Epigenetics and CKD
Masaomi NangakuDivision of Nephrology and Endocrinology
the University of Tokyo Graduate School of Medicine
Nephrology and Transplantation Update Course
I have the following relationships to disclose.Potential Financial Conflicts of Interest
(1)Employment: No
(2)Stock ownership or options: No
(3)Patent royalties/licensing fees: No
(4)Honoraria and advisory fees: Kyowa-Hakko-Kirin, Astellas, Chugai,
GSK, JT, Tanabe-Mitsubishi
(5)Research funding: Kyowa-Hakko-Kirin, Daiichi-Sankyo, Alexion,
Astellas, Takeda, JT
COI disclosure
presenter: Masaomi Nangaku
Epigenetics: the study of persistent
changes in gene expression that does not
involve mutations of the underlying DNA
Long-term benefits of intensive glucose control for
preventing ESKD: ADVANCE-ON
Muh Geot Wong et al. Dia Care 2016
5th September
Seminar 2: Basic Science; Diabetes
Chairs: Josephine Forbes & Melinda Coughlan
Karin Jandeleit-Dahm
Masaomi Nangaku
Carol Pollock
VAP-1 in pericytes enhances neutrophil infiltration
into the IR-injured kidney by generating H2O2
Tanaka, Nangaku et al. Kidney Int 2017
Peritubular ischemia contributes more to tubular damage than
proteinuria in immune-mediated glomerulonephritis
Muh Geot Wong et al. JASN 2008
Double immunostaining showed
pimonidazole-positive tubules
(brown) located close to Kim-1–
positive tubules (pink).
pimonidazole
Hirakawa, Tanaka, Nangaku. J Diabetes Investig 2017
HIF
Nrf2
Defense against hypoxia
Defense against
oxidative stress
Oxygen sensing –
an essential process for
survival
2016 Albert Lasker Basic
Medical Research Award
Gregg L. SemenzaWilliam G. Kaelin, Jr. Peter J. Ratcliffe
D-serine accelerates tubular senescence
SA-βG staining
D-serineControl
γ-H2AX staining
Okada, Nangaku et al.
Sci Rep in press
Chirality
Indoxyl sulfate suppresses HIF activity
Tanaka, Nangaku et al.
FASEB J 2013
reporters
HIF targets
IS: indoxyl sulfate
Indoxyl sulfate induces HIF-inhibiting CITED2 protein
Tanaka, Nangaku et al. FASEB J 2013
Increased CITED2 mRNA
stability by IS
CITED2 mRNA
CITED2 protein
The efficient displacement of HIF-1α from its complex with
CBP/p300 by CITED2 is kinetically driven and proceeds
through a transient ternary intermediate
Berlow et al. Nature 2017
Hirakawa, Nangaku et al. Sci Rep 2015
Measurement of oxygen tension in the kidney
utilizing a novel phosphorescence probe
Hirakawa, Nangaku et al. Sci Rep 2015
Ph
os
ph
oflu
ore
sc
en
ce
life tim
e (µ
s)
Phosphofluorescent probe to detect hypoxia
S1 segment has higher oxygen tension compared
with S2 segment
Hirakawa & Nangaku.
manuscript in submission
U: upstream tubules
D: downstream tubles
Maternal protein restriction diet induced less weaning weight
and hypermethylation of ribosomal DNA in offspring
Holland et al. Science 2016
Ischemia-reperfusion injury decreased the genome-
wide methylation level and the CpG methylation level
Zhao et al. Gene 2017
Aberrant Rasal1 promoter methylation contributes to
sustained fibroblast activation and AKI-to-CKD progression
Tampe et al. Kidney Int 2017
Rasal1: Ras-Gap–like protein-1
A clear advantage of targeting lncRNA rather than epigenetic-
related enzymes or other non-coding RNA such as miRNA is the
direct sequence specificity of the target site.
H3K4me1 normoxia
H3K4me1 hypoxia
H3K27ac normoxia
H3K27ac hypoxia
H3K4me3 normoxia
H3K4me3 hypoxia
HIF1α normoxia
HIF1α hypoxia
-35kb-24kbGLUT3
Epigenetic modification of expression of
glucose transporter 3 (GLUT3) by hypoxia
enhancer mark
active mark
Mimura, Nangaku et al. Mol Cell Biol 2012
0
200
400
600
800
1000
1200
1400
0 h 1 hr 2 hrs 4 hrs 8 hrs 12hrs 24hrs
Histone demethylation by hypoxia
Upregulation of KDM3A
ON
OFF supprssive
H3K9me2
0 1 2 4 8 12 24 hr
1400
1200
1000
800
600
400
200
0
KDM3A = Jmjd1a
demethylase of
H3K9me2
Mimura, Nangaku et al. Mol Cell Biol 2012
Regulation of GLUT3 expression by hypoxia
normoxia hypoxia
GLUT3 GLUT3
Up-regulation
Mimura, Nangaku et al. Mol Cell Biol 2012
Cross-enhancement of ANGPTL4 transcription
by HIF1 and PPAR β/δ
Inoue, Nangaku et al. Genome Biol 2014
HIF1α
PPARβ/δ
OMX super-resolution microscopy showed changes of the
distribution of H3K9Me3 marks in NRK-52e cells by TGF-β1
H3K9Me3: repressive histone mark
NUP62: nuclear envelope protein
nucleoporin 62
Hewitson et al.
Front Pharmacol 2017
curcumin improves nephrosclerosis via suppression of
histone acetylation
Normal
saltHigh
salt
High salt +
curcuminacetylated H3K9
Serum creatinine
Muta et al. NDT 2016
ChIP using anti-acetyl-H3K9
increased EZH2 in the human fibrotic kidney
Zhou et al. JASN 2016
EZH2 (Enhancer of zeste
homolog 2):
histone methyltransferase of
H3K27me
EZH2 mediates kidney
fibrosis by downregulating
expression of Smad7 and
PTEN
Zhou et al. JASN 2016
Dznep: inhibitor of EZH2
Dznep attenuates renal fibrosis in obstructed
kidneys
αSMADznep
I/R
0
10
20
Vehicle群 阻害薬投与群
Control I/RArea(%)
* p<0.001
**
*
0
0.5
1
Vehicle群 阻害薬投与群
*
WB
αSMA Ab
Β-Actin Ab
Vehicle Dznep
Amelioration of AKI-to-CKD transition by Dznep
DznepVehicle
DznepVehicle
Mimura, Hirakawa, Nangaku. manuscript in submission
Vehicle
Vehicle DznepH
3K
27
me
3 (
DA
B)
Nuclear staining of tubular
cells showed decreased
staining of H3K27me3.
Suppression of H3K27me3 by Dznep
Mimura, Hirakawa, Nangaku. manuscript in submission
DZnep 2 control 1 control 2 hypoxia 1 hypoxia 2 DZnep 1 DZnep 20.000.02
2.901.19
3.88
0.000.00
4.2715.59
3.97
47.99
0.040.00
2.80
9.630.040.55
0.00
4.250.96
11.765.80
8.56
3.523.237.89
0.02
0.000.760.00
0.00
0.0013.64
13.716.74
5.52218.61
9.249.84
35.99
46.9910.37
6.17
24.23
88.3937.73
17.98
109.343.977.17
6.84
116.5149.21
7.7017.29
91.66
51.6158.1121.35
11.53
13.385.78
197.57
60.10
12.3528.63
12.4126.92
8.57
8.167.08
13.68
13.22
21.8112.47
16.40
7.99
57.777.09
10.77
4.0918.8014.19
6.68
23.2878.79
20.635.79
4.27
28.2118.047.63
339.67
40.745.267.37
15.75
5.7917.21
55.7115.43
7.06
2.907.795.01
8.86
288.829.85
3.81
7.61
9.2525.67
11.181.28
24.20
501.8812.58
13.420.00
102.5113.56
113.84
84.2429.9419.94
16.34
7.955.80
28.08
14.88
3.066.52
6.924.62
9.62
0.0160.3211.09
25.15
19.516.37
16.45
19.97
5.216.86
34.148.54
4.40
4.1414.68
11.9521.28
11.4516.52
10.97
25.8947.2727.59
14.13
12.84103.2645.78
23.80
14.538.93
90.3422.30
11.36
26.499.90
22.56
37.31
10.1713.61
8.83
19.81
11.7611.54
16.99
32.5810.41
34.2523.25
44.7414.55
16.0515.71
10.84
39.9320.5914.90
25.00
20.029.48
17.40108.37
20.3211.26
11.2811.77
28.32
40.4216.8431.89
14.44
15.9210.83
14.13
27.55
30.6413.63
27.3715.51
23.89
19.6816.34
13.04
12.31
22.1414.20
40.84
22.4119.5411.54
20.84
26.6515.82
21.4823.22
11.809.05
11.7617.69
13.68
11.7113.6731.30
41.88
12.1427.25
13.0514.86
58.76119.59
14.7316.84
14.09
12.4962.31
11.67
18.28
78.039.22
6.80
19.95
5.388.14
26.23
109.7752.51
8.8336.62
9.79
17.204.00
15.41
13.86
9.025.29
18.61
8.56
5.0010.64
3.7756.94
9.269.57
8.168.48
9.59
3.7317.27
13.50
15.81
26.4010.63
8.834.69
7.139.10
11.29
4.75429.31
11.6513.44
21.87
34.6314.6312.47
15.80
9.5812.8924.34
45.73
17.819.11
4.188.06
6.1310.05
5.895.87
7.35
11.273.81
211.48
190.60
1.5211.53
8.460.00
10.078.95
0.02
0.0051.63
9.047.28
8.60
243.61
13.6410.60
3.41
9.4518.385.56
11.24
7.0227.39
186.796.22
9.98
11.6023.306.56
16.43
7.545.38
169.20
6.54
26.8825.34
4.6310.15
4.4623.19
17.89
2.9813.23
10.65284.93
10.86
3.52
15.605.10
77.47
5.0514.123.29
10.98
14.6477.52
5.699.67
136.40
773.160.01
10.02
12.31
4.4193.867.77
9.91
4.9014.36
0.0517.91
4.88
4.7925.31
0.000.43
18.540.34
11.41
4.74
0.006.59
6.70
23.937.342.93
5.40
2.1811.51
9.3010.11
8.19
5.120.006.67
13.16
18.775.21
16.57
4.14
27.6524.95
9.1412.84
7.32
5.317.17
18.7944.52
13.289.98
8.06
0.003.68
17.74
40.05
0.005.07
12.971.70
37.979.89
2.499.58
13.62
5.394.636.72
7.12
10.471.005.09
44.13
8.1013.20
3.1171.86
5.99
1.236.238.11
6.50
5.8522.43
8.69
15.71
0.0014.46
0.00
9.1834.85
7.179.49
5.787.20
10.54
2.94
23.51
6.91
0.00
2.92
16.21
478 g
en
es
VehicleControl
VehicleI/R injury
DznepI/R injury
Group1
Group2
Group3
149 g
en
es
103 g
en
es
226 g
en
es
Low
High
RNA-seq of tubules isolated by laser capture
microdissection
Dznep suppressed TIMP2 expression in the kidney
RNA-seq of the kidney
0
5
10
15
20
Vehicle Ctl Vehicle IR Dznep IR
Timp2
RN
A-s
eq
(rp
km
)
Mimura, Hirakawa, Nangaku. manuscript in submission
0
100
200
300
400
Normoxia
Dznep(-)
Hypoxia
Dznep(-)
Hypoxia
Dznep(+)
TIMP2R
NA
-seq
(rp
km
)
HK2
0
20
40
60
80
Normoxia
Dznep(-)
Hypoxia
Dznep(-)
Hypoxia
Dznep(+)
TIMP2
RPTEC
HK2: human kidney-2 RPTEC: renal proxymal tubular epithelial cell(human primary culture cells)
RNA-seq: in vitro
Mimura, Hirakawa, Nangaku. manuscript in submission
Ischemia/reperfusion Fibrosis↑
Dznep Novel microRNA targets
TIMP2↓MMP↑
TIMP2↑Collagen↑
Fibrosis↓
MMP↓
Collagen↓
Dznep suppresses expression of TIMP2 via microRNA
Identified by small RNA-seq
Ischemia/reperfusion
Mimura, Hirakawa, Nangaku. manuscript in submission
ISN President
LOC chair
Meetings Committee chair
William
Couser
Richard
JohnsonStuart
ShanklandMark
Okusa
David
HarrisKai-Uwe
Eckardt
Juergen
Floege
Motoko
Yanagita