Uncoupling Endothelial Nitric Oxide Synthase Is Ameliorated by Green Tea in Experimental Diabetes by Re-establishing Tetrahydrobiopterin Levels

All reagents were purchased from Sigma (St. Louis, MO), unless otherwise stated.

This study protocol was approved by the local committee for ethics in animal research (CEEA/IB/Unicamp). The SHR rats used in the study were provided by Taconic (Germantown, NY). Experimental diabetes was induced in 12-week-old SHR rats via an intravenous injection of streptozotocin (50 mg/kg in sodium citrate buffer, pH 4.5). The day after diabetes induction, the diabetic rats were randomly assigned to receive or not receive GT (∼5 g/kg body weight/day) instead of drinking water. Japanese GT (Midori Industria de Chá) was prepared daily, as we have described before (5). During the study, the diabetic rats received 2 units of insulin (human insulin HI-0310; Lilly), three times per week, subcutaneously. The control rats only received the vehicle. We chose to induce diabetes in SHR rats because they present a more progressive form of renal disease (19), we have a vast experience with this model (2,5,20), and also because of the frequent association of diabetes with hypertension in human diabetic renal disease. After 12 weeks of diabetes induction, the rats were killed, the kidneys were decapsulated and removed, and a piece of the cortex was used for protein isolation. The remaining kidney cortex was snap-frozen at −80°C for future assays.

The kidney was embedded in paraffin, and 3-μm sections were cut and stained with periodic acid–Schiff. Matrix mesangial expansion, quantified by Leica Application Suite (LAS Image Analysis), was derived from assessment of 30 glomeruli from each rat.

Immortalized human mesangial cells (ihMCs; passage 10 to 20) from Dr. Bernhard Banas (Nephrology Center, Medical Policlinic, Ludwig-Maximilian University of Munich, Germany) were provided by Dr. Nestor Schor (Department of Medicine, Nephrology Division, Federal University of São Paulo, Brazil). The ihMCs were cultured as described previously (21). The cells were kept without serum in normal glucose (NG, 5.5 mmol/L) and HG (30 mmol/L) mediums in the presence of various treatments for an additional 24 h. The concentrations of treatments used in the HG medium in all experiments were chosen after completing a thiazolyl blue tetrazolium bromide assay (data not shown).

The kidney cortex or ihMCs were lysed in a radioimmunoprecipitation assay buffer supplemented with a protease inhibitor cocktail (Complete; Boehringer-Mannheim, Indianapolis, IN). The samples and Western blots were prepared as previously described (5). The Bradford method (22) was used for protein quantification. The following primary antibodies were used: rabbit polyclonal anti-eNOS (Santa Cruz) rabbit polyclonal p-eNOS Thr495, and rabbit polyclonal p-eNOS Ser1177 (Cell Signaling Technology). Equal loading and transfer was achieved by reprobing the membranes for β-actin. To determine eNOS dimer-to-monomer ratio, sample preparation and Western blot was performed as previously described (23).

The analysis of NO end products, such as nitrate and nitrite (NO_X⁻), was evaluated by the Griess reaction, as previously described (24). Briefly, the renal cortex was lysed in 300 μL extraction buffer (50 mmol/L Tris-HCl [pH 7.4], 1 mmol/L EDTA, 10 mmol/L dithiothreitol). The kidney cortex lysate was then deproteinized by 0.6 mol/L trichloroacetic acid for 1 h at 4°C, and the samples were incubated with chloride vanadium in 1:1 proportion for 15 min. Then, 0.1% N-naftil-etilenodiamine and 2% sulfanilamide were added to the samples for 30 min in the dark, at room temperature. The samples were read by a spectrophotometer (Powerwave XS2, Biotek) at 540-nm absorbance.

Oxidized and reduced forms of biopterins were analyzed by the differential oxidation method and were determined according to a previously described method (25), with some modifications (26). The renal cortex and ihMCs were lysed in a 500 μL extraction buffer (50 mmol/L Tris-HCl [pH 7.4], 1 mmol/L EDTA, 10 mmol/L dithiothreitol). The samples were injected into an ultraperformance liquid chromatography system (Waters). BH₄ concentration, expressed as picomoles or nanomoles per milligram of protein, was calculated by subtracting BH₂ plus biopterin from total biopterins. The percentage of BH₄ oxidation was calculated using the following formula: Percentage of BH₄ oxidation = 100 − (BH₄ levels × 100/biopterin levels)

GTPCH activity was measured using the high performance liquid chromatography (HPLC) method, as described previously (27).

Intracellular ROS levels were measured by H₂DCF-DA. Qualitative assessment of ROS was carried out in the ihMCs after they were kept for 24 h with the NG and HG mediums alone or in the presence of GT (100 µg/mL) and different treatments. To quantify the ROS levels, the same procedure used for the qualitative analysis of ROS was applied. Relative fluorescence was measured using a fluorescence plate reader (SynergyMx; Biotek) at excitation and emission wavelengths of 485 and 528 nm, respectively. The relative fluorescence values were corrected by the number of cells in each treatment.

Intracellular NO was measured in ihMCs via diaminorhodamine-4M AM (Alexis Biochemicals, Switzerland). To quantify NO levels, the same procedure used for the qualitative analysis of NO was applied, but using the probe DAF-2DA. Relative fluorescence was measured using a fluorescence plate reader (SynergyMx; Biotek) at excitation and emission wavelengths of 495 and 515 nm, respectively. The relative fluorescence values were corrected by the number of cells in each treatment.

The estimation of EGCG in GT was assessed by HPLC (Waters) using an EGCG standard, as previously reported (28).

All experiments were independently performed three times and the results are expressed as means ± SD. One-way ANOVA, followed by the Bonferroni test, were used to compare the groups. A value of P < 0.05 was considered significant. All analyses were performed using StatView software (SAS Institute, Inc., Cary, NC).

Body weight gain was lower in the diabetic rats than in the control rats. Systolic blood pressure was similar in all groups. Blood glucose concentration was greater in the diabetic rats than in the control rats but was not affected by GT (Table 1).

Matrix mesangial expansion was greater in diabetic SHR rats than in control rats. This abnormality was reversed by GT treatment (P = 0.03; Supplementary Fig. 1A and B).

Kidney homogenates from the diabetic rats had significantly lower levels of NO_X⁻ compared with the control rats (P = 0.02), which was reversed by GT treatment (P = 0.05; Fig. 1A).

Modulation of NO bioavailability was also assessed via analysis of eNOS expression and its phosphorylation status. Renal cortical expression of eNOS did not differ between the studied rats (Fig. 1Band C). Diabetic SHR rats demonstrated increased phospho-Thr495 eNOS expression (P = 0.002), which was reversed by GT treatment (P = 0.005; Fig. 1B and D). Thr495 represents the major negative regulatory site of eNOS and is constitutively phosphorylated in cultures in many endothelial cell types (29). However, the expression of phospho-Ser1177 eNOS in renal cortical homogenates increased in the diabetic SHR rats (P = 0.03), and GT consumption reduced its expression (P = 0.05; Fig. 1B and E). Ser1177 thus appears to be the most important positive regulatory domain in eNOS. Finally, the percentage of the dimer-to-monomer ratio, an indicator of eNOS uncoupling, showed a significant decrease in diabetic rats (P = 0.02), which was re-established by GT treatment (P = 0.05; Fig. 1F and G).

BH₄ and total biopterin levels in renal cortical homogenates (P < 0.002) and urine (P < 0.0001) decreased in the diabetic SHR rats compared with the control SHR rats (Fig. 2A and B). GT significantly abrogated the reduction of BH₄ in the renal cortex (P = 0.0008), and the urinary levels of BH₄ with GT tended to increase in the diabetic SHR rats (Fig. 2Aand B). Similar results were obtained for total biopterin levels (Fig. 2A and B). Moreover, the percentage of oxidation of BH₄ to BH₂ was increased in the diabetic rats compared with the control rats in the renal cortex (P = 0.03) and urine (P = 0.05), whereas GT treatment abolished the oxidation of BH₄ (Fig. 2Cand D).

Qualitative and quantitative analyses of NO production showed that the ihMCs kept in the HG medium for 24 h had a significant reduction (P = 0.03) in fluorescence intensity compared with cells kept in the NG medium, whereas GT (100 μg/mL) reversed the HG-induced reduction in NO (P = 0.02; Fig. 3Aand B). Western blot analysis revealed that the eNOS expression was similar in ihMCs cultured in NG, HG, or HG with GT (Fig. 3C and D). However, the eNOS dimer-to-monomer ratio was decreased in ihMCs cultured under HG (P = 0.04), and this alteration was reversed by GT treatment (P = 0.05; Fig. 3Eand F).

Qualitative and quantitative analyses (Fig. 4A and B) showed a significant rise in ROS production after the ihMCs were exposed to HG levels for 24 h (P = 0.0001) compared with NG levels. To evaluate the sources involved in the HG-induced ROS production in the ihMCs, we analyzed ROS production at HG in the presence of diphenyleneiodonium (DPI; 50 nmol/L, a blocker of NADPH-oxidase), l-N^G-nitro-l-arginine methyl ester (L-NAME; 100 μmol/L, an inhibitor of NOS enzymes), and rotenone (10 μmol/L, an inhibitor of mitochondria electron transport complex I). Fluorescent microscope (Fig. 4A) and fluorometer (Fig. 4B) data showed that DPI reversed HG-induced ROS production to levels lower than those of NG (P = 0.03), suggesting that NADPH oxidase is one main source of superoxide in ihMCs. DPI reduces ROS production even in cells cultured in NG. This observation may explain the reduction of ROS in cells under HG to below control levels (Fig. 4B). Furthermore, incubation of ihMC with L-NAME significantly reduced HG-induced superoxide production (P = 0.04), suggesting that eNOS uncoupling is an important source of ROS production. Finally, incubation with rotenone reduced HG-induced superoxide production, although it failed to reach statistical significance (P = 0.07). Mannitol (30 mmol/L), which was used as an osmotic control, did not alter ROS production. These results imply that NADPH oxidase and uncoupling eNOS are both important sources in the HG-induced ROS production in ihMCs.

We observed a significant reduction in total biopterin and BH₄ levels in ihMCs kept in the HG medium (P = 0.05) compared with cells in the NG medium (Fig. 5A), which was reversed by GT (P = 0.04). Furthermore, there was an increase in oxidation of BH₄ to BH₂ in cells kept in the HG medium (P = 0.01), and this also was reversed after GT treatment (P = 0.01; Fig. 5B). We next assessed the role of BH₄ in ROS and NO production in ihMCs. Exogenous administration of BH₄ in ihMCs kept in the HG medium (Fig. 5C) reduced the HG-induced rise in ROS production in a concentration-dependent manner, although this only reached significance at 100 μmol/L (P = 0.004). Moreover, measurement of NO via DAF-2DA (Fig. 5D) showed that BH₄ at all concentrations used (P = 0.001) reversed the HG-induced decline in NO production (P = 0.007). These findings may imply that a reduction in BH₄ levels may be involved in HG-induced ROS production and a decline in NO formation.

To investigate the mode of action of HG-induced reduction in BH₄ levels, we next used HPLC to assess the activity of GTPCH I. Our results showed that there was a significant reduction in GTPCH I activity (P = 0.03) in ihMCs kept in the HG medium (Fig. 6A) compared with cells kept in the NG medium. Interestingly, GT reversed the HG-induced decline in GTPCH I activity (P = 0.009; Fig. 6A). To further assess the hypothesis that GT re-established GTPCH I activity in ihMCs, we cultured these cells in HG with GT (100 µg/mL), and after 1 h, we supplemented the medium with 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTPCH I. We observed that the presence of the GTPCH I inhibitor abrogated the beneficial effect of GT (i.e., it increases ROS production [Fig. 6E] and decreases NO bioavailability [Fig. 6F]), in a concentration-dependent manner. These observations further support the concept that GT improved NOS uncoupling by re-establishing the GTPCH I activity and synthesis of BH₄.

To test the integrity of BH₂-to-BH₄ recycling in ihMC under HG, the cells were kept with HG in the presence of BH₂, and ROS production was evaluated. Analysis (Fig. 6B and C) of ROS production showed that BH₂ reduced the HG-induced ROS production in a concentration-dependent manner, although this was only significant at 10 μmol/L and 100 μmol/L (P = 0.0001). Analysis of NO production showed that the exogenous administration of BH₂ significantly blunted the HG-induced decline of NO production (P = 0.03; Fig. 6D). These data may further suggest that HG in ihMCs reduces the production of BH₄ via a decline in GTPCH I activity at the de novo synthesis pathway of BH₄ formation. However, HG does not affect BH₄ production via the recycling pathway because BH₂ ameliorated both the HG-induced rise in ROS production and the decline in NO formation.

In ihMCs, assessment of ROS production showed that GT reversed the HG-induced rise in ROS formation to NG levels (Fig. 7Aand B; P = 0.0001). Cotreatment of ihMCs with GT and BH₄ (Fig. 7C) did not confer an additional reduction in ROS production compared with GT treatment alone. This suggests that GT may inhibit HG-induced ROS production through a rise in BH₄ levels.

When compared with the EGCG standard, we identified this polyphenol as an important constituent of GT. The EGCG retention time was 8.891 min (Supplementary Fig. 2A), consistent with the main peak retention time of 8.894 min observed in GT (Supplementary Fig. 2B). Assessment of the effect of EGCG in ihMCs exposed to HG has shown that this component of GT can reduce ROS production (Supplementary Fig. 2C) and increase NO bioavailability (Supplementary Fig. 2D). These observations suggest that the main effect of GT may be attributed, at least partly, to EGCG.

The current study aimed to explore the potential of GT to ameliorate kidney-uncoupling eNOS in diabetic conditions. We also investigated the mechanisms by which GT reversed uncoupling eNOS. We observed, both in vivo and in vitro, an uncoupling of eNOS secondary to a reduction of BH₄ and elevation of its oxidized form with a consequent decrease in NO and an increase in oxidative stress. GT re-established the levels of BH₄, reduced its oxidized form, coupled eNOS, and consequently, increased NO bioavailability and reduced oxidative stress. Our in vitro data also suggest that the re-establishment of GTPCH I activity is the main mechanism by which GT increased BH₄ and coupled eNOS. These observations are of great interest. First, they reinforce the importance of uncoupling eNOS and BH₄ synthesis/oxidation in reducing NO bioavailability, and increase oxidative stress, two conditions involved in the pathogenesis of DN (1–8). In addition, the mechanism by which GT can couple eNOS in diabetes is described for the first time. Together with previous observations that GT can reduce oxidative stress and improve indices of DN in rats (5), data from this translational study demonstrate the possible beneficial use of GT or its main flavonol, EGCG, in patients with DN.

Bioavailability of NO in the diabetic kidney has been a subject of major controversy (7,8). Earlier studies have suggested that NO production increases and contributes to glomerular hyperfiltration in short-term diabetes (30,31). More recently, it has been suggested that endothelial dysfunction, which is often defined as a decrease in the bioavailability of endothelial-derived NO, is a preponderant factor in diabetes and contributes to the pathogenesis of DN (6–8,17). To this end, researchers have demonstrated that the knockout of eNOS in diabetic mice leads to severe histologic lesions in the kidney that resemble the lesions seen in human DN but which are not seen in control wild-type diabetic mice (32). In addition, hypertensive type 2 individuals subjected to an acute reduction in NO bioavailability, by means of blockade of NO synthesis with L-NAME, displayed an elevated albumin excretion rate, a hallmark of DN (33). Therefore, it seems that a reduction in NO bioavailability is the predominant abnormality in DM.

It has become apparent that eNOS phosphorylation at Thr495 or Ser1177, rather than eNOS expression, are crucial parameters in estimating NOS production by eNOS (7,8). Interestingly, in our diabetic rats, we observed an increase in inactivation of eNOS via a rise in phosphorylated eNOS at Thr495, which is known to lead to a reduction in electron transfer in eNOS, thus diminishing NO production (34). Surprisingly, we also observed in the same model a rise in the expression of phosphorylated eNOS at Ser1177, which is known to enhance eNOS activity (8). These observations are in agreement with a recent study showing that phosphorylation at Thr495 results in a less active eNOS, even in the context of phosphorylation of eNOS at Ser1177 (35). In our study, GT reversed diabetes-induced alterations in phosphorylated Th495, Ser1177, eNOS, and the dimer-to-monomer ratio, and hence, improved NO bioavailability.

Previous studies have shown that enzymatic coupling of eNOS by BH₄ plays a critical role in the maintenance of NO bioavailability (6,12,16,36,37); for example, endothelial function improved after the exogenous administration of BH₄ in stroke-prone SHR rats via an improvement of eNOS coupling, and hence, a rise in NO bioavailability (37). In our studies, we observed a reduction in BH₄ levels and a rise in the oxidation rate of BH₄ to BH₂ in the renal cortex and urine of diabetic SHR rats. A recent study suggested that BH₄ oxidation, rather than decreased BH₄, is the main determinant of uncoupling eNOS (11,38). In our study, GT treatment restored the levels of BH₄ and the diabetes-induced oxidation of BH₄ to BH₂ in the renal cortex and urine of diabetic SHR rats. Therefore, GT seems to reverse diabetes-induced eNOS uncoupling, thereby increasing NO bioavailability via a rise in BH₄ availability. This is further supported by our observation that cotreatment of GT and BH₄ did not confer additional protection against HG-induced ROS production compared with GT treatment alone. The ihMCs exposed to HG displayed a rise in ROS production and a decline in intracellular NO production.

Two sources appeared to mediate HG-induced ROS production—NADPH oxidase and uncoupled eNOS—because blocking each one abolished the HG-induced rise in ROS production. Our studies are in agreement with previous work showing that uncoupling eNOS and NADPH oxidase in the glomeruli of rats with experimental DN are the major sources of superoxide mediated by the loss of BH₄ availability (6). In agreement with the importance of reduction in BH₄ availability, exogenous administration of BH₄ in ihMCs abolished ROS production and reversed the decline of NO under HG levels. This finding suggests that ihMCs kept in HG mediums exhibit low levels of BH₄, leading to uncoupling of eNOS, a subsequent rise in ROS, and a decline in NO levels. This is further supported by our finding that BH₄ levels decreased and the oxidation of BH₄ rose to BH₂ in ihMCs kept in the HG medium.

Our present work further showed that ihMCs kept in the HG medium exhibited reduced levels of BH₄ compared with cells kept in the NG medium, due to the diminished de novo synthesis pathway of BH₄ formation, because activity of GTPCH I was reduced in cells cultured in HG. Diminished GTPCH I activity has also been reported in rats with experimental DN, leading to reduced BH₄ formation via the de novo synthesis (39). In addition, the observation that a GTCH I blocker abrogated the effect of GT in ROS and NO production in ihMCs exposed to HG and treated with GT further supports the concept that GT acts by improving GTPCH I activity. Our work also indicated that the recycling pathway of BH₄ is probably preserved in ihMCs exposed to HG because BH₂ supplementation decreased ROS and increased NO bioavailability by enhancing BH₄ levels via DHFR. These findings are in agreement with previous in vivo (11) and in vitro studies (11,38) showing that DHFR plays a key role in regulating the BH₄-to-BH₂ ratio and eNOS coupling under conditions of low total BH₄ availability. For example, in endothelial cells (38,40) expressing eNOS with low BH₄ levels, DHFR inhibition or knockdown further diminished the BH₄-to-BH₂ ratio and exacerbated eNOS uncoupling.

Reduced BH₄ availability in ihMCs could also be attributed to reduced BH₄ stability. Diminished BH₄ stability has been reported in endothelial dysfunction (41) as well as in rats with DN (6). HG levels, as seen in diabetic conditions, increase the formation of superoxide through one main source in the kidney, NADPH oxidase activation. NO produced by eNOS and superoxide combine to form peroxynitrite anions. Oxidation of BH₄ by ROS, such as peroxynitrite, results in BH₂ formation, which inactivates the eNOS cofactor function, suggesting that reduced BH₄ stability uncouples eNOS and leads to reduced NO bioavailability and a further rise in the formation of diabetic glomeruli superoxides.

In conclusion, the current work indicates that GT reverses diabetes-induced uncoupling eNOS as experienced in renal mesangial cells exposed to HG levels and SHR diabetic rats. GT seems to ameliorate uncoupling eNOS via a rise in BH₄ levels/reduction in BH₄ oxidation, which occurs as a result of the de novo synthesis of BH₄. A rise in BH₄ levels would account, then, for reduced eNOS uncoupling leading to the amelioration of oxidative stress and enhanced NO availability.

This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (Grant 2008/57560-0) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). A.M.F. received a scholarship from CNPq.

No potential conflicts of interest relevant to this article were reported.

A.M.F., A.P., and K.C.S. acquired the data and wrote the manuscript. J.M.L.F. contributed to discussion and reviewed the manuscript. J.B.L.F. designed the study, reviewed the data, and wrote, reviewed, and edited the manuscript. J.B.L.F. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

The authors thank Luciana Cristina Teixeira (Department of Medicine, Nephrology Division, Federal University of São Paulo, Brazil) for helping with the culture of the ihMC line and are grateful to Dr. Ricardo Pereira and Dr. Marcelo Ganzarolli de Oliveira (Laboratory of Liquid Chromatography, Institute of Chemistry, University of Campinas [Unicamp], Campinas, SP, Brazil) for their technical and scientific assistance. The authors are very grateful to the personnel from the Renal Pathophysiology Laboratory, Investigation on Diabetes Complications, Faculty of Medical Sciences (FCM), Unicamp, for their invaluable help with this work. In particular, the authors are very grateful to Dr. Elisa E.M. Peixoto (Renal Pathophysiology Laboratory, Unicamp), for blindly estimating the extracellular matrix expansion in kidney slides.