ldquoHydrogen sulfide suppresses oxi-dized low-density lipoprotein (Ox-LDL)-stimulated monocytechemoattractant protein 1 generation frommacrophages via thenuclear factor 120581b (NF-120581B) pathwayrdquo The Journal of BiologicalChemistry vol 289 no 14 pp 9741ndash9753 2014
[7] J Zheng T Zhao Y Yuan N Hu and X Tang ldquoHydrogensulfide (H
through oxidative stress and inflammatory response via Nrf2-NF-120581B pathwaysrdquo Chemico-Biological Interactions vol 242 no1 pp 353ndash362 2015
[8] D L Miller and M B Roth ldquoHydrogen sulfide increasesthermotolerance and lifespan in Caenorhabditis elegansrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 104 no 51 pp 20618ndash20622 2007
[9] Y Wei and C Kenyon ldquoRoles for ROS and hydrogen sulfidein the longevity response to germline loss in Caenorhabditiselegansrdquo Proceedings of the National Academy of Sciences vol113 no 20 pp e2832ndashe2841 2016
[10] Y Zhang Z-H Tang Z Ren et al ldquoHydrogen sulfide thenext potent preventive and therapeutic agent in aging and age-associated diseaserdquo Molecular and Cellular Biology vol 33 no6 pp 1104ndash1113 2013
[11] C Wei Y Zhao L Wang et al ldquoH2S restores the cardiopro-
tection from ischemic post-conditioning in isolated aged ratheartsrdquo Cell Biology International vol 39 no 10 pp 1173ndash11762015
[12] R E Sandu A Buga A T Balseanu M Moldovan and APopa-Wagner ldquoTwenty-four hours hypothermia has temporaryefficacy in reducing brain infarction and inflammation in agedratsrdquo Neurobiology of Aging vol 38 no 1 pp 127ndash140 2016
[13] S Jin S X Pu C L Hou et al ldquoCardiac H2S generation
is reduced in ageing diabetic micerdquo Oxidative Medicine andCellular Longevity vol 2015 Article ID 758358 14 pages 2015
[14] K L Quick and L L Dugan ldquoSuperoxide stress identifiesneurons at risk in a model of ataxia-telangiectasiardquo Annals ofNeurology vol 49 no 5 pp 627ndash635 2001
[15] X Shen C B Pattillo S Pardue S C Bir R Wang and C GKevil ldquoMeasurement of plasma hydrogen sulfide in vivo and invitrordquo Free Radical Biology andMedicine vol 50 no 9 pp 1021ndash1031 2011
[16] B-B Tao S-Y Liu C-C Zhang et al ldquoVEGFR2 functionsas an H
2S-targeting receptor protein kinase with its novel
Cys1045-Cys1024 disulfide bond serving as a specific molecularswitch for hydrogen sulfide actions in vascular endothelialcellsrdquo Antioxidants amp Redox Signaling vol 19 no 5 pp 448ndash464 2013
Oxidative Medicine and Cellular Longevity 13
[17] K J Bitterman R M Anderson H Y Cohen M Latorre-Esteves and D A Sinclair ldquoInhibition of silencing and accel-erated aging by nicotinamide a putative negative regulatorof yeast Sir2 and human SIRT1rdquo The Journal of BiologicalChemistry vol 277 no 47 pp 45099ndash45107 2002
[18] X Zhou Y Feng Z Zhan and J Chen ldquoHydrogen sulfidealleviates diabetic nephropathy in a streptozotocin-induceddiabetic ratmodelrdquoThe Journal of Biological Chemistry vol 289no 42 pp 28827ndash28834 2014
[19] P Wang C K Isaak Y L Siow and K O ldquoDownregulationof cystathionine 120573-synthase and cystathionine 120574-lyase expres-sion stimulates inflammation in kidney ischemiamdashreperfusioninjuryrdquo Physiological Reports vol 24 no 12 article e12251 2014
[20] F Yi and P-L Li ldquoMechanisms of homocysteine-inducedglomerular injury and sclerosisrdquo American Journal of Nephrol-ogy vol 28 no 2 pp 254ndash264 2008
[21] R Xue D-D Hao J-P Sun et al ldquoHydrogen sulfide treat-ment promotes glucose uptake by increasing insulin receptorsensitivity and ameliorates kidney lesions in type 2 diabetesrdquoAntioxidants amp Redox Signaling vol 19 no 1 pp 5ndash23 2013
[22] N Li M-J Wang S Jin et al ldquoThe H2S donor NaHS changes
the expression pattern ofH2S-producing enzymes aftermyocar-
dial infarctionrdquo Oxidative Medicine and Cellular Longevity vol2016 Article ID 6492469 11 pages 2016
[23] U Sen W E Rodriguez N Tyagi M Kumar S Kundu andS C Tyagi ldquoCiglitazone a PPAR120574 agonist ameliorates diabeticnephropathy in part through homocysteine clearancerdquo Ameri-can Journal of PhysiologymdashEndocrinology and Metabolism vol295 no 5 pp E1205ndashE1212 2008
[24] W E Rodriguez U Sen N Tyagi et al ldquoPPAR gammaagonist normalizes glomerular filtration rate tissue levels ofhomocysteine and attenuates endothelial-myocyte uncouplingin alloxan induced diabetic micerdquo International Journal ofBiological Sciences vol 4 no 4 pp 236ndash244 2008
[25] C Hine E Harputlugil Y Zhang et al ldquoEndogenous hydrogensulfide production is essential for dietary restriction benefitsrdquoCell vol 160 no 1-2 pp 132ndash144 2015
[26] S-I Imai ldquoDissecting systemic control ofmetabolism and agingin the NAD World the importance of SIRT1 and NAMPT-mediated NAD biosynthesisrdquo FEBS Letters vol 585 no 11 pp1657ndash1662 2011
[27] N Braidy G J Guillemin H Mansour T Chan-Ling APoljak and R Grant ldquoAge related changes inNAD+metabolismoxidative stress and sirt1 activity in wistar ratsrdquo PLoS ONE vol6 no 4 Article ID e19194 pp 1ndash18 2011
[28] A PurushothamT T SchugQXu S Surapureddi XGuo andX Li ldquoHepatocyte-specific deletion of SIRT1 alters fatty acidmetabolism and results in hepatic steatosis and inflammationrdquoCell Metabolism vol 9 no 4 pp 327ndash338 2009
[29] A K Walker F Yang K Jiang et al ldquoConserved roleof SIRT1 orthologs in fasting-dependent inhibition of thelipidcholesterol regulator SREBPrdquoGenes and Development vol24 no 13 pp 1403ndash1417 2010
[30] X Li S Zhang G Blander J G Tse M Krieger and L Guar-ente ldquoSIRT1 deacetylates and positively regulates the nuclearreceptor LXRrdquoMolecular Cell vol 28 no 1 pp 91ndash106 2007
[31] M M Sachdeva M Cano and J T Handa ldquoNrf2 signaling isimpaired in the aging RPE given an oxidative insultrdquo Experi-mental Eye Research vol 119 no 1 pp 111ndash114 2014
[32] A Loboda M Damulewicz E Pyza A Jozkowicz and JDulak ldquoRole of Nrf2HO-1 system in development oxidative
stress response and diseases an evolutionarily conservedmech-anismrdquo Cellular and Molecular Life Sciences vol 73 no 17 pp3221ndash3247 2016
[33] G Yang K Zhao Y Ju et al ldquoHydrogen sulfide protects againstcellular senescence via S-sulfhydration of Keap1 and activationof Nrf2rdquo Antioxidants amp Redox Signaling vol 18 no 15 pp1906ndash1919 2013
[34] A J Done M J Gage N C Nieto and T TraustadottirldquoExercise-induced Nrf2-signaling is impaired in agingrdquo FreeRadical Biology and Medicine vol 96 no 1 pp 130ndash138 2016
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
NaHS (120583molkgday) NaHS (120583molkgday)
00
05
10
15
Nrf2
GA
PDH
(fol
ds o
f old
cont
rol)
00
05
10
15
20
HO
-1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
1G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
00
05
10
15
SOD
2G
APD
H (f
olds
of o
ld co
ntro
l)
Old Old-10 Old-50 Old-100YoungNaHS (120583molkgday)
(a)
NS
GAPDH
Cyto Nrf2
Young Old-50OldNaHS (120583molkgday)
P lt 0001
00
05
10
15
20
Cyto
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
Nuclear Nrf2
Lamin B
Young Old-50Old
P = 0010 P = 0044
NaHS (120583molkgday)
00
05
10
15
20
Nuc
lear
Nrf2
GA
PDH
(fold
s of o
ld co
ntro
l)
Old Old-50YoungNaHS (120583molkgday)
(b)
Figure 8 The protective effects of the H2S donor on the expression of antioxidant-related proteins in the ageing kidney tissue (a) The
expression change of Nrf2 (119873 = 11) HO-1 (119873 = 10) SOD1 (119873 = 11) and SOD2 (119873 = 10) after 10 weeks of exogenous H2S donor (b) Nrf2
was translocated from cytosol to nucleus Lamin B was used as nuclear control and GAPDH was used as cytosol control (119873 = 6) Values aremean plusmn SE 119875 lt 005 was considered significant
12 Oxidative Medicine and Cellular Longevity
decrease CHOL and TG Therefore H2S may modulate liver
and renal lipidmetabolism through an enhanced SIRT1 signaland contribute to the prevention of renal disease progression
Nrf2 is an important antioxidative molecule that mod-ulates the expression of antioxidant genes [30] and thusprevents age-related oxidative stress [31 32] Here we showedthat the kidney Nrf2 expression decreased during age-ing and that chronic NaHS treatment could rescue Nrf2expression (10 and 50 120583molkgday) and enhance its nucleartranslocation (50120583molkgday) The precise mechanism ofexcessive oxidative stress in ageing is not clear but thedecreased expression of Nrf2antioxidant stress element-linked antioxidant genes is thought to play a role [32ndash34]We found that chronic NaHS treatment could activate Nrf2downstream genes such as HO-1 SOD1 and SOD2 whichconsequently enhanced resistance to oxidative stress in theageing kidney Taken together our data support the idea thatH2S plays a crucial role in protecting the ageing kidney from
antifibrosis and antiapoptosis through the regulation of redoxhomeostasis
5 Conclusion
In summary we demonstrated that 3-MST was expressed inthe ageing kidney and that endogenous H
2S levels decreased
because of the impaired expression ofH2S-producing enzymes
Chronic exogenous H2S treatment could protect the ageing
kidney by reducing oxidative stress decreasing collagendeposition and enhancing Nrf2 nuclear translocation aswell as increasing endogenous H
2S production
Competing Interests
The authors declare no conflict of interests regarding thepublication of this paper
Authorsrsquo ContributionsCui-Lan Hou and Ming-Jie Wang contributed equally to thiswork
AcknowledgmentsThe authors thank Bo Tan for his excellent technical assis-tance in H
2S measurements This present study was sup-
ported by the National Natural Science Foundation of China(NSFC) (no 81230003 to Yi-Chun Zhu no 81670248 toMing-Jie Wang and no 81402917 to Bo Tan) the Ministryof Education (20110071130009 to Yi-Chun Zhu) the Shang-hai Pujiang Program (15PJ1400700 to Ming-Jie Wang) theResearch Center on Ageing and Medicine Fudan University(13dz2260700 to Ming-Jie Wang) and Key Laboratory Pro-gramof the EducationCommission of ShanghaiMunicipality(ZDSYS14005 to Yi-Chun Zhu)
References
[1] J Volkert H SchulzMHarter OWlodarczyk and S AndreasldquoThe prevalence of mental disorders in older people inWesterncountriesmdasha meta-analysisrdquo Ageing Research Reviews vol 12no 1 pp 339ndash353 2013
[2] A Denic R J Glassock and A D Rule ldquoStructural andfunctional changes with the aging kidneyrdquo Advances in ChronicKidney Disease vol 23 no 1 pp 19ndash28 2016
[3] G Yang L Wu B Jiang et al ldquoH2S as a physiologic vasore-
laxant hypertension in mice with deletion of cystathionine 120574-lyaserdquo Science vol 322 no 5901 pp 587ndash590 2008
[4] Y Sun Y Huang R Zhang et al ldquoHydrogen sulfide upreg-ulates KATP channel expression in vascular smooth musclecells of spontaneously hypertensive ratsrdquo Journal of MolecularMedicine vol 93 no 4 pp 439ndash455 2015
[5] C Guo F Liang W Shah Masood and X Yan ldquoHydrogen sul-fide protected gastric epithelial cell from ischemiareperfusioninjury by Keap1 s-sulfhydration MAPK dependent anti-apoptosis and NF-120581B dependent anti-inflammation pathwayrdquoEuropean Journal of Pharmacology vol 725 no 1 pp 70ndash782014
[6] J Du Y Huang H Yan et al ldquoHydrogen sulfide suppresses oxi-dized low-density lipoprotein (Ox-LDL)-stimulated monocytechemoattractant protein 1 generation frommacrophages via thenuclear factor 120581b (NF-120581B) pathwayrdquo The Journal of BiologicalChemistry vol 289 no 14 pp 9741ndash9753 2014
[7] J Zheng T Zhao Y Yuan N Hu and X Tang ldquoHydrogensulfide (H
through oxidative stress and inflammatory response via Nrf2-NF-120581B pathwaysrdquo Chemico-Biological Interactions vol 242 no1 pp 353ndash362 2015
[8] D L Miller and M B Roth ldquoHydrogen sulfide increasesthermotolerance and lifespan in Caenorhabditis elegansrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 104 no 51 pp 20618ndash20622 2007
[9] Y Wei and C Kenyon ldquoRoles for ROS and hydrogen sulfidein the longevity response to germline loss in Caenorhabditiselegansrdquo Proceedings of the National Academy of Sciences vol113 no 20 pp e2832ndashe2841 2016
[10] Y Zhang Z-H Tang Z Ren et al ldquoHydrogen sulfide thenext potent preventive and therapeutic agent in aging and age-associated diseaserdquo Molecular and Cellular Biology vol 33 no6 pp 1104ndash1113 2013
[11] C Wei Y Zhao L Wang et al ldquoH2S restores the cardiopro-
tection from ischemic post-conditioning in isolated aged ratheartsrdquo Cell Biology International vol 39 no 10 pp 1173ndash11762015
[12] R E Sandu A Buga A T Balseanu M Moldovan and APopa-Wagner ldquoTwenty-four hours hypothermia has temporaryefficacy in reducing brain infarction and inflammation in agedratsrdquo Neurobiology of Aging vol 38 no 1 pp 127ndash140 2016
[13] S Jin S X Pu C L Hou et al ldquoCardiac H2S generation
is reduced in ageing diabetic micerdquo Oxidative Medicine andCellular Longevity vol 2015 Article ID 758358 14 pages 2015
[14] K L Quick and L L Dugan ldquoSuperoxide stress identifiesneurons at risk in a model of ataxia-telangiectasiardquo Annals ofNeurology vol 49 no 5 pp 627ndash635 2001
[15] X Shen C B Pattillo S Pardue S C Bir R Wang and C GKevil ldquoMeasurement of plasma hydrogen sulfide in vivo and invitrordquo Free Radical Biology andMedicine vol 50 no 9 pp 1021ndash1031 2011
[16] B-B Tao S-Y Liu C-C Zhang et al ldquoVEGFR2 functionsas an H
2S-targeting receptor protein kinase with its novel
Cys1045-Cys1024 disulfide bond serving as a specific molecularswitch for hydrogen sulfide actions in vascular endothelialcellsrdquo Antioxidants amp Redox Signaling vol 19 no 5 pp 448ndash464 2013
Oxidative Medicine and Cellular Longevity 13
[17] K J Bitterman R M Anderson H Y Cohen M Latorre-Esteves and D A Sinclair ldquoInhibition of silencing and accel-erated aging by nicotinamide a putative negative regulatorof yeast Sir2 and human SIRT1rdquo The Journal of BiologicalChemistry vol 277 no 47 pp 45099ndash45107 2002
[18] X Zhou Y Feng Z Zhan and J Chen ldquoHydrogen sulfidealleviates diabetic nephropathy in a streptozotocin-induceddiabetic ratmodelrdquoThe Journal of Biological Chemistry vol 289no 42 pp 28827ndash28834 2014
[19] P Wang C K Isaak Y L Siow and K O ldquoDownregulationof cystathionine 120573-synthase and cystathionine 120574-lyase expres-sion stimulates inflammation in kidney ischemiamdashreperfusioninjuryrdquo Physiological Reports vol 24 no 12 article e12251 2014
[20] F Yi and P-L Li ldquoMechanisms of homocysteine-inducedglomerular injury and sclerosisrdquo American Journal of Nephrol-ogy vol 28 no 2 pp 254ndash264 2008
[21] R Xue D-D Hao J-P Sun et al ldquoHydrogen sulfide treat-ment promotes glucose uptake by increasing insulin receptorsensitivity and ameliorates kidney lesions in type 2 diabetesrdquoAntioxidants amp Redox Signaling vol 19 no 1 pp 5ndash23 2013
[22] N Li M-J Wang S Jin et al ldquoThe H2S donor NaHS changes
the expression pattern ofH2S-producing enzymes aftermyocar-
dial infarctionrdquo Oxidative Medicine and Cellular Longevity vol2016 Article ID 6492469 11 pages 2016
[23] U Sen W E Rodriguez N Tyagi M Kumar S Kundu andS C Tyagi ldquoCiglitazone a PPAR120574 agonist ameliorates diabeticnephropathy in part through homocysteine clearancerdquo Ameri-can Journal of PhysiologymdashEndocrinology and Metabolism vol295 no 5 pp E1205ndashE1212 2008
[24] W E Rodriguez U Sen N Tyagi et al ldquoPPAR gammaagonist normalizes glomerular filtration rate tissue levels ofhomocysteine and attenuates endothelial-myocyte uncouplingin alloxan induced diabetic micerdquo International Journal ofBiological Sciences vol 4 no 4 pp 236ndash244 2008
[25] C Hine E Harputlugil Y Zhang et al ldquoEndogenous hydrogensulfide production is essential for dietary restriction benefitsrdquoCell vol 160 no 1-2 pp 132ndash144 2015
[26] S-I Imai ldquoDissecting systemic control ofmetabolism and agingin the NAD World the importance of SIRT1 and NAMPT-mediated NAD biosynthesisrdquo FEBS Letters vol 585 no 11 pp1657ndash1662 2011
[27] N Braidy G J Guillemin H Mansour T Chan-Ling APoljak and R Grant ldquoAge related changes inNAD+metabolismoxidative stress and sirt1 activity in wistar ratsrdquo PLoS ONE vol6 no 4 Article ID e19194 pp 1ndash18 2011
[28] A PurushothamT T SchugQXu S Surapureddi XGuo andX Li ldquoHepatocyte-specific deletion of SIRT1 alters fatty acidmetabolism and results in hepatic steatosis and inflammationrdquoCell Metabolism vol 9 no 4 pp 327ndash338 2009
[29] A K Walker F Yang K Jiang et al ldquoConserved roleof SIRT1 orthologs in fasting-dependent inhibition of thelipidcholesterol regulator SREBPrdquoGenes and Development vol24 no 13 pp 1403ndash1417 2010
[30] X Li S Zhang G Blander J G Tse M Krieger and L Guar-ente ldquoSIRT1 deacetylates and positively regulates the nuclearreceptor LXRrdquoMolecular Cell vol 28 no 1 pp 91ndash106 2007
[31] M M Sachdeva M Cano and J T Handa ldquoNrf2 signaling isimpaired in the aging RPE given an oxidative insultrdquo Experi-mental Eye Research vol 119 no 1 pp 111ndash114 2014
[32] A Loboda M Damulewicz E Pyza A Jozkowicz and JDulak ldquoRole of Nrf2HO-1 system in development oxidative
stress response and diseases an evolutionarily conservedmech-anismrdquo Cellular and Molecular Life Sciences vol 73 no 17 pp3221ndash3247 2016
[33] G Yang K Zhao Y Ju et al ldquoHydrogen sulfide protects againstcellular senescence via S-sulfhydration of Keap1 and activationof Nrf2rdquo Antioxidants amp Redox Signaling vol 18 no 15 pp1906ndash1919 2013
[34] A J Done M J Gage N C Nieto and T TraustadottirldquoExercise-induced Nrf2-signaling is impaired in agingrdquo FreeRadical Biology and Medicine vol 96 no 1 pp 130ndash138 2016
decrease CHOL and TG Therefore H2S may modulate liver
and renal lipidmetabolism through an enhanced SIRT1 signaland contribute to the prevention of renal disease progression
Nrf2 is an important antioxidative molecule that mod-ulates the expression of antioxidant genes [30] and thusprevents age-related oxidative stress [31 32] Here we showedthat the kidney Nrf2 expression decreased during age-ing and that chronic NaHS treatment could rescue Nrf2expression (10 and 50 120583molkgday) and enhance its nucleartranslocation (50120583molkgday) The precise mechanism ofexcessive oxidative stress in ageing is not clear but thedecreased expression of Nrf2antioxidant stress element-linked antioxidant genes is thought to play a role [32ndash34]We found that chronic NaHS treatment could activate Nrf2downstream genes such as HO-1 SOD1 and SOD2 whichconsequently enhanced resistance to oxidative stress in theageing kidney Taken together our data support the idea thatH2S plays a crucial role in protecting the ageing kidney from
antifibrosis and antiapoptosis through the regulation of redoxhomeostasis
5 Conclusion
In summary we demonstrated that 3-MST was expressed inthe ageing kidney and that endogenous H
2S levels decreased
because of the impaired expression ofH2S-producing enzymes
Chronic exogenous H2S treatment could protect the ageing
kidney by reducing oxidative stress decreasing collagendeposition and enhancing Nrf2 nuclear translocation aswell as increasing endogenous H
2S production
Competing Interests
The authors declare no conflict of interests regarding thepublication of this paper
Authorsrsquo ContributionsCui-Lan Hou and Ming-Jie Wang contributed equally to thiswork
AcknowledgmentsThe authors thank Bo Tan for his excellent technical assis-tance in H
2S measurements This present study was sup-
ported by the National Natural Science Foundation of China(NSFC) (no 81230003 to Yi-Chun Zhu no 81670248 toMing-Jie Wang and no 81402917 to Bo Tan) the Ministryof Education (20110071130009 to Yi-Chun Zhu) the Shang-hai Pujiang Program (15PJ1400700 to Ming-Jie Wang) theResearch Center on Ageing and Medicine Fudan University(13dz2260700 to Ming-Jie Wang) and Key Laboratory Pro-gramof the EducationCommission of ShanghaiMunicipality(ZDSYS14005 to Yi-Chun Zhu)
References
[1] J Volkert H SchulzMHarter OWlodarczyk and S AndreasldquoThe prevalence of mental disorders in older people inWesterncountriesmdasha meta-analysisrdquo Ageing Research Reviews vol 12no 1 pp 339ndash353 2013
[2] A Denic R J Glassock and A D Rule ldquoStructural andfunctional changes with the aging kidneyrdquo Advances in ChronicKidney Disease vol 23 no 1 pp 19ndash28 2016
[3] G Yang L Wu B Jiang et al ldquoH2S as a physiologic vasore-
laxant hypertension in mice with deletion of cystathionine 120574-lyaserdquo Science vol 322 no 5901 pp 587ndash590 2008
[4] Y Sun Y Huang R Zhang et al ldquoHydrogen sulfide upreg-ulates KATP channel expression in vascular smooth musclecells of spontaneously hypertensive ratsrdquo Journal of MolecularMedicine vol 93 no 4 pp 439ndash455 2015
[5] C Guo F Liang W Shah Masood and X Yan ldquoHydrogen sul-fide protected gastric epithelial cell from ischemiareperfusioninjury by Keap1 s-sulfhydration MAPK dependent anti-apoptosis and NF-120581B dependent anti-inflammation pathwayrdquoEuropean Journal of Pharmacology vol 725 no 1 pp 70ndash782014
[6] J Du Y Huang H Yan et al ldquoHydrogen sulfide suppresses oxi-dized low-density lipoprotein (Ox-LDL)-stimulated monocytechemoattractant protein 1 generation frommacrophages via thenuclear factor 120581b (NF-120581B) pathwayrdquo The Journal of BiologicalChemistry vol 289 no 14 pp 9741ndash9753 2014
[7] J Zheng T Zhao Y Yuan N Hu and X Tang ldquoHydrogensulfide (H
through oxidative stress and inflammatory response via Nrf2-NF-120581B pathwaysrdquo Chemico-Biological Interactions vol 242 no1 pp 353ndash362 2015
[8] D L Miller and M B Roth ldquoHydrogen sulfide increasesthermotolerance and lifespan in Caenorhabditis elegansrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 104 no 51 pp 20618ndash20622 2007
[9] Y Wei and C Kenyon ldquoRoles for ROS and hydrogen sulfidein the longevity response to germline loss in Caenorhabditiselegansrdquo Proceedings of the National Academy of Sciences vol113 no 20 pp e2832ndashe2841 2016
[10] Y Zhang Z-H Tang Z Ren et al ldquoHydrogen sulfide thenext potent preventive and therapeutic agent in aging and age-associated diseaserdquo Molecular and Cellular Biology vol 33 no6 pp 1104ndash1113 2013
[11] C Wei Y Zhao L Wang et al ldquoH2S restores the cardiopro-
tection from ischemic post-conditioning in isolated aged ratheartsrdquo Cell Biology International vol 39 no 10 pp 1173ndash11762015
[12] R E Sandu A Buga A T Balseanu M Moldovan and APopa-Wagner ldquoTwenty-four hours hypothermia has temporaryefficacy in reducing brain infarction and inflammation in agedratsrdquo Neurobiology of Aging vol 38 no 1 pp 127ndash140 2016
[13] S Jin S X Pu C L Hou et al ldquoCardiac H2S generation
is reduced in ageing diabetic micerdquo Oxidative Medicine andCellular Longevity vol 2015 Article ID 758358 14 pages 2015
[14] K L Quick and L L Dugan ldquoSuperoxide stress identifiesneurons at risk in a model of ataxia-telangiectasiardquo Annals ofNeurology vol 49 no 5 pp 627ndash635 2001
[15] X Shen C B Pattillo S Pardue S C Bir R Wang and C GKevil ldquoMeasurement of plasma hydrogen sulfide in vivo and invitrordquo Free Radical Biology andMedicine vol 50 no 9 pp 1021ndash1031 2011
[16] B-B Tao S-Y Liu C-C Zhang et al ldquoVEGFR2 functionsas an H
2S-targeting receptor protein kinase with its novel
Cys1045-Cys1024 disulfide bond serving as a specific molecularswitch for hydrogen sulfide actions in vascular endothelialcellsrdquo Antioxidants amp Redox Signaling vol 19 no 5 pp 448ndash464 2013
Oxidative Medicine and Cellular Longevity 13
[17] K J Bitterman R M Anderson H Y Cohen M Latorre-Esteves and D A Sinclair ldquoInhibition of silencing and accel-erated aging by nicotinamide a putative negative regulatorof yeast Sir2 and human SIRT1rdquo The Journal of BiologicalChemistry vol 277 no 47 pp 45099ndash45107 2002
[18] X Zhou Y Feng Z Zhan and J Chen ldquoHydrogen sulfidealleviates diabetic nephropathy in a streptozotocin-induceddiabetic ratmodelrdquoThe Journal of Biological Chemistry vol 289no 42 pp 28827ndash28834 2014
[19] P Wang C K Isaak Y L Siow and K O ldquoDownregulationof cystathionine 120573-synthase and cystathionine 120574-lyase expres-sion stimulates inflammation in kidney ischemiamdashreperfusioninjuryrdquo Physiological Reports vol 24 no 12 article e12251 2014
[20] F Yi and P-L Li ldquoMechanisms of homocysteine-inducedglomerular injury and sclerosisrdquo American Journal of Nephrol-ogy vol 28 no 2 pp 254ndash264 2008
[21] R Xue D-D Hao J-P Sun et al ldquoHydrogen sulfide treat-ment promotes glucose uptake by increasing insulin receptorsensitivity and ameliorates kidney lesions in type 2 diabetesrdquoAntioxidants amp Redox Signaling vol 19 no 1 pp 5ndash23 2013
[22] N Li M-J Wang S Jin et al ldquoThe H2S donor NaHS changes
the expression pattern ofH2S-producing enzymes aftermyocar-
dial infarctionrdquo Oxidative Medicine and Cellular Longevity vol2016 Article ID 6492469 11 pages 2016
[23] U Sen W E Rodriguez N Tyagi M Kumar S Kundu andS C Tyagi ldquoCiglitazone a PPAR120574 agonist ameliorates diabeticnephropathy in part through homocysteine clearancerdquo Ameri-can Journal of PhysiologymdashEndocrinology and Metabolism vol295 no 5 pp E1205ndashE1212 2008
[24] W E Rodriguez U Sen N Tyagi et al ldquoPPAR gammaagonist normalizes glomerular filtration rate tissue levels ofhomocysteine and attenuates endothelial-myocyte uncouplingin alloxan induced diabetic micerdquo International Journal ofBiological Sciences vol 4 no 4 pp 236ndash244 2008
[25] C Hine E Harputlugil Y Zhang et al ldquoEndogenous hydrogensulfide production is essential for dietary restriction benefitsrdquoCell vol 160 no 1-2 pp 132ndash144 2015
[26] S-I Imai ldquoDissecting systemic control ofmetabolism and agingin the NAD World the importance of SIRT1 and NAMPT-mediated NAD biosynthesisrdquo FEBS Letters vol 585 no 11 pp1657ndash1662 2011
[27] N Braidy G J Guillemin H Mansour T Chan-Ling APoljak and R Grant ldquoAge related changes inNAD+metabolismoxidative stress and sirt1 activity in wistar ratsrdquo PLoS ONE vol6 no 4 Article ID e19194 pp 1ndash18 2011
[28] A PurushothamT T SchugQXu S Surapureddi XGuo andX Li ldquoHepatocyte-specific deletion of SIRT1 alters fatty acidmetabolism and results in hepatic steatosis and inflammationrdquoCell Metabolism vol 9 no 4 pp 327ndash338 2009
[29] A K Walker F Yang K Jiang et al ldquoConserved roleof SIRT1 orthologs in fasting-dependent inhibition of thelipidcholesterol regulator SREBPrdquoGenes and Development vol24 no 13 pp 1403ndash1417 2010
[30] X Li S Zhang G Blander J G Tse M Krieger and L Guar-ente ldquoSIRT1 deacetylates and positively regulates the nuclearreceptor LXRrdquoMolecular Cell vol 28 no 1 pp 91ndash106 2007
[31] M M Sachdeva M Cano and J T Handa ldquoNrf2 signaling isimpaired in the aging RPE given an oxidative insultrdquo Experi-mental Eye Research vol 119 no 1 pp 111ndash114 2014
[32] A Loboda M Damulewicz E Pyza A Jozkowicz and JDulak ldquoRole of Nrf2HO-1 system in development oxidative
stress response and diseases an evolutionarily conservedmech-anismrdquo Cellular and Molecular Life Sciences vol 73 no 17 pp3221ndash3247 2016
[33] G Yang K Zhao Y Ju et al ldquoHydrogen sulfide protects againstcellular senescence via S-sulfhydration of Keap1 and activationof Nrf2rdquo Antioxidants amp Redox Signaling vol 18 no 15 pp1906ndash1919 2013
[34] A J Done M J Gage N C Nieto and T TraustadottirldquoExercise-induced Nrf2-signaling is impaired in agingrdquo FreeRadical Biology and Medicine vol 96 no 1 pp 130ndash138 2016
[17] K J Bitterman R M Anderson H Y Cohen M Latorre-Esteves and D A Sinclair ldquoInhibition of silencing and accel-erated aging by nicotinamide a putative negative regulatorof yeast Sir2 and human SIRT1rdquo The Journal of BiologicalChemistry vol 277 no 47 pp 45099ndash45107 2002
[18] X Zhou Y Feng Z Zhan and J Chen ldquoHydrogen sulfidealleviates diabetic nephropathy in a streptozotocin-induceddiabetic ratmodelrdquoThe Journal of Biological Chemistry vol 289no 42 pp 28827ndash28834 2014
[19] P Wang C K Isaak Y L Siow and K O ldquoDownregulationof cystathionine 120573-synthase and cystathionine 120574-lyase expres-sion stimulates inflammation in kidney ischemiamdashreperfusioninjuryrdquo Physiological Reports vol 24 no 12 article e12251 2014
[20] F Yi and P-L Li ldquoMechanisms of homocysteine-inducedglomerular injury and sclerosisrdquo American Journal of Nephrol-ogy vol 28 no 2 pp 254ndash264 2008
[21] R Xue D-D Hao J-P Sun et al ldquoHydrogen sulfide treat-ment promotes glucose uptake by increasing insulin receptorsensitivity and ameliorates kidney lesions in type 2 diabetesrdquoAntioxidants amp Redox Signaling vol 19 no 1 pp 5ndash23 2013
[22] N Li M-J Wang S Jin et al ldquoThe H2S donor NaHS changes
the expression pattern ofH2S-producing enzymes aftermyocar-
dial infarctionrdquo Oxidative Medicine and Cellular Longevity vol2016 Article ID 6492469 11 pages 2016
[23] U Sen W E Rodriguez N Tyagi M Kumar S Kundu andS C Tyagi ldquoCiglitazone a PPAR120574 agonist ameliorates diabeticnephropathy in part through homocysteine clearancerdquo Ameri-can Journal of PhysiologymdashEndocrinology and Metabolism vol295 no 5 pp E1205ndashE1212 2008
[24] W E Rodriguez U Sen N Tyagi et al ldquoPPAR gammaagonist normalizes glomerular filtration rate tissue levels ofhomocysteine and attenuates endothelial-myocyte uncouplingin alloxan induced diabetic micerdquo International Journal ofBiological Sciences vol 4 no 4 pp 236ndash244 2008
[25] C Hine E Harputlugil Y Zhang et al ldquoEndogenous hydrogensulfide production is essential for dietary restriction benefitsrdquoCell vol 160 no 1-2 pp 132ndash144 2015
[26] S-I Imai ldquoDissecting systemic control ofmetabolism and agingin the NAD World the importance of SIRT1 and NAMPT-mediated NAD biosynthesisrdquo FEBS Letters vol 585 no 11 pp1657ndash1662 2011
[27] N Braidy G J Guillemin H Mansour T Chan-Ling APoljak and R Grant ldquoAge related changes inNAD+metabolismoxidative stress and sirt1 activity in wistar ratsrdquo PLoS ONE vol6 no 4 Article ID e19194 pp 1ndash18 2011
[28] A PurushothamT T SchugQXu S Surapureddi XGuo andX Li ldquoHepatocyte-specific deletion of SIRT1 alters fatty acidmetabolism and results in hepatic steatosis and inflammationrdquoCell Metabolism vol 9 no 4 pp 327ndash338 2009
[29] A K Walker F Yang K Jiang et al ldquoConserved roleof SIRT1 orthologs in fasting-dependent inhibition of thelipidcholesterol regulator SREBPrdquoGenes and Development vol24 no 13 pp 1403ndash1417 2010
[30] X Li S Zhang G Blander J G Tse M Krieger and L Guar-ente ldquoSIRT1 deacetylates and positively regulates the nuclearreceptor LXRrdquoMolecular Cell vol 28 no 1 pp 91ndash106 2007
[31] M M Sachdeva M Cano and J T Handa ldquoNrf2 signaling isimpaired in the aging RPE given an oxidative insultrdquo Experi-mental Eye Research vol 119 no 1 pp 111ndash114 2014
[32] A Loboda M Damulewicz E Pyza A Jozkowicz and JDulak ldquoRole of Nrf2HO-1 system in development oxidative
stress response and diseases an evolutionarily conservedmech-anismrdquo Cellular and Molecular Life Sciences vol 73 no 17 pp3221ndash3247 2016
[33] G Yang K Zhao Y Ju et al ldquoHydrogen sulfide protects againstcellular senescence via S-sulfhydration of Keap1 and activationof Nrf2rdquo Antioxidants amp Redox Signaling vol 18 no 15 pp1906ndash1919 2013
[34] A J Done M J Gage N C Nieto and T TraustadottirldquoExercise-induced Nrf2-signaling is impaired in agingrdquo FreeRadical Biology and Medicine vol 96 no 1 pp 130ndash138 2016
![Hepato- and neuro-protective effects of watermelon juice ... · effects of watermelon juice have been reported [26,27]. Furthermore, the protective effects of watermelon juice against](https://static.documents.pub/doc/80x56/60218d2d8602627ee124664d/hepato-and-neuro-protective-effects-of-watermelon-juice-effects-of-watermelon.jpg)
