High Glucose Inhibits Nitric Oxide Production in Cultured

Rat Mesangial Cells

HOWARD TRACHTMAN, STEPHEN FUTTERWEIT, and DAN L. CRIMMINSDivision of Nephrology, Schneider Children ‘s Hospital, Long Island Jewish Medical Center, Long island

Campus for Albert Einstein College of Medicine, New Hyde Park, New York; and Department of Molecular

Biology and Pharmacology. and Protein and Nucleic Acid Chemistry Laboratory, Washington University

School of Medicine, St. Louis, Missouri.

Abstract. Hyperglycemia directly contributes to the develop-

ment of diabetic nephropathy. A high-serum glucose concen-

tration alters intraglomerular hemodynamics and promotes

deposition of extracellular matrix in the kidney. Nitric oxide

(NO) is a short-lived messenger molecule that participates in

the regulation of renal blood flow, GFR, and mesangial matrix

accumulation. Therefore, in this study it was tested whether

high glucose directly modulates NO synthesis by rat mesangial

cells in vitro by measuring the accumulation of nitrite, the

stable metabolite of NO, in the incubation media. Raising the

external glucose concentration to 33.3 mM for 24 to 72 h

reduced nitrite levels in cell supernatants in a time-dependent

manner to a nadir of 14 ± 3% of the amount in normal glucose

media (5.6 mM) (P < 0.01). The decline in NO synthesis in

high glucose media was paralleled by decreased cyclic

guanosine monophosphate generation; however, there was no

alteration in rat mesangial cell expression of inducible NO

synthase protein. The suppressive effect of high glucose on NO

production by mesangial cells was not modified by inhibition

of protein kinase C (H-7), the addition of antioxidants (vitamin

E or superoxide dismutase), or a pan-specific anti-transforming

growth factor-a antibody. An elevated ambient glucose caused

a time-dependent reduction in mesangial cell L-arginine con-

tent. Addition of L-arginine (10 to 20 mM) to external media

partially reversed the inhibitory effect of high glucose on

mesangial cell NO production in a dose-dependent manner.

The highest dose of L-arginine (20 mM) increased mesangial

cell L-arginine content to comparable levels in normal and high

glucose media. These results indicate that high glucose causes

depletion of L-arginine in mesangial cells and compromises

NO synthesis. Limitation in the metabolic precursor and other,

as yet unidentified, factors act to reduce NO production by

mesangial cells in the presence of an elevated ambient glucose

level, a change that may play a role in the development of

diabetic gbomerulosclerosis. (J Am Soc Nephrol 8: 1276-1282,

1997)

Hyperglycemia is a major factor in the pathogenesis of target

organ complications of diabetes including nephropathy (1). An

elevated glucose concentration enhances single nephron

plasma flow and GFR (2), causes oxidative injury to mesangial

cells (3). and enhances the production of extracellular matrix

(ECM) components by renal cells (4,5).

Nitric oxide (NO) is a volatile, cell-signaling molecule that

is synthesized from the guanidino nitrogen of L-arginine and

dioxygen by at least three different isoforms of the enzyme NO

synthase (NOS) (6). NO participates in the regulation of affer-

ent arteniolar tone in the glomerular circulation (7), tubular

handling of sodium and salt balance (8,9), and mesangial cell

proliferation (10). Mesangial cells and renal tubular epithelial

cells possess inducible NOS (iNOS) and synthesize NO in

response to various cytokines (I 1,12).

There are conflicting data concerning the effect of experi-

Received April 18, 1996. Accepted March 17, 1997.

Correspondence to Dr. Howard Trachtman. Schneider Children’s Hospital,

Division of Nephrology. 269-01 76th Avenue, New Hyde Park, NY I 1040.

l()46-6673/0808- I 276$03.00/0

Journal of the American Society of Nephrology

Copyright U 1997 by the American Society of Nephrology

mental diabetes on renal NO production. Some reports indicate

increased NO synthesis (13,14), whereas others have docu-

mented reduced NO production and decreased NO-dependent

cyclic guanosine monophosphate (cGMP) generation by gb-

meruli isolated from diabetic rats (15-17). To clarify the role

of NO in the pathogenesis of diabetic nephropathy, we exam-

med the effect of high glucose on NO production in cultured rat

mesangial cells (RMC) by measuring the accumulation of

nitrite, the stable metabolite of NO, in the incubation media.

Materials and MethodsCells

RMC were obtained from primary glomerular explants and used

between passages 6 and 10. Using phase-contrast microscopy, the

identity of mesangial cells was confirmed by their elongated, stellate,

or fusiform appearance: in addition, there was no inhibitory effect of

puromycin aminonucleoside or d-valine on cell growth (18). Cells

were plated in 96-well plates, 25 X I0� cells/mb, to assay nitrite

production, an index of NO synthesis ( 19). RMC were grown in

75-cm2 flasks for determination of L-arginine content and Western

analysis of iNOS protein. Cells were incubated at 37#{176}Cin a humid-

ified atmosphere of 10% C02-90% air and maintained in Dulbecco’s

modified Eagle’s medium (DMEM) supplemented with 1(X) �g/ml

streptomycin, 100 p�g/ml penicillin, and 10% fetal bovine serum.

High Glucose Inhibits NO Production in Cultured RMC 1277

Experimental Conditions

At 24 h after plating RMC into 96-well plates or when the cells

reached confluence in 75-cm2 flasks (usually within I wk), cells were

randomly assigned to one of the following two experimental condi-

tions: (1) Control: DMEM containing 5.6 mM glucose; (2) High

glucose: DMEM supplemented with 27.8 mM glucose to a final

concentration of 33.3 mM. Lipopolysacchanide (LPS) ( 10 pg/ml) and

interferon (IFN)-y (50 U/mI) were added to both test media because

NO production was negligible in the absence of the cytokine combi-

nation. A hyperosmolality control medium consisted of DMEM +

27.8 mM mannitol.

Measurement of Nitrite and cGMP Production

Nitrite production was measured using the Griess assay (19).

Briefly, 125 �tl of a solution containing 1% sulfanilamide, 0.1%

naphthylethylene diamine dihydnochloride, and 2.5% phosphoric acid

was added to I 25 j.tl of conditioned media. Samples were incubated at

25#{176}Cfor 10 mm, and absorbance was measured at 550 nm. Nitrite

production was normalized to the number of viable cells and ex-

pressed as a percentage of the value in control media containing a

normal glucose concentration. RMC were grown in 25-cm2 flasks,

and cGMP in the supennatants was measured by enzyme immunoas-

say after acetylation. using a commercially available kit (Cayman

Chemical Co., Ann Arbor, MI).

Cell Proliferation Assay

A colonimetnic method was used to determine the number of viable

RMC in these experiments. A total of 100 jd of phenazine methosul-

fate was added to 2 ml of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-

methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium. inner salt in the

dark. Twenty-five microliters of this mixture was added to each wellin 96-well plates. Plates were wrapped in foil and incubated for I h at

37#{176}Cin the 10% CO,-90% air atmosphere. Absorbance was read at

490 nm in samples and solution blanks.

HPLC Assay for L-Argimne

After incubation in normal and high glucose media that did not

contain LPS and IFN-y, cell monolayers were rinsed twice with

phosphate-buffered saline (PBS). RMC were removed, and the sus-

pension was spun at 10(X) rpm for 5 mm to sediment the cells. They

were resuspended in 500 �l of PBS, and then 1000 �l of 100%

methanol was added. Samples were spun at 10,000 X g for 10 mm,

and the cleared supernatants were analyzed by HPLC.

Vacuum-dried extracts were derivatized with 6-aminoquinolyl-N-

hydroxysuccinimidyl carbamate, using the Waters kit (Millipore, Mil-

fond, MA) with slight modifications (20). The most important change

was reducing the final volume of derivatized sample from 100 to 50�l comprising 10 pJ of 20 mM HCI, 30 �l of borate buffer, and 10 �l

of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent. A

maximum of 20 M1 of the denivatized sample can be injected onto the

HPLC column without obliterating the first one-third to one-half of

the chromatogram. Therefore, this modification allows up to 40% of

the sample to be analyzed. Mobile phase and gradient development

were adapted from Liu (2 1 ) for ultraviolet detection at 248 nm.

Calibration was performed with 250-pmol injections of standard H

(Pierce Chemical Co., Rockford. IL), and a-amino butynic acid was

the internal standard. Day-to-day system performance was reproduc-

ible to <4% variation.

Western Analysis

RMC were harvested in PBS and centrifuged at 1000 rpm for 5 mm

to sediment cells. The pellet was dissolved in 100 �l of lysis buffer

(50 mM Tnis/HC1, pH 7.6, 100 mM NaCl, 2 mM ethylenediami-

netetra-acetic acid, 2 mM ethyleneglycol-bis(�3-aminoethyI ether)-

N,N’-tetra-acetic acid, 1 mM dithiothreitol, I mM phenylmethylsul-

fonyl fluoride, and 1% Triton X-l00). The suspension was freeze-

thawed three times, and equal aliquots ofthe lysates (25 p�g of protein)

were loaded onto a 7.5% acrylamide-sodium dodecyl sulfate gel. Gels

were run at 200 V and 70 mA (milliamps) for 45 mm, and protein was

then transferred from the gel onto a nitroceblulose membrane electro-

phonetically. After blocking the membrane with buffer containing

0.25% gelatin and 0.05% Tween 20, the membrane was exposed to a

primary murine monoclonal antibody to iNOS (Transduction Labo-

ratonies, Lexington, KY) followed by a secondary antibody (horse-

radish peroxidase-binked anti-mouse IgG). Immunoblots of the iNOS

protein were visualized with enhanced chemibuminescence (Amen-

sham, Arlington Heights. IL).

Protein Assay

The protein contents of the cell homogenates were determinedusing a Coomassie blue reagent (BioRad, Richmond, CA).

Reagents

A pan-specific. polyclonal neutralizing anti-transforming growth

factor (TGF)-� antibody, reactive with TGF-� I ,TGF-�2, TGF-� I .2,TGF-/33, and TGF-�5, was purchased from R&D Systems (Minne-

apolis, MN). All other reagents were purchased from Sigma Chemical

Co. (St. Louis, MO).

Statistical Analyses

Results are presented as mean ± SEM. The means between the

experimental groups were compared using ANOVA; post hoc inter-

group comparisons were made using the Bonferroni correction.

ResultsNO Production

There was no measurable NO production by RMC, indicated

by undetectable nitrite levels, in media that did not contain

IFN-y and LPS. In the presence of the cytokine combination,

exposure of RMC to 33.3 mM glucose led to a time-dependent

inhibition of NO production (Figure 1). Thus, after 24 h of

incubation in high glucose media, nitrite accumulation (0.5 to

I .0 nmol/well) per unit number of viable cells was 88 ± 3% of

the amount in normal glucose media (P < 0.05). More pro-

longed exposure to high glucose conditions resulted in a pro-

gressively greater effect, i.e., a decline to 52 ± 4 and 14 ± 3%

of the nitrite levels (1 to 4 nmol/well) observed in control

media after 48 and 72 h, respectively (P < 0.01). Cell viability

was consistently greater in the high glucose media at 24, 48,

and 72 h, i.e., 108 ± 6, 152 ± 8, and 186 ± 11% of the value

in normal glucose media, respectively (P < 0.05). Thus, the

effect of high glucose on nitrite levels was the result of a

combined effect on NO production and improved cell viability.

NO production and cell viability were comparable in the dif-

ferent cell lines and passages used in these experiments.

To determine whether high glucose altered the partitioning

of NO metabolites between nitrate and nitrite, the nitrite level

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RMC supernatants after 48 h of exposure to test media withindicated additive. Samples were analyzed before incubation with

Escherichia co/i reductase (NO, ) or after incubation withEsc/ierichia co!i reductase (NO, + NO� ). RMC, rat mesangialcells.

1278 Journal of the American Society of Nephrology

24H 48H 72H

Figure 1. Effect of high glucose on rat mesangial cell (RMC) nitrite

levels (nmol/unit number of viable cells). Results are expressed as a

percentage of paired normal glucose media (n = 10 for each time

point). */) < 0.05 versus pained normal glucose media. **� < 0.01versus paired normal glucose media.

in conditioned RMC media was determined in normal and high

glucose media, before and after incubation of samples with

Escher#{252}hia co/i reductase ( I 9). The difference between these

two values represents the amount of nitrate in the supernatant.

The ratio of NO,/NO, + NO1 was the same in normal and

high glucose media and was unaffected by supplementation

with 20 mM L-arginine (Table 1).

The inhibition of NO production by RMC during exposure

to high glucose media was paralleled by a reduction in cGMP

generation. Thus, after 24 h, cGMP accumulation in the media

(expressed as pmollnil supernatant per unit number of viable

cells) declined from 3. 1 5 ± 0.30 to 2.9 1 ± 0.43. After 48 h of

incubation in high glucose media, cGMP accumulation was

further reduced by 47%, from 2.71 ± 0.29 in normal glucose

media to 1 .44 ± 0. 13 in high glucose media (ii = 3) (P <

0.05).

Hyperosmolal media containing 27.8 mM mannitol did not

alter RMC NO production (data not shown). There was no

evidence of cytotoxicity as a result of incubation with high

Table 1. N0/NO + NO� ratio in cultured RMC: effect

of normal and high glucose media�’

Group Norma 1 Glucose High Glucose

No additives 0.38 ± 0.05 0.41 ± 0.03

+2 0 mM L-arginine 0.34 ± 0.07 0.35 ± 0.04

glucose media, based on the amount of lactate dehydrogenase

detectable in the conditioned media (data not shown).

The diabetic state is associated with activation of protein

kinase C (PKC) (22). This enzyme can phosphorylate the

neuronal and endothelial isoforms of NOS and reduce their

activity (23,24). Therefore, we examined whether addition of

H-7 (50 �sM), an inhibitor of PKC, could normalize NO syn-

thesis by RMC incubated in high glucose media for 48 h.

Instead, we found that H-7 caused a profound reduction in NO

synthesis per unit number of viable cells to 9 ± 3 and 7 ± 2%

of the control value under normal glucose and high glucose

conditions, respectively (Table 2). The inhibitory effect also

was observed after 24 h of exposure to H-7. Despite the lack of

specificity of H-7 as a PKC inhibitor, the data do not support

a role of PKC activation in reducing NO production by RMC

in high glucose media.

High glucose stimulates gene expression and release of

TGF-/3 by renal cells (25). This cytokine destabilizes iNOS

mRNA, reduces translational efficiency, and enhances iNOS

protein degradation; these changes result in decreased NO

synthesis (26). Therefore, we tested whether a pan-specific,

polyclonal anti-TGF-3 antibody (35 to 200 j�g/ml) would

restore mesangial cell NO synthesis to normal. In fact, there

was no significant effect of anti-TGF-�3 antibody on NO syn-

thesis by RMC that were maintained in normal glucose media

for 48 h. In contrast, RMC exposed to a high, ambient glucose

level for 48 h manifested a dose-dependent inhibition of NO

synthesis by anti-TGF-f3 antibody (Table 2). Nonimmune IgG

(200 pg/ml) had no effect on RMC NO production (data not

shown).

An elevated, ambient glucose exerts a direct oxidant stress

on cultured RMC (3). We examined the effect of the antioxi-

dants vitamin E (100 p�M) and superoxide dismutase (250

U/mb) on NO synthesis by RMC. These agents failed to reverse

Table 2. Nitrite levels in cultured RMC: effect of H-7,

anti-TGF-3 antibody, and antioxidantsa

Group Normal Glucose Hig h Glucose

No additives I 00 ( 12)” 48 ± 4 (12)”

H-7 (50 �M) 9 ± 3 (6)” 7 ± 2 (6)”

Anti-TGF-� antibody

35 p.g/ml 92 (2) 43 (2)

100 �tg/ml 102 (2) 31 (2)

200�g/ml 78(2) 6(2)

Vitamin E ( 100 �M) 90 ± 5 (4) 49 ± 3 (4)�)

SOD (250 U/ml) 66 ± 5 (4)” 35 ± 5 (4)b.c

a The results are mean ± SEM. The numbers in parentheses

indicate number of paired samples. Nitrite levels (nmol/unit numberof viable cells) after 48 h of exposure to the test media withindicated additive is expressed as a percentage of control normalglucose medium containing interferon-y and lipopolysacchanide andno further additives. TGF, transforming growth factor; SOD,superoxide dismutase.

b p < 0.01 versus normal glucose, no additives.

C p < 0.05 versus normal glucose plus SOD.

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the suppressed NO synthesis by RMC incubated in high glu-

cose media to the bevel observed in normal glucose conditions

(Table 2).

Addition of L-arginine caused a dose-dependent increase in

NO synthesis by RMC maintained in normal glucose media for

48 h. Thus, there was a 125 ± 7 and 197 ± 20% increase in

NO synthesis after addition of 10 and 20 mM L-arginine,

respectively, to the external media (P < 0.01) (Figure 2). In

contrast, in the presence of an elevated, external glucose con-

centration, 10 mM L-arginine had no effect on RMC NO

synthesis, whereas 20 mM L-argrnine enhanced NO production

to I 17 ± 2 1% of the value in control normal glucose media

(P < 0.01) (Figure 2). The increased NO synthesis by RMC

exposed to supplemental L-arginine was reduced nearly 80%

after the addition of NG�nitro�L�arginine methyl ester

(L-NAME) (I mM) to normal and high glucose media.

HPLC Analysis of RMC Amino Acid Content

In these experiments, LPS and IFN-y were not present in the

test media. There was no significant change in L-arginine

content in RMC after 24 h of exposure to high glucose (n = 3)

(Figure 3). L-Arginine content varied widely in RMC that were

exposed to high glucose (range, 0.2 to 2.5 nmol/unit number of

viable cells) or normal glucose media (range, 0.5 to 5.6 nmoll

unit number of viable cells) for 48 to 72 h because of the use

of RMC of different passage. Therefore, we expressed

L-arginine content in cells exposed to high glucose as a per-

centage of the paired sample maintained in normal glucose

media. There was a time-dependent decrease in RMC L-argi-

nine content by 34 and 68% after 48 and 72 h exposure to high

glucose, respectively (P < 0.05) (n 3 for each time point).

The reduction in RMC L-arginine content paralleled the decline

in NO production observed in the presence of an elevated,

ambient glucose and the two cytokines (Figures 1 and 3).

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Figure 2. Effect of L-arginine on RMC nitrite levels after 48 h

(nmol/unit number of viable cells). Results are expressed as a per-

centage of paired normal glucose media (it = I 0 for each time point).

Open bars, normal glucose; hatched bars. high glucose. *� < 0.01

versus control normal glucose media, no added L-arginine. **�P <

0.01 versus high glucose media, no added L-argiflifle.

0

Figure 3. Effect of high glucose on RMC L-arginine content (nmol/

unit number of viable cells). Results (mean ± SEM) are expressed asa percentage of paired normal glucose media. *P < 0.05 versus paired

normal gbucose media.

Addition of 20 mM L-arginine (ii = 4 pairs for normal and high

glucose conditions) to incubation media for 48 h significantly

increased RMC L-arginine content 4.6-fold in normal glucose

conditions, from I .6 ± 0.4 to 7.2 ± 2.4 nmol/unit number of

viable cells, and 5.8-fold in high glucose conditions, from

1.1 ± 0.3 to 6.1 ± 1.1 nmol/unit number of viable cells.

Despite the comparable L-arginine content of RMC after ex-

posure to 20 mM L-arginine, NO production was still lower

under high glucose conditions (Figure 2). This suggests that

other, as yet unidentified, factors, in addition to L-arginine

depletion. contribute to lower NO production by RMC in high

glucose media.

Western Analysis

Incubation of RMC with high glucose media for 24 to 48 h

had no demonstrable effect on the expression of the 1 30-kD

iNOS protein (n = 3) (Figure 4). Densitometric analysis re-

vealed no difference in the intensity of the iNOS protein band

in lysates prepared from RMC incubated in normal or high

glucose conditions for 24 or 48 h (data not shown). In addition,

high glucose media had no effect on iNOS protein abundance

in RAW 264.7 cells grown in normal versus high glucose

conditions for 24 h (Figure 4).

DiscussionThe results of this study indicate that high glucose inhibits

NO production by cultured RMC. This change was not due to

alterations in PKC activity, TGF-f3 synthesis, or oxidant injury

to mesangial cells. Instead, reduced NO synthesis in RMC

exposed to high glucose appeared to result from a time-depen-

dent depletion of L-arginine, the metabolic precursor of NO, as

well as other unidentified factors. These findings are in accord

with previous results, in which NO production was reduced in

gbomeruli isolated from rats with streptozocin-induced diabetes

(15-17). NO synthesis is reduced in coronary endothelial cells

of the spontaneously diabetic BB (BioBreeding) rat (27). Ex-

posure of human umbilical vein endothelial cells (28) and

**

0 10mM 20mM

High Glucose Inhibits NO Production in Cultured RMC 1279

1280 Journal of the American Society of Nephrology

C I II lilly vvIcFigure 4. Effect of exposure to high glucose for 24 h on inducible NO synthase (iNOS) protein content assayed by Western analysis. Lane C.

iNOS control specimen; lane I. normal glucose, 24 h: lane II, high glucose. 24 h: lane III. normal glucose, 48 h: lane IV, high glucose. 48 h:

lane V. RAW 264.7 cells. normal glucose, 24 h; lane VI, RAW 264.7 cells, high glucose, 24 h. Arrow indicates the location of iNOS protein

at 130 kD.

SH-SY5Y human neuroblastonla cells to high glucose (29)

inhibits NO production. Finally. a recent study indicates that

high glucose downregulates NO production and iNOS expres-

sion in cultured rat aortic vascular smooth muscle cells, an

effect that was reversed by addition of a PKC inhibitor (30).

However, high glucose does not uniformly inhibit NO produc-

tion. Porcine aortic endothelial cells manifest increased nitrite

accumulation in vitro in response to an elevated, ambient

glucose level (3 1 ). Thus, the effect of high glucose on different

cell types in diabetes may vary from system to system and

requires further study.

Our data are at variance with the work of Sharma et al. (32),

in which exposure of murine mesangial cells to high glucose

for 24 h stimulated iNOS gene expression, iNOS protein syn-

thesis, and NO production. The discrepancy between this re-

port and the study presented here may be due to species

differences (mouse versus rat), the high glucose level (450

mg/dl t’er.sii.s 600 mg/dl). and the method of normalization of

NO production (nmol/mg protein in the study by Sharma et al.

versus nmol/number of viable cells in the report presented

here). The improved cell viability of RMC maintained in high

glucose media underscores the importance of the specific

method used to normalize nitrite levels.

The mechanism of high glucose-induced depletion of

L-arginine in cultured RMC was not addressed in this study.

RMC content of L-arginine represents a balance between up-

take, effiux, and utilization of the amino acid. Exogenous

1-arginine increased L-arginine content in RMC maintained in

high glucose media. suggesting that enhanced glucose uptake

via the facilitative transporters GLUT- 1 and GLUT-2 (33)

interferes with L-arginine transport by RMC. The incremental

effect of L-arginine supplementation on coronary artery vaso-

dilation in the isolated guinea pig heart perfused with high

glucose is consistent with a hyperglycemia-induced decrease in

cell l-arginine content (34). The failure to completely normal-

ize NO production by RMC in high glucose despite virtual

normalization of RMC L-arginine content by the addition of 20

mM L-arginifle suggests that there are derangements in

L-argifline utilization that are not reversed by supplementation.

For example. alterations in cytosolic handling of other amino

acids such as glutamine may augment efflux or utilization of

1-arginine by RMC and reduce cellular levels of the NO

precursor (35).

Activation of PKC and secretion of TGF-� inhibit NOS and

reduce NO production (23,24,26). Moreover, high glucose

stimulates PKC activity and TGF-� release by RMC in vitro

(22. 25). However, the inability to normalize NO production

with a PKC inhibitor or anti-TGF-f3 antibody suggests that

these factors do not account for reduced NO production by

RMC in high glucose media. This contrasts with the recent

demonstration that addition of a PKC inhibitor restores NO

production and iNOS expression in rat aortic vascular smooth

muscle cells (30). High glucose also promotes lipid peroxida-

tion of cultured RMC (3). However, the failure of the antioxi-

dants to reverse the inhibition of NO production by high

glucose suggests that enhanced oxidant stress is not involved in

this response.

There is evidence that NO promotes gbomerular injury and

ECM deposition in the acute experimental glomerulonephritis

that develops after injection of anti-thymocyte serum (36).

However, in rats with extensive renal ablation, a chronic non-

inflammatory process, inhibition of NO synthesis enhances

glomerular injury (37). Moreover. rats with streptozocin-in-

duced diabetes have reduced plasma L-arginine levels, and

dietary L-arginine supplementation ameliorates gbomerular hy-

perfiltration and proteinuria (38). However, additional studies

are needed to determine whether diabetes in experimental

animals or in humans results in altered expression of iNOS in

glomerular mesangial cells.

We recently demonstrated that NO downregulates the syn-

thesis of collagen and fibronectin and stimulates the production

of laminin by RMC (39). In addition, NO stimulates the

activity of a 72-kD neutral matrix metalboproteinase in these

gbomerular cells (40). Thus, by inhibiting ECM synthesis and

facilitating matrix degradation, NO may act as an antifibrotic

modulator within the gbomerulus. This renoprotective role of

NO may be more pronounced in chronic, noninflammatory

kidney diseases such as the 5/6 nephrectomy or experimental

diabetes. We speculate that gbomerular NO deficiency in dia-

betes may contribute to the accumulation of ECM proteins in

the mesangium and may be an important factor in the devel-

opment of gbomerulosclerosis in diabetic nephropathy.

AcknowledgmentsThis study was supported by Research Grant 95-()07 from the

Genentech Foundation for Growth and Development to Dr. Tracht-

man. The authors thank Dr. Pravin C. Singhal (Department of Med-

icine, Long Island lewish Medical Center, New Hyde Park. NY) for

providing RMC.

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