Puerarin attenuates diabetic kidney injury through the suppression of NOX4 expression in podocytes

Radix puerariae, a traditional Chinese herbal medication, has been used to treat patients with diabetic nephropathy (DN). Several studies demonstrated that puerarin, the active compound of radix puerariae, reduces diabetic injury in streptozotocin (STZ)-induced diabetic rodent models. However, as STZ injection alone results in mild kidney injury, the therapeutic benefit afforded by puerarin in DN remained inconclusive. Thus we sought to clarify the role of puerarin by employing an accelerated DN model, STZ-induced diabetes in the endothelial nitric oxide synthase-null (eNOS−/−) mice. Puerarin treatment of diabetic eNOS−/− mice significantly attenuated albuminuria and diabetic kidney injury, which were associated with reduced oxidative stress and reduced NAPDH oxidase 4 (NOX4) in glomeruli of diabetic eNOS−/− mice. Puerarin treatment of murine podocytes culture in high glucose conditions led to reduced superoxide production and NOX4 expression. We further determined that that puerarin treatment increased both mRNA and protein levels of SIRT1 in podocytes and that puerarin led to SIRT1-mediated deacetylation of NF-κB and suppression of NOX4 expression. Our findings confirm the renoprotective effects of puerarin in an experimental model of advanced DN and provide a molecular mechanism by which puerarin exerts the anti-oxidative effects in podocytes in the diabetic milieu.

Puerarin reduces oxidative stress in diabetic mice. We next determined the effects of puerarin on the extent of oxidative stress in the diabetic glomeruli. Immunohistochemical staining showed a strong upregulation of nitrotyrosine expression in the glomeruli of vehicle-treated diabetic mice, which was not observed in nondiabetic control kidneys ( Fig. 2A). Puerarin treatment led to a marked decrease of nitrotyrosine staining in the diabetic mouse kidneys ( Fig. 2A). The attenuation of oxidative stress by puerarin was further confirmed by immunofluorescence staining of 8-oxoguanine (8-oxoG), a common DNA lesion resulting from oxidative stress, with co-staining of WT-1, a podocyte marker (Fig. 2B). A large extent of 8-oxoG-positive cells was also WT-1-positive, confirming an increase of oxidative stress in podocytes in diabetic milieu. We did note however that 8-oxoG-positive cells were not limited to podocytes, but were present in other glomerular cells in the diabetic eNOS −/− kidneys. Puerarin treatment significantly decreased the number of 8-oxoG-positive cells, suggesting that puerarin improves DN through the inhibition of oxidative stress. Figure 2C shows the quantification of the 8-oxoG and WT-1 staining in the nondiabetic and diabetic kidneys.
Because NOX4 is a main contributor of reactive oxygen species in the diabetic kidneys 15,16 , we next determined the mRNA and protein levels of NOX4 in the glomeruli of diabetic and nondiabetic mice treated with puerarin or vehicle. We found that NOX4 expression was indeed increased in vehicle-treated diabetic mice, but suppressed in puerarin-treated diabetic mice ( Fig. 3A-C), indicating that puerarin attenuates oxidative stress through the inhibition of NOX4 expression.
Puerarin reduces oxidative stress in conditionally immortalized murine podocytes. In order to confirm the above in vivo findings, we tested in vitro whether puerarin can directly reduce the superoxide production in podocytes cultured under the high glucose condition. Reactive oxygen species were detected using the cell  Table 2. Kidney-to-body weight ratio of mice at 18 weeks post-STZ/vehicle injection. *P < 0.05 and ***P < 0.001 when compared to control eNOS −/− mice; ### P < 0.001 when compared to vehicle-treated diabetic eNOS −/− mice (n = 6 in each group).
Puerarin regulates NOX4 expression through SIRT1-NF-κB pathway in podocytes. We previously found that the expression of NAD + -dependent histone deacetylase SIRT1 is reduced in human DN glomeruli 27 . Interestingly, we observed that the high-glucose-mediated suppression of SIRT1 protein expression (A) Both diabetic and non-diabetic mice were treated with either vehicle or puerarin when mice developed albuminuria for a total of 9 weeks. Urine samples were collected weekly after starting the treatments for determination of urinary albumin/creatinine ratio. Significant differences were observed starting at 2 weeks after puerarin treatment compared with vehicle-treated diabetic mice. *p < 0.05 and *p < 0.001 compared to vehicle-treated diabetic mice, n = 6. (B) Representative image of periodic acid-Schiff (PAS) stained kidneys. Scale bar: 20 μm. (C,D) Morphometric analysis was performed to determine the glomerular area (C) and % of mesangial area (D) in the glomerular cross sections. **p < 0.01 and ***p < 0.001, n = 6.
in podocytes was alleviated by puerarin treatment at both low and high doses (1 and 10 μM, respectively) ( Fig. 5A,B). Moreover, SIRT1 expression was inversely correlated with NOX4 expression (Fig. 5A,B), suggesting that SIRT1 negatively regulates NOX4 expression. Our earlier work showed that the activity of NF-κB is increased in podocytes in DN and that SIRT1 inhibits NF-κB activity through deacetylation 28 . We also observed that puerarin treatment led to the decreased NF-κB acetylation, likely through the stimulation of SIRT1 expression (Fig. 5A,B). In support of this, overexpression of SIRT1 in podocytes attenuated both NF-κB acetylation and NOX4 expression (Fig. 5C,D). Promoter analysis of NOX4 confirmed the presence of putative NF-κB binding sites, and others have shown in non-renal cells that NF-κB directly binds to the NOX4 promoter and regulates NOX4 expression 29 . Our previous studies indicated that NF-κB mutant with a lysine to arginine mutation of its acetylation site at Lys310 (p65 K310R ) serves as a dominant negative to block the activity of endogenous NF-κB 28 . We found that the overexpression of p65 K310R mutant (K310R) in podocytes led to the reduced NOX4 expression as compared to overexpression of wildtype p65 (WT) (Fig. 6A,B), strongly suggesting that NF-κB acetylation is required for NOX4 expression and that SIRT1 suppresses NOX4 expression through deacetylation of NF-κB. We further confirmed that the overexpression of SIRT1 exhibited similar inhibition of superoxide production in podocytes as puerarin (Fig. 6C). Lastly, we found that puerarin also increased SIRT1 mRNA levels in podocytes, suggesting that puerarin stimulates SIRT1 expression at the transcriptional level (Fig. 6D).

Discussion
In the current study, we confirmed that puerarin mitigates albuminuria and kidney injury in STZ-induced diabetic mice with eNOS-deficiency, a model which better mimics the advanced human DN. In addition, we showed that the anti-oxidative effect of puerarin is mediated through the suppression of NOX4 expression in diabetic kidney in vivo and in cultured murine podocytes in vitro. We also demonstrated that puerarin suppressed NOX4 expression through the upregulation of SIRT1, resulting in increased deacetylation of NF-κB, thus uncovering a new molecular mechanism by which puerarin exerts renoprotection in kidney cells under diabetic conditions. Lastly, we have shown that puerarin stimulates SIRT1 expression at the transcriptional level.  Puerarin is a major isoflavonoid component from the root of pueraria candollei of Leguminosae family. Puerarin has a structure of 7-hydroxy-3-(4-hydroxyphenyl)-1-benzopyran-4-one-8-β-D-glucopyranoside 30 . Although large randomized clinical trials are lacking, several small clinical studies suggest that puerarin treatment significantly reduced albuminuria in patients with the stage 3 DN 18 . The renoprotective effects of puerarin have been also reported in several animal studies [19][20][21] . However, these studies were reported in STZ-induced diabetic rodent models with very mild kidney injury. In this study, we took advantage of the accelerated DN in absence of eNOS in STZ-induced diabetic eNOS −/− mice 31 , which is a better-suited DN model to mimic the  Anti-oxidative activity of puerarin has been demonstrated in cardiovascular 32 and neurological diseases 33 . Several studies also suggest that puerarin has anti-oxidative effects in kidney cells 19,20,22,23 . However, the mechanism by which puerarin exerts these effects remained unclear. Here, we report that the anti-oxidative effect of puerarin to confer renoprotection is in part mediated through the suppression of NOX4 expression in podocytes. Previous studies have shown that NOX4 expression is increased in diabetic kidneys and that podocyte-specific knockout of NOX4 attenuates DN 15,16 , underscoring the importance of NOX4 regulation in DN. In addition to NOX4 suppression, our results demonstrated that puerarin upregulates SIRT1 expression. The role of SIRT1 in DN has been well documented 28,34,35 . We have previously demonstrated the anti-inflammatory role of SIRT1 via deacetylation of NF-κB in DN 28 . Our data now suggest that puerarin decreases NOX4 expression through the inhibition of NF-κB activity by SIRT1 upregulation. Our promoter analysis further confirmed that NOX4 has binding sites for NF-κB. Consistent with our data, previous studies suggest that NF-κB directly mediates NOX4 expression 36,37 .
In line with these findings, recent studies reported that SIRT1 expression is increased in the diabetic kidney from mice treated with puerarin 20 . However, it remained unclear how puerarin regulates SIRT1 in the kidney cells. Here, we found that puerarin can upregulate both mRNA and protein levels of SIRT1 in podocytes, suggesting a transcriptional regulation of SIRT1. Since SIRT1 regulates multiple biological pathways such as aging, 38 , metabolism 39 , cancer and inflammation 40 , we believe that puerarin and its analogues could have a broader application in its therapeutic usage. Moreover, the renoprotective effects of puerarin may not be limited to anti-oxidative effects. It has been shown that puerarin can attenuate the apoptosis of proximal tubular cells through the restoration of mitochondrial function 25,41 . We have previously shown that puerarin improves DN through regulation of metalloproteinase 9 (MMP9) in podocytes 19 .
In summary, we confirmed the renoprotective effects of puerarin treatment in a mouse model with established DN. We demonstrated that puerarin exerts anti-oxidative effects through the activation of SIRT1-mediated NF-κB deacetylation and that puerarin is a stimulator of SIRT1 expression in podocytes. Our study suggests that puerarin may be a potential drug to treat patients with DN. However, future clinical studies are required to confirm the therapeutic effects of puerarin in patients with DN.

Methods
Animal studies. 8-week old eNOS-null male mice on a C57BL/6 background were purchased from The Jackson Laboratory (Bar Habor, ME). Diabetes was induced by intraperitoneal injection of freshly prepared streptozotocin (STZ) (Sigma-Aldrich, Saint Louis, MO) dissolved in 0.1 M citrate buffer, pH 4.5 at 50 mg/kg after 4-6 hours of food withdrawal for 5 consecutive days. Control mice were injected with sodium citrate buffer. Blood glucose was measured every week. 2 weeks after diabetes was confirmed in mice (blood glucose > 250 mg/ dl), mice were given puerarin (Sigma-Aldrich, Saint Louis MO) dissolved in 5% DMSO by oral gavage at a dose of 20 mg/kg body weight/day, or 5% DMSO vehicle as control, for 8 weeks. Urine samples were collected every week until they were sacrificed. All experimental methods were performed in accordance with the approved guidelines as described in the Guide for the Care and Use of Laboratory Animals 42 . All animal studies were approved by the Institutional Animal Care and Use Committee at the Icahn School of Medicine at Mount Sinai, New York, NY.
Urine albumin and creatinine measurement. Urine albumin was measured using an ELISA kit (Bethyl Laboratory, Houston, TX), and urine creatinine was measured using a colorimetric assay kit (Cayman, Ann Arbor, MI).
Kidney histology. Kidney samples were fixed in 10% formalin at room temperature for 16 hours and embedded in paraffin. Tissues were cut into 4 μm thick sections for periodic acid-Schiff (PAS) staining. Glomerular area and percentage of glomerular area were calculated as described (2).
Immunohistochemistry and immunofluorescence staining. Paraffin-embedded kidney sections were deparaffinized and rehydrated for immunostaining. Specific antibodies used in this study are as following: anti-nitro-tyrosine from Santa Cruz (SC-32757) at 1:100 dilution, anti-8-oxo-G from Japan Institute for the Control of Aging (N45.1) at 1:50 dilution, and anti-WT-1 antibody was from Santa Cruz at 1:50 dilution. The secondary and tertiary antibodies were obtained from Jackson Immunoresearch, Inc. and used at 1:200 dilution. Glomerular RNA isolation. Mice glomeruli were isolated as previously reported (1). Total RNA was extracted from glomeruli using Trizol method. Purified RNA underwent DNA digestion using RNase-free DNase set (79254, Qiagen, Germantown, MD).
Cell culture. Conditionally immortalized murine podocytes were obtained from Dr. Peter Mundel and cultured at 37 °C for 7 days for full differentiation before being used as described 43 .
Reactive oxygen species measurement. Differentiated podocytes cultured with normal glucose (glucose 5.5 mM, supplemented with 24.5 mM mannitol as high osmolarity control) and high glucose (30 mM) were treated with 5% DMSO or puerarin for 24 hours. Cells were then incubated with 20 μm of DCFDA (Abcam, Cambridge, MA) for 30 min at 37 °C. Cells were viewed and photographed with a fluorescence microscopy with excitation and emission spectra of 495 nm and 529 nm respectively. DAPI was used for visualization of nuclear or phase-contrast pictures to visualize cells.
NADPH oxidase activity measurement. The  4 mM, Na 2 HPO 4 0.4 mM, D-Glucose 5.5 mM, pH 7.2) and the culture plate was snap-frozen. Cells were scraped with ice-cold HBSS containing protease inhibitor cocktail. Cell suspensions were homogenized with 100 strkes in a Dounce homozinizer on ice. Protein content was quantified. 30ug of proteins were transferred to each well followed by adding 25uM Lucigenin (Santa Cruz, Lake Forest, CA) and 200 uM NADPH (Cayman Chemical, Ann Arbor, MI). After 10 minutes at 37 C for dark adaptation, the light emission was recorded. The NOX activity was expressed as relative light units per milligram of cellular protein in cells.  Statistical analysis. Data are expressed as mean ± SEM. The unpaired t-test was used to comparison between groups or ANOVA followed by Bonferroni correction was used when comparing between groups for treatment conditions using the GraphPad Prism software. P-value < 0.05 was considered statistically significant.