Pitavastatin ameliorates albuminuria and renal mesangial expansion by downregulating NOX4 in db/db mice
2007; Elsevier BV; Volume: 72; Issue: 4 Linguagem: Inglês
10.1038/sj.ki.5002366
ISSN1523-1755
AutoresMasakazu Fujii, Toyoshi Inoguchi, Yasutaka Maeda, S. Sasaki, Fumi Sawada, Rintaro Saito, Kunihisa Kobayashi, Hideki Sumimoto, R. Takayanagi,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoRecent studies have uncovered various pleiotrophic effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase-inhibiting drugs (statins). Several studies have identified a beneficial effect of statins on diabetic nephropathy; however, the molecular mechanisms are unclear. In this study, we show that statin ameliorates nephropathy in db/db mice, a rodent model of type 2 diabetes, via downregulation of NAD(P)H oxidase NOX4, which is a major source of oxidative stress in the kidney. Pitavastatin treatment for 2 weeks starting at 12 weeks of age significantly reduced albuminuria in the db/db mice concomitant with a reduction of urinary 8-hydroxy-2′-deoxyguanosine and 8-epi-prostaglandin F2α. Immunohistochemical analysis found increased amounts of 8-hydroxy-2′-deoxyguanosine and NOX4 protein in the kidney of db/db mice. Quantitative reverse transcription-polymerase chain reaction also showed increased levels of NOX4 mRNA. Pitavastatin normalized all of these changes in the kidneys of diabetic animals. Additionally, 12-week treatment with the statin completely normalized the levels of transforming growth factor-β1 and fibronectin mRNA as well as the mesangial expansion characteristic of diabetic nephropathy. Our study demonstrates that pitavastatin ameliorates diabetic nephropathy in db/db mice by minimizing oxidative stress by downregulating NOX4 expression. These findings may provide insight into the mechanisms of statin therapy in early stages of diabetic nephropathy. Recent studies have uncovered various pleiotrophic effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase-inhibiting drugs (statins). Several studies have identified a beneficial effect of statins on diabetic nephropathy; however, the molecular mechanisms are unclear. In this study, we show that statin ameliorates nephropathy in db/db mice, a rodent model of type 2 diabetes, via downregulation of NAD(P)H oxidase NOX4, which is a major source of oxidative stress in the kidney. Pitavastatin treatment for 2 weeks starting at 12 weeks of age significantly reduced albuminuria in the db/db mice concomitant with a reduction of urinary 8-hydroxy-2′-deoxyguanosine and 8-epi-prostaglandin F2α. Immunohistochemical analysis found increased amounts of 8-hydroxy-2′-deoxyguanosine and NOX4 protein in the kidney of db/db mice. Quantitative reverse transcription-polymerase chain reaction also showed increased levels of NOX4 mRNA. Pitavastatin normalized all of these changes in the kidneys of diabetic animals. Additionally, 12-week treatment with the statin completely normalized the levels of transforming growth factor-β1 and fibronectin mRNA as well as the mesangial expansion characteristic of diabetic nephropathy. Our study demonstrates that pitavastatin ameliorates diabetic nephropathy in db/db mice by minimizing oxidative stress by downregulating NOX4 expression. These findings may provide insight into the mechanisms of statin therapy in early stages of diabetic nephropathy. Diabetic nephropathy is a leading cause of end-stage renal disease worldwide. Establishment of therapeutic strategies targeted at the causative mechanisms of diabetic nephropathy has become increasingly urgent. In recent years, oxidative stress has emerged as an important pathogenic factor in the development of diabetic vascular complications, including nephropathy.1Oberley L.W. Free radicals and diabetes.Free Radic Biol Med. 1988; 5: 113-124Crossref PubMed Scopus (898) Google Scholar, 2Baynes J.W. Role of oxidative stress in development of complications in diabetes.Diabetes. 1991; 40: 405-412Crossref PubMed Scopus (0) Google Scholar, 3Wolff S.P. Jiang Z.Y. Hunt J.V. Protein glycation and oxidative stress in diabetes mellitus and ageing.Free Radic Biol Med. 1991; 10: 339-352Crossref PubMed Scopus (776) Google Scholar, 4Ha H. Kim C. Son Y. et al.DNA damage in the kidneys of diabetic rats exhibiting microalbuminuria.Free Radic Biol Med. 1994; 16: 271-274Crossref PubMed Scopus (104) Google Scholar, 5Kakimoto M. Inoguchi T. Sonta T. et al.Accumulation of 8-hydroxy-2′-deoxyguanosine and mitochondrial DNA deletion in kidney of diabetic rats.Diabetes. 2002; 51: 1588-1595Crossref PubMed Scopus (171) Google Scholar Accumulating evidence has shown that many protein, lipid, and DNA markers of oxidative stress are increased in vascular tissues and kidney from animals and from patients with diabetes.4Ha H. Kim C. Son Y. et al.DNA damage in the kidneys of diabetic rats exhibiting microalbuminuria.Free Radic Biol Med. 1994; 16: 271-274Crossref PubMed Scopus (104) Google Scholar, 5Kakimoto M. Inoguchi T. Sonta T. et al.Accumulation of 8-hydroxy-2′-deoxyguanosine and mitochondrial DNA deletion in kidney of diabetic rats.Diabetes. 2002; 51: 1588-1595Crossref PubMed Scopus (171) Google Scholar, 6Dandona P. Thusu K. Cook S. et al.Oxidative damage to DNA in diabetes mellitus.Lancet. 1996; 347: 444-445Abstract Full Text PDF PubMed Scopus (668) Google Scholar Although multiple pathways may be involved in the generation of reactive oxygen species (ROS),7Mullarkey C.J. Edelstein D. Brownlee M. Free radical generation by early glycation products: a mechanism for accelerated atherogenesis in diabetes.Biochem Biophys Res Commun. 1990; 173: 932-939Crossref PubMed Scopus (625) Google Scholar, 8Williamson J.R. Hyperglycemic pseudohypoxia and diabetic complications.Diabetes. 1993; 42: 801-813Crossref PubMed Scopus (0) Google Scholar, 9Nishikawa T. Edelstein D. Du X.L. et al.Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage.Nature. 2000; 404: 787-790Crossref PubMed Scopus (3441) Google Scholar we and other investigators have shown that nonphagocytic NAD(P)H oxidases may be a major source of increased ROS production in vascular tissues in diabetes.10Inoguchi T. Li P. Umeda F. et al.High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C – dependent activation of NAD(P)H oxidase in cultured vascular cells.Diabetes. 2000; 49: 1939-1945Crossref PubMed Scopus (1187) Google Scholar, 11Hink U. Li H. Mollnau H. et al.Mechanisms underlying endothelial dysfunction in diabetes mellitus.Circ Res. 2001; 88: e14-e22Crossref PubMed Google Scholar, 12Kim Y.K. Lee M.S. Son S.M. et al.Vascular NADH oxidase is involved in impaired endothelium-dependent vasodilation in OLETF rats, a model of type 2 diabetes.Diabetes. 2002; 51: 522-527Crossref PubMed Scopus (139) Google Scholar, 13Inoguchi T. Sonta T. Tsubouchi H. et al.Protein kinase C-dependent increase in reactive oxygen species (ROS) production in vascular tissues of diabetes: role of vascular NAD (P) H oxidase.J Am Soc Nephrol. 2003; 14: S227-S232Crossref PubMed Google Scholar, 14Sonta T. Inoguchi T. Tsubouchi H. et al.Evidence for contribution of vascular NAD (P) H oxidase to increased oxidative stress in animal models of diabetes and obesity.Free Radic Biol Med. 2004; 37: 115-123Crossref PubMed Scopus (157) Google Scholar, 15Guzik T.J. Mussa S. Gastaldi D. et al.Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase.Circulation. 2002; 105: 1656-1662Crossref PubMed Scopus (853) Google Scholar The phagocytic NAD(P)H oxidase comprises two plasma membrane-associated proteins, gp91phox (NOX2) and p22phox, and several cytosolic regulatory subunits, p47phox, p67 phox, p40phox, and small GTP-binding protein Rac1 or Rac2. Nonphagocytic NAD(P)H oxidases are isoforms of the phagocytic oxidase. In the kidney, the catalytic subunit gp91phox is replaced with a homolog of gp91phox termed NOX4. Human NOX4 exhibits 39% identity with human gp91phox, with several conservations in membrane-spanning regions and binding sites for heme, flavin adenine dinucleotide, and NAD(P)H, indicative of its function as a superoxide-producing NAD(P)H oxidase.16Lassegue B. Clempus R.E. Vascular NAD(P)H oxidases: specific features, expression, and regulation.Am J Physiol Regul Integr Comp Physiol. 2003; 285: R277-R297Crossref PubMed Scopus (881) Google Scholar, 17Shiose A. Kuroda J. Tsuruya K. et al.A novel superoxide-producing NAD(P)H oxidase in kidney.J Biol Chem. 2001; 276: 1417-1423Crossref PubMed Scopus (421) Google Scholar, 18Geiszt M. Kopp J.B. Varnai P. et al.Identification of Renox, an NAD(P)H oxidase in kidney.Proc Natl Acad Sci USA. 2000; 97: 8010-8014Crossref PubMed Scopus (671) Google Scholar It has been implicated that NOX4, as a major source of ROS production in the kidney, could have a role under pathological conditions.19Gorin Y. Block K. Hernandez J. et al.Nox4 NAD (P) H oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney.J Biol Chem. 2005; 280: 39616-39626Crossref PubMed Scopus (423) Google Scholar,20Geiszt M. Leto T.L. The Nox family of NAD (P) H oxidases: host defense and beyond.J Biol Chem. 2004; 279: 51715-51718Crossref PubMed Scopus (365) Google Scholar We previously reported that increased expression of NOX4 might play an important role in increased ROS production in the kidney of streptozotocin-induced diabetic rats.21Etoh T. Inoguchi T. Kakimoto M. et al.Increased expression of NAD(P)H oxidase subunits, NOX4 and p22phox, in the kidney of streptozotocin-induced diabetic rats and its reversibity by interventive insulin treatment.Diabetologia. 2003; 46: 1428-1437Crossref PubMed Scopus (212) Google Scholar This notion was supported by a recent report showing that downregulation of NOX4 induced by antisense oligonucleotides completely attenuated oxidative stress in the kidneys of streptozotocin-induced diabetic rats concomitant with the normalization of renal hypertrophy and increased fibronectin expression.19Gorin Y. Block K. Hernandez J. et al.Nox4 NAD (P) H oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney.J Biol Chem. 2005; 280: 39616-39626Crossref PubMed Scopus (423) Google Scholar Thus, NAD(P)H oxidase NOX4 might be a therapeutic target for attenuating ROS production in the kidney and preventing the development of diabetic nephropathy. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are widely used as cholesterol-lowering agents. Accumulating evidence has revealed that they have anti-inflammatory and antioxidative actions that are independent of their cholesterol-lowering effect.22Munford R.S. Statins and the acute-phase response.N Engl J Med. 2001; 244: 2016-2018Crossref Scopus (139) Google Scholar, 23Takemoto M. Liao J.K. Pleiotropic effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.Arterioscler Thromb Vasc Biol. 2001; 21: 1712-1719Crossref PubMed Scopus (1193) Google Scholar, 24Ridker P.M. Rifai N. Clearfield M. et al.Measurement of C-reactive protein for the targeting of statin therapy in the primary prevention of acute coronary events.N Engl J Med. 2001; 344: 1959-1965Crossref PubMed Scopus (1431) Google Scholar, 25Rikitake Y. Liao J.K. Rho GTPases, statins, and nitric oxide.Circ Res. 2005; 97: 1232-1235Crossref PubMed Scopus (387) Google Scholar Recently, several reports have suggested that statins may have a beneficial effect on diabetic nephropathy through these pleiotropic actions.26Ota T. Takamura T. Ando H. et al.Preventive effect of cerivastatin on diabetic nephropathy through suppression of glomerular macrophage recruitment in a rat model.Diabetologia. 2003; 46: 843-851Crossref PubMed Scopus (83) Google Scholar, 27Usui H. HMG-CoA reductase inhibitor ameliorates diabetic nephropathy by its pleiotropic effects in rats.Nephrol Dial Transplant. 2003; 18: 265-272Crossref PubMed Scopus (153) Google Scholar, 28Danesh F.R. Sadeghi M.M. Amro N. et al.3-Hydroxy-3-methylglutaryl CoA reductase inhibitors prevent high glucose-induced proliferation of mesangial cells via modulation of Rho GTPase/ p21 signaling pathway: implications for diabetic nephropathy.Proc Natl Acad Sci USA. 2002; 99: 8301-8305Crossref PubMed Scopus (215) Google Scholar Usui H et al.27Usui H. HMG-CoA reductase inhibitor ameliorates diabetic nephropathy by its pleiotropic effects in rats.Nephrol Dial Transplant. 2003; 18: 265-272Crossref PubMed Scopus (153) Google Scholar reported that cerivastatin ameliorated nephropathy in streptozotocin-induced diabetic rats through its anti-inflammatory action. However, the precise molecular mechanisms remain unclear. Notably, statins have been reported to inhibit superoxide production in vascular cells via inhibition of angiotensin II-induced NAD(P)H oxidase activation.29Wassmann S. Laufs U. Baumer A.T. et al.Inhibition of geranylgeranylation reduces angiotensin II-mediated free radical production in vascular smooth muscle cells: involvement of angiotensin AT1 receptor expression and Rac1 GTPase.Mol Pharmacol. 2001; 59: 646-654Crossref PubMed Scopus (333) Google Scholar, 30Wagner A.H. Kohler T. Ruckschloss U. et al.Improvement of nitric oxide-dependent vasodilatation by HMG-CoA reductase inhibitors through attenuation of endothelial superoxide anion formation.Arterioscler Thromb Vasc Biol. 2000; 20: 61-69Crossref PubMed Scopus (483) Google Scholar, 31Vecchione C. Brandes R.P. Withdrawal of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors elicits oxidative stress and induces endothelial dysfunction in mice.Circ Res. 2002; 91: 173-179Crossref PubMed Scopus (164) Google Scholar In addition, we reported that pitavastatin attenuated high glucose-induced and diabetes-induced oxidative stress in vitro and in vivo evaluated by electron spin resonance measurements, which was mediated by inhibition of NAD(P)H oxidase activity.32Tsubouchi H. Inoguchi T. Sonta T. et al.Statin attenuates high glucose-induced and diabetes-induced oxidative stress in vitro and in vivo evaluated by electron spin resonance measurement.Free Radic Biol Med. 2005; 39: 444-452Crossref PubMed Scopus (59) Google Scholar In this study, we investigated whether statin treatment ameliorates nephropathy in db/db mice, a rodent model of type 2 diabetes. In addition, to explore the underlying molecular mechanisms, the effect of statin on the expression of NAD(P)H oxidase NOX4, which may be a major source of ROS production in the kidney, was examined. The body weights and blood glucose levels of db/db and db/+ mice are summarized in Table 1. At baseline (12 weeks of age), both body weight and blood glucose level were significantly higher in db/db mice than in db/+ mice. Two weeks after the start of treatment, pitavastatin had not significantly affected body weights or blood glucose level in db/db or db/+ mice. As shown in Table 2, pitavastatin also did not significantly affect serum levels of total cholesterol, triglyceride, or high-density lipoprotein-cholesterol in the db/db and db/+ mice.Table 1Body weights and blood glucose levels in db/+ and db/db mice at baseline and at 2 weeks after treatment Open table in a new tab Table 2Serum TC, TG, and HDL-cholesterol concentration in db/+ and db/db mice at 2 weeks after treatment Open table in a new tab Urinary albumin excretion was significantly higher in nontreated db/db mice than in nontreated db/+ mice at 14 weeks of age (242.4±50.3 vs 22.8±2.3 μg/day, P<0.01). Pitavastatin treatment significantly reduced urinary albumin excretion in db/db mice. (242.4±50.3 vs 56.9±4.1 μg/day, P<0.01) (Figure 1). Urinary 8-OHdG excretion and 8-epi-PGF2α excretion were significantly higher in nontreated db/db mice than in nontreated db/+ mice at 14 weeks of age (133.2±26. 7 vs 74.8±13.5 ng/day, P<0.05; 175.4±19.0 vs 39.4±3.9 ng/day, P<0.001, respectively). Pitavastatin treatment reduced both urinary 8-OHdG excretion and urinary 8-epi-PGF2α excretion in db/db mice to the control level (Figure 2). We next evaluated oxidative stress in renal tissue by immunostaning analysis of 8-OHdG. The staining intensities of 8-OHdG in the db/db mice were apparently stronger in both renal tubules and to a lesser extent in the glomeruli than those in db/+ mice. Positive 8-OHdG stain was increased mainly in the cytosol and to a lesser extent in the nuclei of the renal cells of db/db mice. Pitavastatin treatment restored the intensity of 8-OHdG staining in db/db mice to the control level (Figure 3). Positive staining of NOX4 was observed in the renal tubules and faint staining was detected in the glomeruli of nontreated db/+ mice at 14 weeks of age. In the kidneys of nontreated db/db mice, the staining intensities of NOX4 protein were apparently stronger in both renal tubules and the glomeruli than those in db/+ mice (Figure 4). The localization and staining intensities of NOX4 were in accordance with those of 8-OHdG staining. Pitavastatin treatment restored the intensity of NOX4 staining to the control level in both the tubules and the glomeruli, in parallel with the changes of 8-OHdG staining (Figure 4). NOX4 mRNA levels were quantified by real-time reverse trancription-polymerase chain reaction (RT-PCR). They were significantly increased in both the cortices and the medullae of nontreated db/db mice (315.7±143.6%, P<0.01; 212.6±43.0%, P<0.01; respectively) compared with nontreated db/+ mice. Consistent with the immunostaining analysis, pitavastatin treatment normalized NOX4 mRNA in db/db mice to the control level in both the cortices and the medullae (146.1±69.2%, not significant (NS) vs control; 122.9±60.0%, NS vs control; respectively) (Figure 5). To investigate the effect of pitavastatin treatment on mesangial expansion, which is one of the most striking characteristics of diabetic nephropathy, pitavastatin was given orally (5 mg/kg/day) for 12 weeks starting at 12 weeks of age. The glomerular structure in db/db mice showed accelerated mesangial expansion characterized by an increase in periodic acid-Sciff (PAS)-positive mesangial matrix area compared with that observed in db/+ mice at 24 weeks of age (Figure 6a–d). Mesangial expansion was semiquantified by measuring the PAS-positive mesangial matrix area as described in the Materials and Methods. The PAS-positive and nuclei-free mesangial area was markedly increased in the glomeruli of nontreated db/db mice (342.5±98.6%, P<0.001). Pitavastatin treatment prevented mesangial expansion in db/db mice, which were not significantly different from the controls (128.5±43.3%, NS vs control) (Figure 6e). In addition, the levels of transforming growth factor β1 (TGF-β1) and fibronectin mRNA were significantly increased in both cortices and medullae of db/db mice (TGF-β1: 669.6±113.9%, P<0.001, 262.0±77.0%, P<0.01, respectively; fibronectin: 329.6±59.0%, P<0.001, 252.5±52.6%, P<0.001, respectively) compared with control mice. Pitavastatin treatment normalized both levels of TGF-β1 and fibronectin mRNA in db/db mice to the control level (Figure 7). In this study, we showed that pitavastatin treatment ameliorated albuminuria concomitantly with a reduction of urinary and renal tissue oxidative stress markers. In addition, its long-term treatment also normalized the increased expression of TGF-β1 and fibronection, and normalized a renal mesangial expansion, which is one of the most striking morphologic characteristics of diabetic nephropathy in db/db mice, a rodent model of type 2 diabetes. Furthermore, this study showed for the first time that the underlying mechanism of these beneficial effects may be mediated by downregulation of NAD(P)H oxidase NOX4 expression. Either antioxidant supplementation such as vitamin E or lipoic acid, or overexpression of superoxide dismutase has been reported to attenuate renal injury in experimental models of diabetes.33Koya D. Prevention of glomerular dysfunction in diabetic rats by treatment with d-alpha-tocopherol.J Am Soc Nephrol. 1997; 8: 426-435PubMed Google Scholar, 34Melhem M.F. Craven P.A. Liachenko J. et al.Lipoic acid attenuates hyperglycemia and prevents glomerular mesangial matrix expansion in diabetes.J Am Soc Nephrol. 2002; 13: 108-116PubMed Google Scholar, 35Craven P.A. Melhem M.F. Phillips S.L. et al.Overexpression of Cu2+/Zn2+ superoxide dismutase protects against early diabetic glomerular injury in transgenic mice.Diabetes. 2001; 50: 2114-2125Crossref PubMed Scopus (131) Google Scholar These findings support an important role for oxidative stress in the pathogenesis of diabetic nephropathy. However, the precise molecular mechanism for increased oxidative stress in diabetic kidneys remained to be elucidated. The NAD(P)H oxidase isoform NOX4 was cloned from the kidney and found to be highly expressed there.16Lassegue B. Clempus R.E. Vascular NAD(P)H oxidases: specific features, expression, and regulation.Am J Physiol Regul Integr Comp Physiol. 2003; 285: R277-R297Crossref PubMed Scopus (881) Google Scholar, 17Shiose A. Kuroda J. Tsuruya K. et al.A novel superoxide-producing NAD(P)H oxidase in kidney.J Biol Chem. 2001; 276: 1417-1423Crossref PubMed Scopus (421) Google Scholar, 18Geiszt M. Kopp J.B. Varnai P. et al.Identification of Renox, an NAD(P)H oxidase in kidney.Proc Natl Acad Sci USA. 2000; 97: 8010-8014Crossref PubMed Scopus (671) Google Scholar It has been suggested that NOX4, as a major source of ROS production in the kidney, could have a role under pathological conditions. Although the nature of the sources of ROS overproduction in diabetes is not precisely defined, we and other investigators have suggested that nonphagocytic NAD(P)H oxidases may be major sources in vascular tissues in diabetes.10Inoguchi T. Li P. Umeda F. et al.High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C – dependent activation of NAD(P)H oxidase in cultured vascular cells.Diabetes. 2000; 49: 1939-1945Crossref PubMed Scopus (1187) Google Scholar, 11Hink U. Li H. Mollnau H. et al.Mechanisms underlying endothelial dysfunction in diabetes mellitus.Circ Res. 2001; 88: e14-e22Crossref PubMed Google Scholar, 12Kim Y.K. Lee M.S. Son S.M. et al.Vascular NADH oxidase is involved in impaired endothelium-dependent vasodilation in OLETF rats, a model of type 2 diabetes.Diabetes. 2002; 51: 522-527Crossref PubMed Scopus (139) Google Scholar, 13Inoguchi T. Sonta T. Tsubouchi H. et al.Protein kinase C-dependent increase in reactive oxygen species (ROS) production in vascular tissues of diabetes: role of vascular NAD (P) H oxidase.J Am Soc Nephrol. 2003; 14: S227-S232Crossref PubMed Google Scholar, 14Sonta T. Inoguchi T. Tsubouchi H. et al.Evidence for contribution of vascular NAD (P) H oxidase to increased oxidative stress in animal models of diabetes and obesity.Free Radic Biol Med. 2004; 37: 115-123Crossref PubMed Scopus (157) Google Scholar, 15Guzik T.J. Mussa S. Gastaldi D. et al.Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase.Circulation. 2002; 105: 1656-1662Crossref PubMed Scopus (853) Google Scholar We previously reported that, in the kidneys of streptozotocin-induced diabetic rats, increased expression of NOX4 might play an important role in increased ROS production.21Etoh T. Inoguchi T. Kakimoto M. et al.Increased expression of NAD(P)H oxidase subunits, NOX4 and p22phox, in the kidney of streptozotocin-induced diabetic rats and its reversibity by interventive insulin treatment.Diabetologia. 2003; 46: 1428-1437Crossref PubMed Scopus (212) Google Scholar Gorin et al.19Gorin Y. Block K. Hernandez J. et al.Nox4 NAD (P) H oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney.J Biol Chem. 2005; 280: 39616-39626Crossref PubMed Scopus (423) Google Scholar also reported increased expression of NOX4 in the diabetic kidney, and further showed that downregulation of NOX4 induced by antisense oligonucleotides completely attenuated oxidative stress in the kidneys of streptozotocin-induced diabetic rats. This is in agreement with the present finding that downregulation of NOX4 induced by pitavastatin completely attenuated oxidative stress in the kidneys of db/db mice. Taken together, these results suggest a role for NOX4 as the major source of ROS production in the diabetic kidney. We previously reported that high glucose levels stimulate superoxide production via protein kinase C-dependent activation of NAD(P)H oxidases in cultured aortic endothelial cells and smooth muscle cells.10Inoguchi T. Li P. Umeda F. et al.High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C – dependent activation of NAD(P)H oxidase in cultured vascular cells.Diabetes. 2000; 49: 1939-1945Crossref PubMed Scopus (1187) Google Scholar The mechanism underlying protein kinase C-dependent activation of NAD(P)H oxidase was supposed to be protein kinase C-dependent activation of small GTPase Rac-1, which is an important regulator of NAD(P)H oxidase activation.32Tsubouchi H. Inoguchi T. Sonta T. et al.Statin attenuates high glucose-induced and diabetes-induced oxidative stress in vitro and in vivo evaluated by electron spin resonance measurement.Free Radic Biol Med. 2005; 39: 444-452Crossref PubMed Scopus (59) Google Scholar Stains inhibit the synthesis of various isoprenoids such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate in addition to inhibiting the synthesis of cholesterol.36Goldstein J.L. Brown M.S. Regulation of the mevalonate pathway.Nature. 1990; 343: 425-430Crossref PubMed Scopus (4356) Google Scholar Farnesyl pyrophosphate and geranylgeranyl pyrophosphate are important attachments for the post-translational modification of small GTPases such as Rho and Rac.37Van Aelst L. D'Souza-Schorey C. Rho GTPases and signaling networks.Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2048) Google Scholar The pleiotropic effects are supposed to be at least partly mediated by small GTPases. Indeed, we found that pitavastatin attenuated high glucose-induced superoxide production in vascular cells by inhibiting NAD(P)H oxidases, which was mediated by inhibition of Rac-1.32Tsubouchi H. Inoguchi T. Sonta T. et al.Statin attenuates high glucose-induced and diabetes-induced oxidative stress in vitro and in vivo evaluated by electron spin resonance measurement.Free Radic Biol Med. 2005; 39: 444-452Crossref PubMed Scopus (59) Google Scholar However, recent studies have suggested that NOX4 functions independent of the presence of cytosolic regulatory subunits, including Rac, in contrast to other isoforms of NAD(P)H oxidase.38Martyn K.D. Frederick L.M. von Loehneysen K. et al.Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases.Cell Signal. 2006; 18: 69-82Crossref PubMed Scopus (583) Google Scholar NOX4 activity may depend on its own expression. In this respect, the molecular mechanism of the effect of pitavastatin on NOX4 expression remains unclear and should be clarified in further studies. Although the detailed mechanism of the regulation of NOX4 expression is unknown, it is speculated that high glucose levels and angiotensin II might be involved in the increased expression of NOX4 in the diabetic kidney. Previous reports showed that angiotensin II induces protein synthesis and hypertrophy via NOX4-derived ROS in renal mesangial cells.39Gorin Y. Ricono J.M. Kim N.H. et al.Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesangial cells.Am J Physiol Renal Physiol. 2003; 285: F219-F229Crossref PubMed Scopus (237) Google Scholar,40Gorin Y. Ricono J.M. Wagner B. et al.Angiotensin II-induced ERK1/ERK2 activation and protein synthesis are redox-dependent in glomerular mesangial cells.Biochem J. 2004; 381: 231-239Crossref PubMed Scopus (106) Google Scholar We also reported that angiotensin II type 1 receptor blocker normalized oxidative stress concomitant with downregulation of NOX4 expression in streptozotocin-induced diabetic mice.41Sonta T. Inoguchi T. Matsumoto S. et al.In vivo imaging of oxidative stress in the kidney of diabetic mice and its normalization by angiotensin II type 1 receptor blocker.Biochem Biophys Res Commun. 2005; 330: 415-422Crossref PubMed Scopus (56) Google Scholar High glucose levels or diabetes may enhance the local renin-angiotensin system in the diabetic kidney, and increased angiotensin II might induce NOX4 expression. A previous report showed that NOX4-derived ROS-mediated kidney hypertrophy and fibronectin expression.19Gorin Y. Block K. Hernandez J. et al.Nox4 NAD (P) H oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney.J Biol Chem. 2005; 280: 39616-39626Crossref PubMed Scopus (423) Google Scholar This is in agreement with our findings that pitavastatin ameliorated renal mesangial expansion concomitant with a reduction of oxidative stress that is mediated by the downregulation of NOX4 expression. However, immunostaining analysis showed that NOX4 expression and 8-OHdG were abundant in renal tubules as well as in glomeruli. It has been suggested that the rate of functional deterioration correlates with the degree of tubulointerstitial fibrosis in diabetic nephropathy. It is tempting to speculate that NOX4-derived ROS may contribute to tubulointerstial injury as well as glomerular lesions in the diabetic kidney. This notion is supported by the previous finding that pitavastatin reduced L-FABP, which is a marker of tubulointerstitial injury, concomitant with a reduction of urinary 8-OHdG in patients with early diabetic nephropathy.42Nakamura T. Sugaya T. Kawagoe Y. et al.Effect of pitavastatin on urinary liver-type fatty acid-binding protein levels in patients with early diabetic nephropathy A table elsewhere in this issue shows conventional and System International (SI) units and conversion factors for many substances.Diabetes Care. 2005; 28: 2728-2732Crossref PubMed Scopus (82) Google Scholar The relationship between NOX4 and tubulointerstitial injury and the effect of statins on it should be clarified in future studies. In conclusion, we showed for the first time that pitavastatin ameliorated diabetic nephropathy via inhibition of oxidative stress mediated by downregulation of NOX4 expression in a rodent model of type 2 diabetes. These findings may provide a new insight into the efficacy of statin therapy in the early stage of diabetic nephropathy. Male C57BL/KsJ db/db mice and their age-matched lean littermates, db/+ mice, were purchased from Clea Japan Inc. (Tokyo, Japan). All mice were bred under pathogen-free conditions at Kyusyu University Animal Center (Fukuoka, Japan). The animals had free access to tap water and standard chow (Clea Japan Inc., Tokyo, Japan) containing 50.1 carbohydrates, 25.1 proteins, 7.1 minerals, 4.5 fat, and 4.3% cellulose. At 12 weeks of age, half of the db/db and db/+ mice were randomly chosen to receive pitavastatin (kindly provided by Kowa Pharmaceutical Co. Ltd, Tokyo, Japan). Pitavastatin was dissolved in 0.5% carboxymethylcellulose-Na solution and orally given to the db/db (n=8) and db/+ (n=8) mice (5 mg/kg) once daily for 2 weeks. The other half of the db/db and db/+ mice were given the same volume of 0.5% carboxymethylcellulose-Na solution without pitavastatin. After 2 weeks of treatment, blood samples were obtained from the retro-orbital venous plexus for determination of the plasma concentration of total cholesterol, high-density lipoprotein-cholesterol, and triglyceride. A 24-h urine sample was collected using metabolic cages (one mouse per cage) for the last 3 days of the 2-week treatment. The well-mixed urine was centrifuged at 7500 × g for 5 min, purged of air with a steam of nitrogen to prevent artificial formation of 8-hydroxy-2′-deoxyguanosine (8-OHdG), and then stored at -80°C until analysis. After these procedures, all mice were anesthetized with pentobarbital (0.1 mg/g intraperitoneally) and killed. Both kidneys were rapidly dissected and separated into cortices and medullae for the following experiments. The samples were frozen in liquid nitrogen and kept at -80°C until use. All protocols were reviewed and approved by the Committee on the Ethics of Animal Experiments, Graduate School of Medical Science, Kyusyu University. Plasma concentrations of total cholesterol, high-density lipoprotein-cholesterol, and triglyceride were measured using a commercially available kit (KAINOS Laboratories Inc., Tokyo, Japan). Urinary albumin concentrations were measured using a Mouse Albumin ELISA Kit (AKRAL-121; Shibayagi, Gunma, Japan). The detection range was 50-1000 ng/ml. Urinary albumin excretion was expressed as the total amount excreted in 24 h. After proper dilution, urine 8-OHdG concentrations were measured using a competitive enzyme-linked immunosorbent assay kit (8OHdG Check; Japan Institute for the Control of Aging, Fukuroi, Japan) as described previously.5Kakimoto M. Inoguchi T. Sonta T. et al.Accumulation of 8-hydroxy-2′-deoxyguanosine and mitochondrial DNA deletion in kidney of diabetic rats.Diabetes. 2002; 51: 1588-1595Crossref PubMed Scopus (171) Google Scholar The detection range was 0.5-200 ng/ml. Urinary 8-OHdG excretion was expressed as the total amount excreted in 24 h. Urinary 8-epi-PGF2α concentrations were measured using a Urinary Isoprostane ELISA Kit (MED.DIA s.r.l., San Germano Vercellese, Italy). The detection range was 0.05-50 ng/ml. Urinary 8-epi-PGF2α excretion was expressed as the total amount excreted in 24 h. Immunostaining for 8-OHdG in the kidney was performed as described previously.21Etoh T. Inoguchi T. Kakimoto M. et al.Increased expression of NAD(P)H oxidase subunits, NOX4 and p22phox, in the kidney of streptozotocin-induced diabetic rats and its reversibity by interventive insulin treatment.Diabetologia. 2003; 46: 1428-1437Crossref PubMed Scopus (212) Google Scholar Briefly, the kidneys were fixed in 10% formaldehyde and embedded in paraffin. Paraffin sections were cut at 3 μm and deparaffinized. After inactivation of endogenous peroxidase with 10% H2O2 in methanol for 20 min at room temperature, the sections were preincubated for 30 min with 1% bovine serum albumin in phosphate-buffered saline. The samples were then incubated with anti-8-OHdG mouse monoclonal antibody (4 μg/ml; Japan Institute for the Control of Aging) overnight at 4°C, washed in phosphate-buffered saline, and probed with anti-mouse IgG antibody labeled with peroxidase (Histofine Simple Stain MAX PO(M); Nichirei, Tokyo, Japan) for 30 min at room temperature. The peroxidase was then visualized with diaminobenzidine (Nichirei). The primary antibody was replaced with mouse IgG as a negative control. To explore the mechanism underlying increased oxidative stress in the diabetic kidney, immunostaining of the NAD(P)H oxidase component NOX4 was performed as described previously.21Etoh T. Inoguchi T. Kakimoto M. et al.Increased expression of NAD(P)H oxidase subunits, NOX4 and p22phox, in the kidney of streptozotocin-induced diabetic rats and its reversibity by interventive insulin treatment.Diabetologia. 2003; 46: 1428-1437Crossref PubMed Scopus (212) Google Scholar The samples were incubated with anti-human NOX4 goat polyclonal antibody (2 μg/ml) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C, washed in phosphate-buffered saline, and probed with anti-goat IgG antibody labeled with peroxidase (Histofine Simple Stain MAX PO(G); Nichirei) for 30 min at room temperature. The primary antibody was replaced with sera obtained from a goat before immunization as a negative control. The peroxidase was then visualized with diaminobenzidine. Total RNA was extracted from the frozen kidney samples using Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. The extracted RNA (4 μg) was converted into single-stranded cDNA by a reverse transcriptase procedure with Superscript II (Invitrogen, Carlsbad, CA, USA). mRNA levels were quantified by quantitative RT-PCR using a LightCycler 2.0 instrument (Roche Diagnostics GmbH, Mannheim, Germany) as described previously.43Ago T. Kitazono T. Ooboshi H. et al.Nox4 as the major catalytic component of an endothelial NAD (P) H oxidase.Circulation. 2004; 109: 227-233Crossref PubMed Scopus (415) Google Scholar Briefly, 1 μl of cDNA was placed in a 20 μl reaction volume containing 1 μl of each primer and 2 μl of LightCycler-FastStart DNA Master SYBR green I (Roche Diagnostics GmbH). Nucleotide, Taq DNA polymerase, and buffer were included in the LightCycler-FastStart DNA Master SYBR green I. The amplification conditions comprised an initial denaturation step at 95°C for 10 min, followed by amplification of the target DNA for 40 cycles of 95°C (0 s) and 60°C (15 s), and extension at 72°C (36 s). Melting curve analysis was performed immediately after amplification at a linear temperature transition rate of 0.1°C/s from 65°C to 95°C with continuous fluorescence acquisition. The following primer pairs were used: NOX4, 5′-ATTTGGATAGGCTCCAGGCAAAC-3′ (sense) and 5′-CACATGGGTATAAGCTTTGTGAGCA-3′ (antisense); TGF-β1, 5′-AACAACGCCATCTATGAG-3′ (sense) and 5′-TATTCCGTCTCCTTGGTT-3′ (antisense); fibronectin, 5′-TGGCTGCCTTCAACTTCTCCT-3′ (sense) and 5′-TGTTTGATCTGGACTGGCAGTTT-3′ (antisense); β-actin, 5′-TGACAGGATGCAGAAGGAGA-3′ (sense) and 5′-GCTGGAAGGTGGACAGTGAG-3′ (antisense). The linearity of the amplifications as a function of the cycle number was tested in preliminary experiments, and NOX4/TGF-β1/fibronectin mRNA expression was normalized to expression of the housekeeping gene β-actin. PCR products were separated on a 4% agarose gel containing ethidium bromide. To examine the effect of pitavastatin on mesangial matrix expansion, db/db mice, and age-matched db/+ mice were given the drug orally, mixed in standard chow (5 mg/kg/day) for 12 weeks (from 12 to 24 weeks of age). After 12 weeks of treatment, the mice were anesthetized and killed. The kidneys were rapidly dissected, fixed in 10% formaldehyde, and embedded in paraffin. Paraffin sections were cut at 3 μm, perpendicular to the long axis of the kidney, for morphometric analysis. For analysis of the glomeruli, sections were stained with PAS. To semiquantify mesangial expansion, sections were coded and read by an observer unaware of the experimental protocol applied. In each animal of the four experimental groups, 30 glomeruli were used and averaged for morphometric analysis. The extent of increase in the mesangial matrix (defined as the mesangial area) was determined by the presence of PAS-positive and nuclei-free areas in the mesangium, which were traced along the outline of the capillary loop using Scion imaging software (Scion, Frederick, MD, USA). Data were expressed as the mean±s.e. Differences between groups were analyzed using Student's t-test with a two-tailed test of significance. Multiple comparisons among the groups were conducted by one-way analysis of variance with Fisher's probable least-squares difference test for post hoc analysis. P-values of <0.05 were considered significant. This work was in part supported by a Grant-in-Aid for Scientific Research (No.16590888) from the Ministry of Education, Science and Culture, Japan.
Referência(s)