Title: Increased podocyte Sirtuin-1 function attenuates diabetic kidney injury
Abstract: Podocyte injury and loss contribute to the progression of glomerular diseases, including diabetic kidney disease. We previously found that the glomerular expression of Sirtuin-1 (SIRT1) is reduced in human diabetic glomeruli and that the podocyte-specific loss of SIRT1 aggravated albuminuria and worsened kidney disease progression in diabetic mice. SIRT1 encodes an NAD-dependent deacetylase that modifies the activity of key transcriptional regulators affected in diabetic kidneys, including NF-κB, STAT3, p53, FOXO4, and PGC1-α. However, whether the increased glomerular SIRT1 activity is sufficient to ameliorate the pathogenesis of diabetic kidney disease has not been explored. We addressed this by inducible podocyte-specific SIRT1 overexpression in diabetic OVE26 mice. The induction of SIRT1 overexpression in podocytes for six weeks in OVE26 mice with established albuminuria attenuated the progression of diabetic glomerulopathy. To further validate the therapeutic potential of increased SIRT1 activity against diabetic kidney disease, we developed a new, potent and selective SIRT1 agonist, BF175. In cultured podocytes BF175 increased SIRT1-mediated activation of PGC1-α and protected against high glucose-mediated mitochondrial injury. In vivo, administration of BF175 for six weeks in OVE26 mice resulted in a marked reduction in albuminuria and in glomerular injury in a manner similar to podocyte-specific SIRT1 overexpression. Both podocyte-specific SIRT1 overexpression and BT175 treatment attenuated diabetes-induced podocyte loss and reduced oxidative stress in glomeruli of OVE26 mice. Thus, increased SIRT1 activity protects against diabetes-induced podocyte injury and effectively mitigates the progression of diabetic kidney disease. Podocyte injury and loss contribute to the progression of glomerular diseases, including diabetic kidney disease. We previously found that the glomerular expression of Sirtuin-1 (SIRT1) is reduced in human diabetic glomeruli and that the podocyte-specific loss of SIRT1 aggravated albuminuria and worsened kidney disease progression in diabetic mice. SIRT1 encodes an NAD-dependent deacetylase that modifies the activity of key transcriptional regulators affected in diabetic kidneys, including NF-κB, STAT3, p53, FOXO4, and PGC1-α. However, whether the increased glomerular SIRT1 activity is sufficient to ameliorate the pathogenesis of diabetic kidney disease has not been explored. We addressed this by inducible podocyte-specific SIRT1 overexpression in diabetic OVE26 mice. The induction of SIRT1 overexpression in podocytes for six weeks in OVE26 mice with established albuminuria attenuated the progression of diabetic glomerulopathy. To further validate the therapeutic potential of increased SIRT1 activity against diabetic kidney disease, we developed a new, potent and selective SIRT1 agonist, BF175. In cultured podocytes BF175 increased SIRT1-mediated activation of PGC1-α and protected against high glucose-mediated mitochondrial injury. In vivo, administration of BF175 for six weeks in OVE26 mice resulted in a marked reduction in albuminuria and in glomerular injury in a manner similar to podocyte-specific SIRT1 overexpression. Both podocyte-specific SIRT1 overexpression and BT175 treatment attenuated diabetes-induced podocyte loss and reduced oxidative stress in glomeruli of OVE26 mice. Thus, increased SIRT1 activity protects against diabetes-induced podocyte injury and effectively mitigates the progression of diabetic kidney disease. Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease in the United States.1United States Renal Data System. Annual Data Report: Atlas of End-Stage-Renal-Disease in the United States. Published 2011. Available at: https://www.usrds.org.Google Scholar The current therapy for DKD remains limited to the renin-angiotensin system blockade, which only provides partial renoprotection. Thus, many patients on angiotensin converting enzyme inhibitors or angiotensin receptor blockades continue to progress to end stage renal disease.2de Zeeuw D. Unmet need in renal protection–do we need a more comprehensive approach?.Contrib Nephrol. 2011; 171: 157-160Crossref PubMed Scopus (20) Google Scholar Therefore there is a large unmet need to develop more potent and safer therapies for patients with DKD. The sirtuin family of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases plays an important role in aging,3Tissenbaum H.A. Guarente L. Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans.Nature. 2001; 410: 227-230Crossref PubMed Scopus (1577) Google Scholar metabolism,4Moynihan K.A. Grimm A.A. Plueger M.M. et al.Increased dosage of mammalian Sir2 in pancreatic beta cells enhances glucose-stimulated insulin secretion in mice.Cell Metab. 2005; 2: 105-117Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar cancer, and inflammation.5Yang H. Bi Y. Xue L. et al.Multifaceted modulation of SIRT1 in cancer and inflammation.Crit Rev Oncog. 2015; 20: 49-64Crossref PubMed Scopus (81) Google Scholar Sirtuin-1 (SIRT1) in renal tubular cells has been shown to protect renal tubular cells from cellular stresses associated with aging, cisplatin, and hypoxia.6He W. Wang Y. Zhang M.Z. et al.Sirt1 activation protects the mouse renal medulla from oxidative injury.J Clin Invest. 2010; 120: 1056-1068Crossref PubMed Scopus (249) Google Scholar, 7Hasegawa K. Wakino S. Yoshioka K. et al.Kidney-specific overexpression of Sirt1 protects against acute kidney injury by retaining peroxisome function.J Biol Chem. 2010; 285: 13045-13056Crossref PubMed Scopus (182) Google Scholar, 8Kume S. Uzu T. Horiike K. et al.Calorie restriction enhances cell adaptation to hypoxia through Sirt1-dependent mitochondrial autophagy in mouse aged kidney.J Clin Invest. 2010; 120: 1043-1055Crossref PubMed Scopus (486) Google Scholar Our previous studies have demonstrated that SIRT1 protein expression is reduced in podocytes and in glomerular cells of human diabetic kidneys, which was consistent with reduced SIRT1 mRNA expression in microdissected glomeruli of diabetic patients.9Chuang P.Y. Yu Q. Fang W. et al.Advanced glycation endproducts induce podocyte apoptosis by activation of the FOXO4 transcription factor.Kidney Int. 2007; 72: 965-976Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 10Liu R. Zhong Y. Li X. et al.Role of transcription factor acetylation in diabetic kidney disease.Diabetes. 2014; 63: 2440-2453Crossref PubMed Scopus (152) Google Scholar We have further shown that the global reduction of SIRT1 accelerated DKD progression in db/db mice10Liu R. Zhong Y. Li X. et al.Role of transcription factor acetylation in diabetic kidney disease.Diabetes. 2014; 63: 2440-2453Crossref PubMed Scopus (152) Google Scholar and that podocyte-specific knockout of Sirt1 similarly accelerated DKD in streptozotocin-induced diabetic mice.10Liu R. Zhong Y. Li X. et al.Role of transcription factor acetylation in diabetic kidney disease.Diabetes. 2014; 63: 2440-2453Crossref PubMed Scopus (152) Google Scholar, 11Chuang P.Y. Xu J. Dai Y. et al.In vivo RNA interference models of inducible and reversible Sirt1 knockdown in kidney cells.Am J Pathol. 2014; 184: 1940-1956Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar More recently, renal tubular SIRT1 expression was reported to mitigate diabetic glomerular injury.12Hasegawa K. Wakino S. Simic P. et al.Renal tubular Sirt1 attenuates diabetic albuminuria by epigenetically suppressing Claudin-1 overexpression in podocytes.Nat Med. 2013; 19: 1496-1504Crossref PubMed Scopus (310) Google Scholar However, whether the increased glomerular SIRT1 expression, particularly in the podocytes, is sufficient to attenuate DKD has not been addressed previously. On the cellular level, SIRT1 has been shown to regulate autophagy,13Salminen A. Kaarniranta K. SIRT1: regulation of longevity via autophagy.Cell Signal. 2009; 21: 1356-1360Crossref PubMed Scopus (171) Google Scholar energetic homeostasis,14Rodgers J.T. Lerin C. Haas W. et al.Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1.Nature. 2005; 434: 113-118Crossref PubMed Scopus (2563) Google Scholar mitochondrial biogenesis,15Scarpulla R.C. Metabolic control of mitochondrial biogenesis through the PGC-1 family regulatory network.Biochim Biophys Acta. 2011; 1813: 1269-1278Crossref PubMed Scopus (845) Google Scholar and apoptosis.16Luo J. Nikolaev A.Y. Imai S. et al.Negative control of p53 by Sir2alpha promotes cell survival under stress.Cell. 2001; 107: 137-148Abstract Full Text Full Text PDF PubMed Scopus (1884) Google Scholar SIRT1 exerts these biological effects through deacetylation of transcription factors and consequently regulating their activities.17Nakagawa T. Guarente L. Sirtuins at a glance.J Cell Sci. 2011; 124: 833-838Crossref PubMed Scopus (232) Google Scholar Among these substrates of SIRT1 are key transcription factors that are implicated in kidney disease progression, such as NF-κB p65 (RelA), STAT3, p53, FOXO, and PGC-1α.18Yacoub R. Lee K. He J.C. The role of SIRT1 in diabetic kidney disease.Front Endocrinol (Lausanne). 2014; 5: 166Crossref PubMed Scopus (58) Google Scholar Systems analysis has revealed that JAK-STAT and NF-κB are key pathways activated in diabetic kidneys,19Berthier C.C. Zhang H. Schin M. et al.Enhanced expression of Janus kinase-signal transducer and activator of transcription pathway members in human diabetic nephropathy.Diabetes. 2009; 58: 469-477Crossref PubMed Scopus (234) Google Scholar, 20Schmid H. Boucherot A. Yasuda Y. et al.Modular activation of nuclear factor-kappaB transcriptional programs in human diabetic nephropathy.Diabetes. 2006; 55: 2993-3003Crossref PubMed Scopus (335) Google Scholar and we have recently shown that SIRT1 deacetylates STAT3 and NF-κB p65 to protect kidney from inflammation-induced kidney injury.10Liu R. Zhong Y. Li X. et al.Role of transcription factor acetylation in diabetic kidney disease.Diabetes. 2014; 63: 2440-2453Crossref PubMed Scopus (152) Google Scholar We also demonstrated that the attenuation of proteinuria and podocyte injury in diabetic db/db mice by pyridoxamine treatment was associated with restored SIRT1 expression and reduced NF-κB p65 and STAT3 acetylation and activation.21Chuang P.Y. Dai Y. Liu R. et al.Alteration of forkhead box O (foxo4) acetylation mediates apoptosis of podocytes in diabetes mellitus.PLoS One. 2011; 6e23566Crossref PubMed Scopus (110) Google Scholar A large body of evidence also suggests that p53 mediates apoptosis of podocytes and tubular epithelial cells in DKD.22Niranjan T. Bielesz B. Gruenwald A. et al.The Notch pathway in podocytes plays a role in the development of glomerular disease.Nat Med. 2008; 14: 290-298Crossref PubMed Scopus (323) Google Scholar, 23Tikoo K. Tripathi D.N. Kabra D.G. et al.Intermittent fasting prevents the progression of type I diabetic nephropathy in rats and changes the expression of Sir2 and p53.FEBS Lett. 2007; 581: 1071-1078Crossref PubMed Scopus (97) Google Scholar, 24Brezniceanu M.L. Liu F. Wei C.C. et al.Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice.Kidney Int. 2007; 71: 912-923Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar SIRT1 has been shown to promote cell survival by suppressing p53-dependent apoptosis in response to DNA damage and oxidative stress,16Luo J. Nikolaev A.Y. Imai S. et al.Negative control of p53 by Sir2alpha promotes cell survival under stress.Cell. 2001; 107: 137-148Abstract Full Text Full Text PDF PubMed Scopus (1884) Google Scholar and recent data suggests that the interplay of SIRT1-p53 pathway controls cellular senescence.25Tran D. Bergholz J. Zhang H. et al.Insulin-like growth factor-1 regulates the SIRT1-p53 pathway in cellular senescence.Aging Cell. 2014; 13: 669-678Crossref PubMed Scopus (104) Google Scholar Furthermore, SIRT1 was also shown to modulate PGC-1α activity and to attenuate aldosterone-induced mitochondrial damage and podocyte injury.26Yuan Y. Huang S. Wang W. et al.Activation of peroxisome proliferator-activated receptor-gamma coactivator 1alpha ameliorates mitochondrial dysfunction and protects podocytes from aldosterone-induced injury.Kidney Int. 2012; 82: 771-789Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar Thus, together with the observation that SIRT1 expression is significantly reduced in glomeruli of mouse and human diabetic kidneys and that SIRT1 is a regulator of the previously mentioned transcription factors whose roles are implicated in DKD progression, we posited that the increased glomerular expression and/or activity of SIRT1 would confer therapeutic benefit against the disease progression. We have tested this hypothesis in vivo by employing both genetic and pharmacological approaches with the use of the inducible podocyte-specific SIRT1 overexpression mice and a novel SIRT1 agonist, respectively, in OVE26 type 1 diabetic mice. To generate transgenic mice with tetracycline-inducible SIRT1 overexpression, human SIRT1 cDNA (Addgene, Cambridge, MA) was subcloned into pLP-TRE2 vector (Clontech Laboratories, Mountain View, CA), resulting in pLP-TRE2-SIRT1 expression construct as described in the Materials and Methods section. Doxycycline (Dox)-inducible expression of pLP-TRE2-SIRT1 construct was first confirmed in vitro in U2OS cell line (Supplementary Figure S1A) and used for microinjection to generate the TRE-SIRT1 transgenic mice in the FVB/N background (TRE-SIRT1OV). Inducible SIRT1 overexpression in vivo was first tested in TRE-SIRT1OV mice bred with transgenic mice with the universal expression of reverse tetracycline-controlled transactivator transgene under the cytomegalovirus early enhancer element and chicken beta-actin promoter (CAG-rtTA). Upon Dox supplementation (625 mg/kg in chow), there was a robust expression of SIRT1 in kidney cortices of CAGs-rtTA;TRE-SIRT1OV mice (Supplementary Figure S1B). TRE-SIRT1OV mice were subsequently crossed with podocin-rtTA transgenic mice to generate podocin-rtTA;TRE-SIRT1OV (referred to hereafter as Pod-SIRT1OV) for podocyte-specific SIRT1 expression. Dox-dependent induction of SIRT1 overexpression in podocytes was confirmed by Western blot analysis of primary podocytes isolated from Pod-SIRT1OV mice with or without Dox supplementation (Supplementary Figure S1C). In order to ascertain whether the podocyte-specific overexpression of SIRT1 can mitigate the diabetic kidney injury, Pod-SIRT1OV mice were crossed with type 1 diabetic OVE26 mice27Zheng S. Noonan W.T. Metreveli N.S. et al.Development of late-stage diabetic nephropathy in OVE26 diabetic mice.Diabetes. 2004; 53: 3248-3257Crossref PubMed Scopus (134) Google Scholar to generate OVE26;Pod-SIRT1OV. Their littermates without SIRT1 transgene (OVE26;WT) and age-matched healthy FVB mice were used as controls. Consistent with previous reports,27Zheng S. Noonan W.T. Metreveli N.S. et al.Development of late-stage diabetic nephropathy in OVE26 diabetic mice.Diabetes. 2004; 53: 3248-3257Crossref PubMed Scopus (134) Google Scholar, 28Xu J. Huang Y. Li F. et al.FVB mouse genotype confers susceptibility to OVE26 diabetic albuminuria.Am J Physiol Renal Physiol. 2010; 299: F487-F494Crossref PubMed Scopus (32) Google Scholar OVE26 mice exhibited significant albuminuria by 16 weeks of age. Thus, we next determined whether the overexpression of SIRT1 in podocytes in OVE26 mice starting at 16 weeks of age can curtail the progress of an established DKD. Healthy nondiabetic littermate control, OVE26;WT, and OVE26;Pod-SIRT1OV mice were given Dox-supplemented chow starting at 16 weeks of age for 6 weeks as outlined in Figure 1a. Blood glucose and urinary albumin levels were monitored starting at 8 weeks until 22 weeks of age when they were killed. OVE26 mice displayed pronounced hyperglycemia at 8 weeks of age that was sustained throughout the duration of the study (Figure 1b). Dox-induced SIRT1 overexpression in OVE26;Pod-SIRT1OV mice did not affect blood glucose levels or overall body weight in comparison to OVE26;WT mice (Figure 1b and c). However, kidney-to-body weight ratio, which was found to be significantly increased in OVE26;WT mice compared with healthy controls, were suppressed in OVE26;Pod-SIRT1OV mice (Figure 1d). Weekly spot collection of urine showed apparent albuminuria in both OVE26;WT and OVE26;Pod-SIRT1OV mice at 8 weeks of age, which had further escalated by 22 weeks of age in OVE26;WT but was significantly curtailed in OVE26;Pod-SIRT1OV mice (Figure 1e). The reduction in albuminuria by SIRT1 overexpression was further confirmed by 24-hour urine excretion at 22 weeks of age, which showed a marked reduction in OVE26;Pod-SIRT1OV mice as compared with OVE26;WT (Figure 1f). Histologically, although both diabetic groups displayed glomerular hypertrophy and mesangial matrix expansion in comparison with nondiabetic controls, they were both significantly attenuated in OVE26;Pod-SIRT1OV compared with OVE26;WT (Figure 2a–c). Consistently, electron microscopic analysis showed significant reduction in podocyte foot process effacement and glomerular basement membrane (GBM) thickening in OVE26;Pod-SIRT1OV mice compared with OVE26;WT mice (Figure 2d). Together, our data suggest that the overexpression of SIRT1 in podocytes in diabetic kidneys mitigates diabetes-induced podocyte injury and effectively blunts the progression of DKD in OVE26 mice. Given that podocyte-specific overexpression of SIRT1 for 6 weeks in OVE26 mice with established DKD was sufficient to mitigate the disease progression, we sought for a pharmacological means of SIRT1 activation as a therapeutic approach against DKD. Although several studies have shown the beneficial effects of SIRT1 agonist resveratrol against DKD injury,18Yacoub R. Lee K. He J.C. The role of SIRT1 in diabetic kidney disease.Front Endocrinol (Lausanne). 2014; 5: 166Crossref PubMed Scopus (58) Google Scholar resveratrol is also associated with renal toxicity29Crowell J.A. Korytko P.J. Morrissey R.L. et al.Resveratrol-associated renal toxicity.Toxicol Sci. 2004; 82: 614-619Crossref PubMed Scopus (252) Google Scholar and reported to have SIRT1-independent effects.30Beher D. Wu J. Cumine S. et al.Resveratrol is not a direct activator of SIRT1 enzyme activity.Chem Biol Drug Des. 2009; 74: 619-624Crossref PubMed Scopus (373) Google Scholar, 31Pacholec M. Bleasdale J.E. Chrunyk B. et al.SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1.J Biol Chem. 2010; 285: 8340-8351Crossref PubMed Scopus (750) Google Scholar Thus, in order to improve the specificity, bioavailability and to lower toxicity, we have synthesized several boron-containing compounds based on the structure of resveratrol using the target-to-hit and structure-activity relationships (SAR) approaches and with computer-aided drug design approach. The introduction of boron atoms into small molecules presents several advantages in that it enhances the interaction with the target molecule and facilitates the molecule’s movement across the cell membrane through their interactions with cell surface glycoproteins.32Das B.C. Thapa P. Karki R. et al.Boron chemicals in diagnosis and therapeutics.Future Med Chem. 2013; 5: 653-676Crossref PubMed Scopus (171) Google Scholar We recently employed a similar approach toward a potent inhibitor of homeodomain-interacting protein kinase-2 (HIPK2) that was shown to be highly effective against renal fibrosis,33Liu R. Das B. Xiao W. et al.A novel inhibitor of homeodomain interacting protein kinase 2 mitigates kidney fibrosis through inhibition of the TGF-beta1/Smad3 pathway.J Am Soc Nephrol. 2017; 8: 2133-2143Crossref Scopus (35) Google Scholar as well as toward a novel retinoic acid alpha agonists for CKD treatment.34Zhong Y. Wu Y. Liu R. et al.Novel retinoic acid receptor alpha agonists for treatment of kidney disease.PLoS One. 2011; 6e27945Crossref PubMed Scopus (33) Google Scholar Currently existing SIRT1 agonists SRT1720, SRT2183, SRT1460, and SRT2104 are high molecular weight amides containing imidazothiazole based pharmacophore groups35Hubbard B.P. Sinclair D.A. Small molecule SIRT1 activators for the treatment of aging and age-related diseases.Trends Pharmacol Sci. 2014; 35: 146-154Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar (Supplementary Figure S2A). Since amide bonds are prone to proteolytic cleavage, leading to increased toxicity and to off-target effects, we first performed the SAR study based on the structure of resveratrol (Supplementary Figure S2B) and synthesized a small library of compounds. Potential SIRT1 agonists were screened for stimulation of SIRT1 activity, and we found BF175 to be a potent SIRT1 activator (Supplementary Figure S3A). BF175 was a more potent activator of SIRT1 than resveratrol at same concentration of 10 μM in cultured human podocytes (Supplementary Figure S3B). Using the drug affinity responsive target stability (DARTS) assay,33Liu R. Das B. Xiao W. et al.A novel inhibitor of homeodomain interacting protein kinase 2 mitigates kidney fibrosis through inhibition of the TGF-beta1/Smad3 pathway.J Am Soc Nephrol. 2017; 8: 2133-2143Crossref Scopus (35) Google Scholar, 36Lomenick B. Hao R. Jonai N. et al.Target identification using drug affinity responsive target stability (DARTS).Proc Natl Acad Sci U S A. 2009; 106: 21984-21989Crossref PubMed Scopus (560) Google Scholar, 37Pai M.Y. Lomenick B. Hwang H. et al.Drug affinity responsive target stability (DARTS) for small-molecule target identification.Methods Mol Biol. 2015; 1263: 287-298Crossref PubMed Scopus (164) Google Scholar which is based on the principle that binding of a small molecule compound to the target protein changes the protein conformation to increase its stability and confer protection against proteolysis, we also confirmed that BF175 directly binds to SIRT1 (Supplementary Figure S3C). To test the biological effects of BF175 on SIRT1 activity, we next examined the SIRT1-mediated modulation of PGC-1α pathway in cultured human podocytes. Podocytes were transduced with lentivirus expressing either a control scrambled shRNA (shScr) or shRNA against SIRT1 (shSIRT1). As expected, stable transduced shSIRT1 podocytes showed significantly reduced SIRT1 expression compared with shScr podocytes (Supplementary Figure S4). Exposure to high-glucose media led to elevated levels of acetylated PGC-1α in both shScr and shSIRT1 podocytes compared with high-mannitol control (Figure 3a and b). Concomitant incubation with BF175 significantly reduced the high glucose-induced PGC-1α in shScr podocytes, but not in shSIRT1 podocytes (Figure 3a and b). Consistently, high-glucose treatment led to the reduced expression of PGC-1α-dependent expression of downstream mitochondrial gene expression, NRF-1, and TFAM (Figure 3c). BF175 treatment significantly restored their expression in shScr podocytes in presence of high glucose, but this protective effect was abrogated in shSIRT1 podocytes (Figure 3c). Similarly, BF175 reduced the superoxide production under high-glucose conditions in shScr podocytes, but not in shSIRT1 podocytes (Figures 3d and e). Consistent with these observations, the amount of mtDNA that was decreased in high-glucose conditions was also significantly restored by BF175 treatment in shScr podocytes, but not in shSIRT1 podocytes (Figure 3f). Together, these results indicate that BF175 is a potent activator of SIRT1 and that it protects the podocytes from high-glucose–induced injury by improving the mitochondrial function and homeostasis in a SIRT1-dependent manner. Therefore, we next explored whether BF175 could attenuate DKD progression in OVE26 mice. Diabetic OVE26 mice at 16 weeks of age received i.p. injection of either BF175 (0.4 mg/kg body weight) or vehicle (5% DMSO in saline) each day for 6 weeks, as outlined in Figure 4a. Similar to what was observed in OVE26;Pod-SIRT1OV mice, BF175 treatment did not affect blood glucose levels or change their body weight for the duration of the treatment (Figure 4b and c). However, BF175 significantly reduced the kidney-to-body weight ratio in OVE26 mice (Figure 4d). Importantly, BF175 markedly reduced albuminuria (Figure 4e and f), glomerular hypertrophy, and mesangial matrix expansion, (Figure 5a–c), similarly to what was observed in OVE26;Pod-SIRT1OV. Consistent with these findings, the extent of podocyte foot process effacement and GBM thickening were markedly diminished with BF175 treatment for 6 weeks (Figure 5d–f). Together, our data indicate that BF175 effectively mitigated diabetic glomerular injury in OVE26 mice.Figure 5BF175 treatment attenuates glomerular injury in diabetic OVE26 mice. (a) Representative images of mouse kidneys stained with periodic acid–Schiff at 22 weeks of age are shown (top panels: original magnification ×200, bar = 50 μm; bottom panels: original magnification ×400, bar = 20 μm). (b,c) Quantification of glomerular volume (b) and fraction of mesangial area (c) are shown. (d) Electron microscopy was performed to assess ultrastructural changes in podocyte morphology (top panels: original magnification ×12,000, bar = 2 μm; bottom panels: original magnification ×40,000, bar = 500 nm). Representative images are shown. (e) Semiquantification of podocyte effacement (n = 5). (f) Measurements of glomerular basement membrane (GBM) thickness (n = 5). P values between groups from 1-way analysis of variance are indicated. Ctr, control. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We further observed that the podocyte-specific induction of SIRT1 overexpression or BF175 administration in OVE26 mice significantly restored the expression of podocyte marker nephrin in OVE26 mice (Figure 6a). Quantification of podocytes by p57 immunostaining showed a marked decrease in podocyte number in OVE26 mice compared with healthy controls (Figure 6b–c). Both OVE26;Pod-SIRT1OV and mice treated with BF175 showed increased podocyte number compared with control OVE26, indicating that increased SIRT1 function protected against podocyte loss in OVE26 glomeruli. Given that BF175 significantly improved mitochondrial function and reduced the superoxide production in cultured podocytes exposed to high glucose, we next examined the effects of increased podocyte SIRT1 expression or BF175 on the extent of oxidative stress in the diabetic glomeruli. Immunohistochemical staining showed a strong upregulation of nitrotyrosine expression in the glomeruli of OVE26;WT and in vehicle-treated OVE26 mice, which was markedly suppressed in glomeruli of both OVE26;Pod-SIRT1OV and in BF175-treated OVE26 mice (Figure 7). These results suggest that the renoprotection observed by increased SIRT1 function is mediated in part through the improved mitochondrial function and reduced oxidative stress in diabetic glomeruli of OVE26 mice. In sum, our data demonstrate that increased podocyte-specific expression of SIRT1 effectively thwarts the DKD progression and that BF175 is a novel SIRT1 agonist that can be further developed as a potential therapy against DKD. Many studies have shown that the loss of SIRT1 expression and/or activity worsens kidney disease, including DKD.12Hasegawa K. Wakino S. Simic P. et al.Renal tubular Sirt1 attenuates diabetic albuminuria by epigenetically suppressing Claudin-1 overexpression in podocytes.Nat Med. 2013; 19: 1496-1504Crossref PubMed Scopus (310) Google Scholar, 18Yacoub R. Lee K. He J.C. The role of SIRT1 in diabetic kidney disease.Front Endocrinol (Lausanne). 2014; 5: 166Crossref PubMed Scopus (58) Google Scholar Our previous work has supported an important role of SIRT1 in protection of podocytes against diabetic injury, as loss of SIRT1 led to aggravated disease progression in murine models of DKD.10Liu R. Zhong Y. Li X. et al.Role of transcription factor acetylation in diabetic kidney disease.Diabetes. 2014; 63: 2440-2453Crossref PubMed Scopus (152) Google Scholar, 11Chuang P.Y. Xu J. Dai Y. et al.In vivo RNA interference models of inducible and reversible Sirt1 knockdown in kidney cells.Am J Pathol. 2014; 184: 1940-1956Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar We now show that the induction of SIRT1 expression in podocytes is sufficient to attenuate DKD progression in OVE26 mice, further corroborating an essential role of SIRT1 against diabetes-induced podocyte injury and glomerulopathy. In addition, our findings also provide a strong basis for SIRT1 as a potential drug target for treatment of DKD. Toward this end, we also report in vitro and in vivo findings of a novel SIRT1 activator BF175. We found that BF175 led to improved mitochondrial function and reduced oxidative stress in podocytes under high glucose conditions, and 6 weeks of BF175 treatment in vivo significantly hindered the progression of DKD in OVE26 mice. Although resveratrol is a well-known SIRT1 activator, the use of resveratrol has been associated with renal toxicity in rodents,29Crowell J.A. Korytko P.J. Morrissey R.L. et al.Resveratrol-associated renal toxicity.Toxicol Sci. 2004; 82: 614-619Crossref PubMed Scopus (252) Google Scholar and recent reports question its specificity of SIRT1 activation.30Beher D. Wu J. Cumine S. et al.Resveratrol is not a direct activator of SIRT1 enzyme activity.Chem Biol Drug Des. 2009; 74: 619-624Crossref PubMed Scopus (373) Google Scholar, 31Pacholec M. Bleasdale J.E. Chrunyk B. et al.SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1.J Biol Chem. 2010; 285: 8340-8351Crossref PubMed Scopus (750)