Tetrahydrobiopterin reverses the inhibition of nitric oxide by high glucose in cultured murine mesangial cells

Sharma S. Prabhakar

Division of Nephrology, Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Alterations of intrarenal nitric oxide (NO) synthesis play an important role in the pathogenesis and progression of diabetic nephropathy. We tested the hypothesis that hyperglycemia modulates intrarenal NO synthesis, which might mediate the mesangial cell proliferation and matrix production. Murine mesangial cells were grown in media containing varying glucose concentrations, and cytokine-induced NO synthesis was assayed by chemiluminescence using an NO analyzer. High media glucose (25 mM) inhibited NO synthesis in a time-dependent fashion. This inhibition was posttranslational as revealed by analysis of inducible nitric oxide synthase (iNOS) gene and protein expression. L-Arginine supplementation partially reversed the inhibition whereas addition of tetrahydrobiopterin (BH4), a cofactor for NOS, restored the inducibility of NO synthesis. The in vitro [3H]citrulline assay for iNOS activity indicated that high glucose decreased BH4 availability whereas examination of the BH4 synthetic pathway suggested decreased BH4 stability rather than synthesis, a defect that was corrected by ascorbic acid. We conclude that hyperglycemia inhibits NO synthesis in mesangial cells by a posttranslational defect that might involve the stability and hence availability of BH4.

hyperglycemia; inducible nitric oxide synthase; ascorbic acid


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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DIABETIC NEPHROPATHY IS THE most common cause of end-stage renal failure in the United States and probably in the entire Western world. Recent large-scale clinical studies have demonstrated that poor glycemic control, activation of the renin-angiotensin axis, and hypertension play an important role in the progression of diabetic nephropathy (17, 34). Several autocrine and vasoactive factors including angiotensin, eicosonoids, endothelin, and nitric oxide (NO) have been implicated in modifying the rate of progression of diabetic renal disease. NO could potentially play a major role in mediating the effects of hyperglycemia, hypertension, and activation of angiotensin. However, the potential role of NO has received comparatively less investigative attention. We examined the effects of high glucose (HG) on NO synthesis by mesangial cells in culture and further attempted to define the mechanisms involved by studying inducible nitric oxide synthase (iNOS) gene and protein expression. The results indicated that HG inhibited NO synthesis at a posttranslational step by interfering with the availability of tetrahydrobiopterin (BH4), a cofactor of the NOS enzyme.


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Cell culture. Murine mesangial cells (MMC) obtained from the American Type Culture Collection were cultured in DMEM (GIBCO-BRL, Life Technologies, Grand Island, NY). The medium was reconstituted to contain 10% fetal bovine serum, 10 U/ml penicillin, 10 µg/ml streptomycin, and 25 µg/ml amphotericin B. Medium was customized to contain L-glutamine but minimal nitrate to avoid interference with nitrate assay. Experiments for assay of NO synthesis were performed by subculturing MMC in 96-well plates using 5 × 104 cells/wells from passages 4-10 and incubating them at 37°C for 20 h. NO synthesis was induced by bacterial lipopolysaccharide (LPS) derived from Escherichia coli serotype 026:B6 (Sigma, St. Louis, MO) at a concentration of 10 µg/ml and murine recombinant tumor necrosis factor (TNF) at a concentration of 100 ng/ml. For iNOS gene expression and iNOS protein expression, cells were cultured in 75-cm2 flasks.

Chemicals. L-Arginine, murine recombinant TNF, E. coli-derived LPS, BH4, sepiapterin, N-acetyl seratonin (NAS), ascorbic acid, and a NOS enzyme assay kit were obtained from Sigma. DMEM, endotoxin-free FCS (heat inactivated), and L-glutamine were purchased from GIBCO. 2,4-Diamino-6-hydroxy-pyramidine (DAHP) was obtained from Aldrich, Milwaukee, WI. Reconstituted medium and the stock solutions of LPS and TNF were stored at 4°C until needed.

NO measurement. The supernatant from the medium in each well was used for the measurement of NO synthesis in MMC by chemiluminescence. A NO analyzer (NOA 280, Seivers Instruments, Boulder, CO) was used for this purpose. The details of the technique and methodology have been previously published (25). Each sample was measured three times, and the mean of the three readings was taken as the result. The NO activity of the untreated medium was subtracted from each sample to reflect the true NO content of each sample.

NOS assay. NOS enzyme activity was assayed by citrulline assay as described by Bredt and Snyder (3). Briefly, after the cells were incubated under experimental conditions for 18 h, they were washed in 1× PBS, harvested in trypsin-EDTA, and centrifuged at full speed for 5 min at room temperature, and the supernatant was discarded. The pellet was resuspended in 100 µl of 1× homogenization buffer, and cells were disrupted by repeated pipetting. The cells and the supernatant were centrifuged at full speed for 5 min, and the supernatant was collected in a tube and kept on ice until used for the NOS assay. The protein concentration of the samples was measured by spectrophotometry at 595 nm, and the sample volume was adjusted to contain 5 µg of protein/µl. A reaction mixture containing 10 mM NADPH, [3H]arginine was prepared in a microcentrifuge tube and placed on ice, and appropriate sample volumes (usually 1-20 µl) containing 5 µg of protein were added to 40 µl of reaction mixture in corresponding tubes. A positive control was prepared using rat cerebellar extract supplied with the NOS assay kit, and a negative control was prepared using NG-nitro-L-arginine methyl ester, a NOS inhibitor. All the samples were incubated at 37°C for 30 min, and the reaction was stopped by a stop buffer containing EDTA. Then, 100 µl of resuspended equilibrated buffer were added to each sample, which were then put into spin cups. The samples were then spun at full speed for 30 s, and the elute was transferred into a scintillation vial containing 5 mls of scintillation fluid. The radioactivity was then measured in a liquid scintillation counter and expressed as counts per minute per milligram of protein per minute.

RNA isolation. When the experimentation with cells was completed, the cells were suspended in 1 ml of TRIzol and incubated at room temp for 5 min. Then 200 µl of CH3Cl were added, and cells were incubated at RT for 3 min and then transferred to a phase-lock tube. The tubes were centrifuged at maximum g for 15 min. The incubation and centrifugation with CH3Cl was repeated twice. The top liquid layer was transferred to a microtube, and an equal volume (usually 500 µl) of 100% isopropanol was added and mixed by inverting the tube. The tubes were incubated at RT for 10 min and centrifuged at maximum g for 10 min. The supernatant was then removed, and the pellet was washed with 500 µl of 70% ethanol. After vortexing, the tubes were centrifuged at maximum g for 10 min, and the supernatant was then removed and the pellet was dried. The ETOH was pipetted off, 20 µl of 1,2-dihexadecyl-sn-glycero-3-phosphocholine water were added, and the pellet was allowed to dry. The tubes were then labeled, and RNA was stored at -70°C.

RT-PCR. One microliter of RNA was added to 19 µl of master mix 1 (5 µl of H2O, 2 µl of PCR buffer, 2 µl of MgCl2, 8 µl of dNTP, 0.5 µl of RNAse inhibitor, 1 µl of random hexamers, and 0.5 µl of Superscript II) in 0.2-ml PCR tubes. After being vortexed and spun, the tubes were placed in a PCR machine, and an RNA2 cycle (23°C × 10 min, 37°C × 15 min, 42°C × 15°C min, 99°C × 5 min, and 4°C × infinity ) was performed. For PCR, 18 µl of master mix 2 (2 µl PCR buffer, 14.45 µl H2O, 0.65 µl MgCl2, 0.8 µl primer, and 0.1 µl Taq) were pipetted into 0.2-ml PCR tubes. Two microliters of cDNA were added to each tube, vortexed, and spun. The PCR tubes were then placed in the PCR machine, and the PCR 1 cycle was run. This consists of 2 cycles of 94°C × 20 s, 60°C × 30 s, 72°C × 90 s and 35 cycles of 94°C × 20 s, 60°C × 30 s, and 72°C × 90 s followed by 72°C × 7 min and 4°C × infinity . The PCR results were then viewed by running an agarose gel. A 1% agarose gel in Tris, acetic acid, and EDTA was made with a total weight of 30 g, and 1 µl of ethidium bromide was added and poured into a gel caster. After the gel hardened, the samples (2 µl of cDNA with 8 µl of loading buffer) were loaded into each well. The gel was run at 120 V for 30 min and viewed in a transilluminator, and autoradiographs were obtained. A housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase, and negative controls omitting RT were used for quality control in all studies.

Western blot analysis. MMC were incubated in six-well plates at 105 cells/well for 24 h in various experimental conditions. The cells were dissociated by 500 µl of SDS lysis buffer. One hundred twenty-five microliters of loading buffer were added and frozen until processed. The concentration of total protein in all samples was assayed by spectrophotometry at 595 nm, and 20 µg of protein from each sample were loaded into microwells in SDS page gels and electrophoresed for one-half hour at 200 V. The gel was then transferred onto a polyvinylidene difluoride membrane (Bio-Rad). The membrane was presoaked in 100% methanol, water, and transfer buffer. The gel was attached to filter paper and placed on a cassette, and the membrane was placed over the gel and covered with filter paper. The gel was transferred onto the membrane, and electrophoresis was carried out at 87 V in a cold room for 2 h. The membrane was placed in a plastic container containing 25 ml of blocking buffer (1.5% BSA in TST) and rocked for 1 h. The membrane was then incubated in 25 ml of blocking buffer containing primary antibody, 1:10,000 dilution, and left overnight in a cold room (-20°C). After the buffer was removed, the membrane was washed with TST sequentially five times in periods varying from 5-15 min. The secondary antibody (anti-mouse IgG tagged to horseradish myeloperoxidase) in 1.5% BSA buffer was added over the membrane and incubated in a rocker for 4 h. The membrane was then washed as above and incubated in reagents 1 and 2 sequentially for 5 min each. The membrane was then dried on a paper towel and placed on a cassette and exposed in a dark room. The density of the bands on the autoradiographs was measured using a commercially available software (Visuage 2000 blot scanning and analysis, Fujitsu America, San Jose, CA), and the integrated optical density was expressed in arbitrary units and as a percentage of control groups.

MMC proliferation studies. MMC were grown in 75-cm2 flasks for 2, 4, or 7 days, dissociated at the end of these periods from the flask using the cell dissociation buffer, centrifuged, washed in PBS twice to clear the dissociation buffer, and resuspended in 5 ml of media. A portion of these samples (0.1 ml) was added to 0.8 ml of PBS and 0.1 ml of trypan blue and vortexed for uniform distribution. Trypan blue stained the dead cells blue. The cell density was then measured by examining the number of viable cells only, using the Nubeaur counting chamber.

Statistical analysis. All results are expressed as means ± SE. The data were analyzed using commercially available statistical software, Sigma Plot 5 (SPSS, Richmond CA). A comparison among groups was performed by independent t-test or repeated measures ANOVA, as appropriate. The difference was considered significant when P < 0.05. In the case of RT-PCR and Western blot analysis, the most representative of several experiments is illustrated.


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ABSTRACT
INTRODUCTION
METHODS
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REFERENCES

Induction of NO synthesis. We first attempted to establish the conditions for NO induction in MMC because it has not been as well described as in rat mesangial cells. Cells were incubated in DMEM media customized to contain minimal nitrite to avoid interference with NO assay. NO synthesis was induced with LPS derived from E. coli (026:B6) at a final concentration of 10 µg/ml, murine recombinant TNF at a final concentration of 100 ng/ml, and a combination of LPS and TNF at the above-mentioned concentrations. NO synthesis was assayed by measuring the accumulation of end products, i.e., nitrites and nitrates, in the media [designated as metabolites nitrate and nitrite (NOx)]. Although LPS and TNF caused significant NO synthesis (see Fig. 1) independently, (5.86 ± 1.38 and 7.97 ± 1.23 µM, respectively, vs. control 2.83 ± 0.58 µM, P <0.05, n = 12 in each group), the combination of LPS and TNF induced the highest NOx accumulation (19.81 ± 2.41 vs. 2.83 ± 0.58 µM in control P < 0.01, n = 12). We employed this combination in our further experiments. We then examined the time course of NOx accumulation in MMC on stimulation with LPS and TNF. Ten microliters of media were recovered at each time point for analysis, and NOx was assayed at 0, 6, 12, 18, 24, and 48 h (Fig. 2). The results indicated that although detectable NOx was found as early as 6 h, the amount steadily increased and peaked at 18 h, then plateaued and started declining after 24 h. We therefore incubated MMC for 20 h before NOx was assayed in all subsequent experiments.


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Fig. 1.   Induction of nitric oxide (NO) synthesis in murine mesangial cells (MMC). Comparison is shown between effect of different stimulating agents on NO synthesis as measured by accumulation of metabolites nitrate and nitrite (NOx) in media. The combination of lipopolysaccharide (LPS) and tumor necrosis factor (TNF) caused the highest NOx accumulation among all groups. *P value <0.05, **P < 0.001 compared with control (n = 12 in all groups).



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Fig. 2.   Time course of NOx accumulation in MMC. The peak levels are seen between 18 and 24 h, (n = 24 in each group). In all further experiments, a 20-h incubation was used.

Effect of HG in the media on NO synthesis. Next, the effects of increasing glucose concentrations on NO synthesis in MMC were examined. The standard medium or low glucose (LG) contained 100 mg/dl of glucose. The glucose concentration was raised in the test groups to contain 450 mg/dl or 25 mM HG. Our results demonstrated that MMC exposed to LG demonstrated a sixfold increase in NOx accumulation, whereas HG in the media significantly inhibited NO synthesis in MMC (see Fig. 3) after 20 h of incubation. Exposure to HG resulted in only a twofold increase; however, incubation of MMC in equimolar (25 mM) concentration of mannitol demonstrated NOx accumulation comparable to LG groups, indicating that HG inhibited NO synthesis in MMC by mechanisms independent of osmolality.


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Fig. 3.   Effects of high glucose (HG) on NOx accumulation in MMC. T and L denote TNF and LPS. M, mannitol. *P < 0.05, **P < 0.001, compared with unstimulated cells. #P < 0.05 compared with low glucose (LG); (n = 24 in each group).

To test whether NO inhibition by HG was related to duration of exposure to HG, we incubated MMC in LG or HG for 7 days and then stimulated them with LPS and TNF (Fig. 4). Although cells incubated in LG retained the ability to induce NOx accumulation (18.89 ± 2.12 vs. 2.89 ± 1.05 in unstimulated cells, P < 0.001), cells exposed to HG demonstrated near-total inhibition of NOx accumulation [4.12 ± 0.9 vs. 3.53 ± 0.89 µM in unstimulated MMC, P value not significant (NS)]. These data suggest that the HG-induced incubation of NO synthesis in MMC becomes total with prolonged exposure.


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Fig. 4.   Effects of prolonged exposure to HG on NOx accumulation in MMC. **P < 0.001 as seen in LG+L+T compared with LG and HG+L+T vs. LG+L+T; n = 24 in each group.

Effects of L-arginine supplementation on NO synthesis. Hyperglycemia has been shown to inhibit cellular uptake of L-arginine (8), the substrate for NO, thereby limiting the ability to synthesize NO. To examine the potential role of this phenomenon, we increased the media L-arginine from 0.5 to 10 mM (a 20-fold increase) and examined NOx accumulation after 20 h of incubation of MMC with L-arginine-supplemented media (Fig. 5). Although cells in LG demonstrated a sixfold increase with stimulation (25.42 ± 3.35 vs. 4.02 ± 0.92 µM in unstimulated cells), cells exposed to HG, arginine-enriched media demonstrated a threefold increase (15.32 ± 1.82 vs. 5.85 ± 0.98 µM), as opposed to a twofold increase in standard arginine concentration (8.18 ± 1.67 vs. 4.12 ± 1.01 µM). These data suggest that although L-arginine supplementation significantly increased the NOx accumulation in media it was not sufficient to overcome the inhibition of NO synthesis by HG. Compared with the LG group, cells exposed to HG even with L-arginine supplementation continued to demonstrate significantly (P < 0.05) lower NO synthesis.


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Fig. 5.   Effects of L-arginine on NOx accumulation in MMC. A in the test groups represents L-arginine. *P < 0.05 as between HG+L+T vs. HG and HG+A+L+T vs. HG+A. **P < 0.001 between LG and L+T. The P value for HG+L+T vs. HG+A+L+T was also <0.05, n = 12 in each group.

Effects of HG on MMC proliferation. The decreased NO synthesis in HG media might be secondary to decreased viability and proliferation of MMC. To examine this possibility, we measured the rate of cell proliferation in LG and HG (Fig. 6). Cells exposed to HG demonstrated significantly higher survival and proliferation than in LG (P < 0.01) at days 2, 4, and 7. The decreased total NOx accumulation together with increased cell proliferation suggest a dramatic decrease in NO release per viable cell.


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Fig. 6.   MMC glucose growth curve. Effects of glucose concentration on cell growth are shown. The number of viable cells per well were counted manually on a Neubar cell counting chamber, and the mean of the 6 wells with error bars is represented on the y-axis. Compared with LG, cells grown in HG were significantly more (P < 0.01) at days 2, 4, and 7.

Effects of HG in media on iNOS expression. We then examined iNOS gene expression in MMC by RT-PCR using a 226-bp iNOS mRNA primer, and iNOS protein expression by Western analysis using rat iNOS antibody. There was no difference in the expression of iNOS mRNA and iNOS protein in cells exposed to standard glucose and HG (see Figs. 7 and 8). Image analysis of the bands on the Western blots showed that the integrated optical density was 4.825 ± 0.41 U (standard glucose) vs. 5.125 ± 0.32 U (HG), whereas the positive iNOS control was 5.32 ± 0.53 U (n = 4, P = NS among all three groups). These data suggested that the inhibition of NO synthesis by HG was at a posttranslational level.


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Fig. 7.   RT-PCR for iNOS mRNA in MMC. Effects of HG on iNOS gene expression are shown. The RT-PCR results shown are representative of 3 other similar experiments. Lane 1: 100-bp ladder mass molecular marker controls; lane 2: LG + GAP (-) (RT-) control; lane 3: LG + L + T + iNOS (RT-) control; lane 4: LG+L+T GAP(+); lane 5: LG+L+T iNOS(+); lane 6: HG+GAP (RT-) control; lane 7: HG+iNOS (RT-) control; lane 8: HG+L+T GAP(+); lane 9: HG L+T iNOS (+); and lane 10: 100-bp ladder. The positive band in lanes 5 and 9 represents a 226-bp primer for iNOS mRNA. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.



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Fig. 8.   Western blotting showing effects of HG on iNOS protein expression. The immunoblot shown above represents similar results from 3 other experiments. Densitometric image analysis revealed no significant differences between the bands seen in lane LG+(L+T) and lane HG+(L+T).

Effects of BH4 on iNOS in MMC exposed to HG. Next, we evaluated the effects of BH4, a cofactor for iNOS that activates the enzyme at a posttranslational step. MMC were incubated in HG-containing media with or without BH4 for 20 h, and the effects on NO induction were evaluated. In micromolar concentrations, BH4 reversed HG-induced NO inhibition (Fig. 9), suggesting that HG interfered with the availability and/or binding of iNOS with BH4. These data further suggested that the level of inhibition of NO by HG was at a posttranslational level.


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Fig. 9.   Effects of tetrahydrobiopterin (BH4) on NOx accumulation in MMC. **P < 0.001. L+T vs. LG and HG+BH4+L+T vs. HG+L+T, n = 12 in each group.

Evaluation of iNOS activity in MMC lysates treated with HG. To determine whether the iNOS inhibition was due to reduced binding or availability of BH4, the iNOS activity was examined in lysates of MMC treated with standard and HG-containing media. The lysates were then incubated in a reaction mixture for 30 min at 37°C, as described in METHODS. During the incubation, a second set of lysates from MMC treated with normal and HG were exposed to saturating concentrations of BH4. As shown in Fig. 10, LPS and TNF treatment resulted in significantly higher NOS activity in lysates from MMC treated with standard glucose (498 ± 57.1 vs. control 182.4 ± 32.2 cpm · mg protein-1 · min-1, where cpm is counts per minute, n = 6, P < 0.01), whereas no such increase was seen in MMC treated with HG (209.7 ± 35.3 vs. control 167 ± 29.1 cpm · mg protein-1 · min-1, n = 6, P = NS). These observations confirmed the NOx measurements in intact MMC under similar conditions. When lysates from MMC treated with standard and HG were incubated with BH4, the iNOS activity was similar in both groups (548 ± 52.2 vs. 524 ± 47.1 cpm · mg protein-1 · min-1, n = 6, P = NS). These results indicate that the reduced iNOS activity mediated by HG is related to reduced availability of BH4. A similar increase in iNOS activity was seen in intact MMC treated with HG, and 10 µM of BH4 and lysates examined subsequently (484 ± 46.8 cpm · mg protein-1 · min-1, n = 6). These data reflecting iNOS activity are consistent with the NOx measurements from intact cells described earlier.


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Fig. 10.   NO synthase (NOS) assay using L-[3H]arginine to L-[3H]citrulline conversion. Effects of BH4 on NOS activity in MMC lysates treated with LG and HG are shown. A: control. B: LPS + TNF. C: HG. D: HG +L +T. E: HG (Ly) +L+T. F: LG (Ly) L+TG. HG (IC)+L+T. HG(Ly) denotes lysates treated with BH4 whereas HG (IC) indicates intact cells incubated with BH4. *P < 0.01 compared with control, n = 6 in all groups.

Examination of BH4 synthetic pathway. To determine the mechanism of the decreased availability of BH4 resulting from HG, we examined the critical steps in the BH4 synthetic pathway (Fig. 11). Incubation of HG-treated MMC with dibutyryl cAMP (100 µM) that stimulates GTP-cyclohydrolase I (GTP-CH I) activity, did not increase iNOS activity as measured by NOx in MMC (9.81 ± 2.27 vs. 8.92 ± 2.16 µM, n = 12), suggesting that HG-mediated inhibition of iNOS activity was not due to suppression of GTP-CH activity (Fig. 12). If iNOS inhibition in HG-treated cells was mediated through inhibition of the de novo BH4 synthetic pathway, sepiapterin treatment would restore iNOS activity by a salvage pathway of BH4 synthesis. The NOx measurement in LPS- and TNF-stimulated MMC treated with HG and sepiapterin (10 µM) were not different from stimulated cells treated with HG alone (12.1 ± 2.16 vs. 8.92 ± 2.17 µM, n = 12, P = NS). However, a 10-fold higher concentration of sepiapterin (100 µM) restored the iNOS activity in these cells (49.3 ± 4.32 vs. 8.92 ± 2.17 µM, n = 12, P < 0.01), suggesting that iNOS inhibition in HG-treated MMC was dependent on the cellular concentration of BH4.


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Fig. 11.   BH4 biosynthesis. In addition to de novo synthesis from GTP, BH4 can be regenerated from preexisting dihydropterins by a salvage pathway. The interrupted arrows are inhibitory in action. BH4 is oxidized to BH2 during NOS activation that is recycled to BH4. Ascorbic acid is believed to interfere with BH4 oxidation and thereby maintain it in a reduced state.



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Fig. 12.   NOx accumulation in MMC: mechanism of BH4 effects. A: control. B: LPS + TNF C: HG. D: HG +L +T. Groups E-H all contain HG +L+T. In addition, they also have the following. E: BH4 (10 µM). F: sepiapterin (10 µM). G: dibutyryl cAMP (100 µM). H: sepiapterin (100 µM), n = 12 in all groups, *P < 0.01 compared with control.

Effects of ascorbic acid on HG-mediated iNOS inhibition. Alternatively, the decreased BH4 availability could result from reduced stability or increased degradation. Recent studies have shown that abnormalities in NO-mediated vascular reactivity in diabetic subjects are improved with ascorbic acid (35) and furthermore that ascorbic acid enhances endothelial NOS activity by increasing intracellular BH4 levels (12). In view of these recent findings, we examined the effect of ascorbic acid on HG-mediated iNOS inhibition (Fig. 13). Ascorbic acid (100 µM) significantly increased NOx accumulation in stimulated MMC treated with HG and was comparable to stimulated MMC treated with standard glucose (46.81 ± 7.9 vs. 48.6 ± 5.67 µM, n = 12, P = NS). Furthermore, to establish that the iNOS-potentiating effect of ascorbic acid was mediated by BH4, the effects of inhibition of the BH4 synthetic pathway on ascorbic acid effects were studied. This was accomplished by using NAS, which inhibits sepiapterin reductase and DAHP, which inhibits GTP-CH I (Fig. 13). The effect of ascorbic acid on iNOS activity in HG-treated MMC was examined in the presence of NAS and DAHP. Ascorbic acid (100 µM) failed to increase the NOx accumulation in LPS- and TNF-stimulated MMC treated with HG and incubated in 5 mM DAHP and 5 mM NAS (13.84 ± 2.16 vs. 7.12 ± 1.98 µM, n = 12, P = NS). This indicates that the iNOS activation seen with ascorbic acid is mediated by increased BH4 activity, by either increasing the stability or inhibiting the oxidation of BH4 to BH2, an NOS inhibitor. Further, ascorbic acid coadministration restored iNOS activity in stimulated MMC treated with HG and 10 µM sepiapterin (43.7 ± 6.91 vs. 7.12 ± 1.98 µM, n = 12, P < 0.01), supporting the hypothesis that ascorbic acid may enhance BH4 levels by decreased degradation rather than increased synthesis.


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Fig. 13.   NOx accumulation in MMC-effects of ascorbic acid. A: control. B: LPS + TNF. C: HG. D: HG +L +T. Groups E-G all contain HG +L+T. In addition they also have the following. E: ascorbic acid (100 µM), F: ascorbic acid (100 µM), N-acetyl seratonin (5 mM), and 2,4-diamino-6-hydroxy-pyramidine (DAHP). G: sepiapterin (10 µM) and ascorbic acid (100 µM), n = 12 in all groups * P < 0.01 compared with control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the Western Hemisphere, diabetic nephropathy has become the leading cause of end-stage renal disease in the recent past. Hypertension (both systemic and glomerular), uncontrolled hyperglycemia, and activation of the renin-angiotensin axis all play a major role in the development and progression of diabetic nephropathy (17, 34). Alterations of intrarenal NO synthesis potentially modulate these three pathogenic factors and therefore might impact progression of diabetic nephropathy (5, 6, 7, 18, 23). Progressive loss of renal function is associated with incremental downregulation of NO synthesis in the kidney (14, 41, 43, 44). Urinary excretion of nitrates is diminished in patients with advanced diabetic nephropathy (7, 31). Indeed, recent data suggest that inhibition of NO synthesis by NG-monomethyl-L-arginine (7) accelerates, and supplementation of L-arginine retards, the progression of glomerular damage, proteinuria, and renal failure in diabetic rats (19, 29). These findings emphasize the importance of NO abnormalities in the progression of diabetic nephropathy.

Hyperglycemia mediates the development and progression of diabetic nephropathy by several mechanisms such as hyperfiltration, development of advanced glycosylation end products, and activation of protein kinase C, etc. (9-10, 20, 32, 34, 43). Ambient media glucose has been shown to modulate NO synthesis by renal mesangial cells in culture (30, 36, 39) but published data have been inconsistent. Sharma et.al. (30) showed that exposure of mouse mesangial cells to HG (25 mM) stimulated iNOS gene expression, iNOS protein expression, and enhanced NO synthesis. On the other hand, Trachtman et al. (36) more recently showed that rat mesangial cells, when exposed to HG (33.5 mM), decreased NO synthesis as reflected by a decrease in nitrite levels in the media. Nevertheless, iNOS protein expression by Western analysis was similar in cells incubated in LG and HG. Our results (26) are at variance with those of Sharma et al. (30) but support the findings of Trachtman et al. (36). Although the latter have argued that the variance of their data from Sharma et. al. (30) was due to differences in species and ambient glucose levels, it seems more reasonable that differences may be due to different experimental techniques. Although the other two studies employed the Griess assay for NO measurement that measures only nitrite in the media, our assay measured both nitrites and nitrates in the media, making it more accurate and 100-fold more sensitive.

Our studies showed that NO synthesis progressively diminished on exposure to HG, so that at 7 days of incubation there was a total inhibition of NO synthesis. Although these results are in concurrence with those of Trachtman et al. (36), the latter included data at 72 h only. More recently, high media glucose has been shown to directly or indirectly modulate NO synthesis in microvascular endothelial cells (1, 41). Several studies have indicated that transforming growth factor-beta (TGF-beta ) mediates the effects of HG, including mesangial expansion (2, 28), as these effects could be replicated by the addition of TGF-beta to LG (46). TGF-beta is also a potent inhibitor of iNOS (15). Thus it is possible that TGF-beta activation may be involved in HG-induced inhibition of iNOS in MMC.

Hyperglycemia decreases the cellular content of L-arginine (8, 36). This may be due to interference with transport of L-arginine into the cells by mechanisms hitherto undefined. It has been suggested that increased glucose uptake by mesangial cell transporters glutathione-S-transferase fusion protein-1 and -2 interferes with the cellular uptake of L-arginine (8). More recently, high ambient glucose has been shown to decrease cellular uptake of glucose (11). These data suggest a generalized interruption of cellular transporters on exposure to HG. On the basis of these findings, we examined the effects of supplementation of the media with L-arginine on NO synthesis in MMC exposed to HG. Our results indicated that despite a 20-fold increase in substrate concentration in the medium, the cellular NO generation increased partially but was not sufficient to overcome the inhibition by HG (Fig. 5). These data are in agreement with the findings of Trachtman et al. (36), who demonstrated that L-arginine supplementation increased cellular arginine content and nitrite levels but did not reverse the inhibition of NO synthesis by HG. It is likely that the enhanced cellular levels of L-arginine may increase the maximum velocity of the enzyme iNOS initially, but they may not be sufficient to overcome the other mechanisms by which HG inhibits iNOS.

HG has previously been shown to result in mesangial matrix expansion (27, 40). The available evidence suggests that matrix protein production may be mediated by alterations of NO (39). However, published data are inconsistent with respect to the effects of ambient glucose on mesangial cell proliferation (8, 10, 21, 22, 45). Our results (Fig. 6) clearly demonstrate that HG enhances cell proliferation rate in MMC. Because the total NOx accumulation in MMC exposed to HG is significantly diminished, the NO synthesis per viable cell exposed to HG is decreased by an even greater magnitude.

The present studies show that HG in the media did not interfere with expression of the iNOS gene or protein as reflected by RT-PCR and Western blot analysis (Figs. 7 and 8). These data are in agreement with findings of both Sharma et al. (30) and Trachtman et al. (36). However, although the former reported enhanced NO production, the latter found decreased NO synthesis. The results in the present studies are more comparable to the observations of Trachtman et al., implying that HG inhibited iNOS by mechanisms operating at a posttranslational level (24).

Several factors modify NOS at a posttranslational level, functioning as cofactors, and these include NADPH, oxygen, flavin adenine dinucleotide, and BH4. BH4 is pyrazino-pyramidine formed enzymatically from GTP. The requirement for BH4 for maximal catalytic activity of all isoforms of NOS has been well established (16, 33). BH4 is believed to participate in NOS reaction by activation of dioxygen or posttranslational allosteric modulation of NOS. Recent studies (13) have shown that systemic administration of BH4 in animal models of ischemic renal failure before clamping of renal arteries ameliorated renal failure and injury by restoration of endothelial NO synthesis. In view of the significance of BH4 in the posttranslational activation of the enzyme NOS, we examined the effects of supplementation of BH4 on NO synthesis in MMC exposed to HG. Our results indicated that BH4 restored the ability of MMC cultured in HG to synthesize maximal NO as in cells grown in standard or LG (Fig. 9). These data suggest that high ambient concentrations of glucose in the media inhibited the availability or binding of BH4 with NOS at a posttranslational level.

The in vitro iNOS assay examining L-[3H]arginine conversion to L-[3H]citrulline indicated that BH4 availability rather than binding would explain the iNOS inhibition in MMC treated with HG (Fig. 10). The increase in iNOS activity seen with BH4 enrichment of MMC lysates is comparable to similar manipulation in intact cells, confirming that decreased availability and not decreased binding accounted for iNOS inhibition in HG-treated MMC.

The experimental data presented suggest that decreased availability is unlikely due to interference with BH4 synthesis (Fig. 12). Neither the augmentation of GTP-CH activity with dibutyryl cAMP nor sepiapterin in conventional doses restored iNOS activity in MMC treated with HG. However, the observation that maximal supplementation of sepiapterin increased NO production indicates that higher BH4 concentrations may be necessary for effective iNOS activation in MMC treated with HG than those treated with standard glucose. Alternatively, it is likely that HG treatment may increase the BH4 oxidation to BH2, resulting in a lowered BH4/BH2 ratio. It is to be noted that maximal NOS activity depends not only on a critical BH4 concentration but also on an optimal BH4/BH2 ratio because BH2 can depress NOS activity. Thus the impaired NOS activity resulting from HG may be a consequence of disturbance of BH4 stability.

Recent literature suggests that altered NO-mediated endothelial vascular reactivity seen in diabetic subjects can be corrected by ascorbic acid (35). Furthermore, ascorbic acid has been shown to increase intracellular BH4 levels (12) by mechanisms that are unclear at present. A proposed mechanism involves chemical stabilization by maintenance of BH4 in a continuously reduced state. Based on these recent observations, we examined the effects of ascorbic acid on HG-mediated iNOS inhibition in MMC. The data presented (Fig. 13) demonstrate that when BH4 synthesis is blocked by DAHP and NAS, ascorbic acid failed to activate iNOS activity, clearly suggesting that iNOS potentiation by ascorbic acid is mediated by a BH4-dependent mechanism. Thus although the exact nature of the defect in BH4 metabolism in HG-treated MMC is unclear, it is corrected by ascorbic acid.

In summary, the findings in the present study clearly demonstrated that a high ambient glucose level in the medium downregulated NO synthesis in MMC in culture. Furthermore, we have shown that the mechanism of such inhibition may involve a posttranslational defect of iNOS that pertains to the availability of BH4, a cofactor for iNOS. Evaluations of BH4 biosynthetic pathways indicate that the defect may possibly be related to stability rather than synthesis of BH4 and can be corrected by ascorbic acid. Further studies are needed to evaluate the specific defects in BH4 metabolic pathways in diabetes. These findings may be very significant in understanding the altered regulation of NO synthesis in diabetic nephropathy and its relevance to disease progression.


    ACKNOWLEDGEMENTS

We thank Dr. Peter Syapin, Department of Pharmacology, Texas Tech University Health Sciences Center, for providing technical help with Western analysis experiments. Our special thanks to Dr. Donald E. Wesson for valuable suggestions during the study and for carefully reading the manuscript. We also acknowledge the technical help rendered by Melody Wainscott, Mercedes Sayago, Alexia Rendon, and Geraldine Tasby in conducting these studies. Finally, we thank Dr. Thomas Kahn, Bronx Veterans Affairs Medical Center, for the support rendered during the initial phase of this project.


    FOOTNOTES

Parts of this work were presented at the annual meetings of the Southern Society of Clinical Investigation in New Orleans, LA, February 2000, and the American Society of Nephrology meetings in Toronto, ONT, October 2000.

These studies are partly funded by intramural grants from Texas Tech University Health Sciences Center and a departmental seed grant.

Address for reprint requests and other correspondence: S. S. Prabhakar, Div. of Nephrology, Dept. of Internal Medicine, Texas Tech Univ. Health Sciences Ctr., Lubbock, TX 79430 (E-mail: medssp2{at}ttuhsc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3 May 2000; accepted in final form 6 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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