Apocynin attenuates tubular apoptosis and tubulointerstitial fibrosis in transgenic mice independent of hypertension
2008; Elsevier BV; Volume: 75; Issue: 2 Linguagem: Inglês
10.1038/ki.2008.509
ISSN1523-1755
AutoresFang Liu, Chih‐Chang Wei, Shyh-Jong Wu, Isabelle Chénier, Shao‐Ling Zhang, János G. Filep, Julie R. Ingelfinger, John S.D. Chan,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoAngiotensin II stimulates the formation of reactive oxygen species by increased NADPH oxidase activity, which contributes to proapoptotic and profibrotic mechanisms critical in renal injury. Here we determine if apocynin, an inhibitor of NADPH oxidase, interferes with the action of the intrarenal renin–angiotensin system to minimize the progression of renal disease. Transgenic mice that overexpress rat angiotensinogen in their proximal tubule cells were given either apocynin, perindopril, or hydralazine while untreated or apocynin-treated non-transgenic littermates served as controls. Untreated transgenic mice had significant elevations of their systolic blood pressure, albuminuria, reactive oxygen species production, NADPH oxidase activity, tubular apoptosis, active caspase-3, Bax, transforming growth factor-β1, plasminogen activator inhibitor-1, extracellular matrix proteins, collagen type IV, and phosphorylated p47phox expression compared to untreated non-transgenic mice. Apocynin and perindopril blunted these changes; however, apocynin had no effect on the systolic blood pressure whereas hydralazine prevented hypertension and tubulointerstitial fibrosis but not proximal tubule cell apoptosis. Our study shows that the intrarenal renin–angiotensin system stimulates proximal tubule cell apoptosis and tubulointerstitial fibrosis, in part, by enhanced NADPH oxidase activity and reactive oxygen species generation independent of systemic hypertension. Angiotensin II stimulates the formation of reactive oxygen species by increased NADPH oxidase activity, which contributes to proapoptotic and profibrotic mechanisms critical in renal injury. Here we determine if apocynin, an inhibitor of NADPH oxidase, interferes with the action of the intrarenal renin–angiotensin system to minimize the progression of renal disease. Transgenic mice that overexpress rat angiotensinogen in their proximal tubule cells were given either apocynin, perindopril, or hydralazine while untreated or apocynin-treated non-transgenic littermates served as controls. Untreated transgenic mice had significant elevations of their systolic blood pressure, albuminuria, reactive oxygen species production, NADPH oxidase activity, tubular apoptosis, active caspase-3, Bax, transforming growth factor-β1, plasminogen activator inhibitor-1, extracellular matrix proteins, collagen type IV, and phosphorylated p47phox expression compared to untreated non-transgenic mice. Apocynin and perindopril blunted these changes; however, apocynin had no effect on the systolic blood pressure whereas hydralazine prevented hypertension and tubulointerstitial fibrosis but not proximal tubule cell apoptosis. Our study shows that the intrarenal renin–angiotensin system stimulates proximal tubule cell apoptosis and tubulointerstitial fibrosis, in part, by enhanced NADPH oxidase activity and reactive oxygen species generation independent of systemic hypertension. Although the progression of chronic renal failure may be initiated by glomerular injury, studies over the past three decades have demonstrated that tubulointerstitial injury, characterized by interstitial fibrosis and tubular atrophy, may be a better predictor of renal disease progression than glomerular pathology.1.Eddy A.A. Molecular insights into renal interstitial fibrosis.J Am Soc Nephrol. 1996; 7: 2495-2508Crossref PubMed Google Scholar, 2.Gilbert R.E. Cooper M.E. The tubulointerstitium in progressive diabetic kidney disease: more than an aftermath of glomerular injury?.Kidney Int. 1999; 56: 1627-1637Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 3.Eddy A.A. Molecular basis of renal fibrosis.Pediatr Nephrol. 2000; 15: 290-301Crossref PubMed Scopus (532) Google Scholar, 4.Risdon R.A. Sloper J.C. De Wardener H.E. Relationship between renal function and histological changes found in renal-biopsy specimens from patients with persistent glomerular nephritis.Lancet. 1968; 2: 363-366Abstract PubMed Google Scholar, 5.Schainuck L.I. Striker G.E. Cutler R.E. et al.Structural-functional correlations in renal disease.Hum Pathol. 1970; 1: 631-641Abstract Full Text PDF PubMed Scopus (424) Google Scholar, 6.Bohle A. MacKensen-Haen S. Von Gise H. Significance of tubulointerstitial changes in the renal cortex for the secretory function and concentration ability of the kidney: a morphometric contribution.Am J Nephrol. 1988; 7: 421-433Crossref Scopus (262) Google Scholar Tubular atrophy has been observed in various experimental models of nephropathy as well as in human renal diseases,7.Marcussen N. Olsen T.S. Atubular glomeruli in patients with chronic pyelonephritis.Lab Invest. 1990; 62: 467-473PubMed Google Scholar, 8.Markowitz G.S. Radhakrishnan J. Kambham N. et al.Lithium nephrotoxicity: a progressive combined glomerular and tubulointerstitial nephropathy.J Am Soc Nephrol. 2000; 11: 1439-1448PubMed Google Scholar, 9.Benigni A. Gagliardini E. Remuzzi A. et al.Angiotensin-converting enzyme inhibition prevents glomerular-tubule disconnection and atrophy in passive Heymann nephritis, an effect not observed with a calcium antagonist.Am J Pathol. 2001; 159: 1743-1750Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 10.Cosio F.G. Grande J.P. Larson T.S. et al.Kidney allograft fibrosis and atrophy early after living donor transplantation.Am J Transplant. 2005; 5: 1130-1136Crossref PubMed Scopus (106) Google Scholar, 11.Horiguchi H. Oguma E. Kayama F. Cadmium and cisplatin damage erythropoietin-producing proximal renal tubular cells.Arch Toxicol. 2006; 80: 680-686Crossref PubMed Scopus (29) Google Scholar, 12.Kimura M. Asano M. Abe K. et al.Role of atrophic changes in proximal tubular cells in the peritubular deposition of type IV collagen in a rat renal ablation model.Nephrol Dial Transplant. 2005; 20: 1559-1565Crossref PubMed Scopus (35) Google Scholar including diabetic nephropathy.13.Najafian B. Kim Y. Crosson J.T. et al.Atubular glomeruli and glomerulotubular junction abnormalities in diabetic nephropathy.J Am Soc Nephrol. 2003; 14: 908-917Crossref PubMed Scopus (89) Google Scholar, 14.Najafian B. Crosson J.T. Kim Y. et al.Glomerulotubular junction abnormalities are associated with proteinuria in type 1 diabetes.J Am Soc Nephrol. 2006; 17: S53-S60Crossref PubMed Scopus (49) Google Scholar However, the pathogenesis of tubular atrophy remains poorly understood. Apoptosis, postulated to be involved in the pathogenesis of tubular atrophy, mediates renal cell death in various renal diseases, including glomerulosclerosis,15.Sugiyama M. Kashihara N. Makino H. et al.Apoptosis in glomerular sclerosis.Kidney Int. 1996; 49: 103-111Abstract Full Text PDF PubMed Scopus (251) Google Scholar polycystic kidney disease,16.Woo D. Apoptosis and loss of renal tissue in polycystic kidney diseases.N Engl J Med. 1995; 333: 18-25Crossref PubMed Scopus (290) Google Scholar ischemic renal disease,17.Schumer M. Colombel M.C. Sawczuk I.S. et al.Morphologic, biochemical, and molecular evidence of apoptosis during reperfusion phase after brief periods of renal ischemia.Am J Pathol. 1992; 140: 831-838PubMed Google Scholar allograft nephropathy,18.Noronha I.L. Oliveira S.G. Tavares T.S. et al.Apoptosis in kidney and pancreas allograft biopsies.Transplantation. 2005; 79: 1231-1235Crossref PubMed Scopus (24) Google Scholar and diabetes.19.Gilbert R.E. Cooper M.E. The tubulointerstitium in progressive diabetic kidney disease: more than an aftermath of glomerular injury?.Kidney Int. 1999; 56: 1627-1637Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar The concept that apoptosis may mediate tubular atrophy is appealing because, unlike ischemia, which commonly leads to necrosis, apoptosis promotes cell removal with minimal inflammation.20.Nagata S. Apoptosis by death factor.Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4455) Google Scholar The prevalence of apoptotic renal proximal tubular cells (RPTCs) without concomitant RPTC hyperplasia suggests that net RPTC deletion by apoptosis may be important in the pathogenesis of tubular atrophy in chronic kidney diseases. Enhanced reactive oxygen species (ROS) production also has been implicated in the progression of various renal diseases,21.Shimizu M.H. Coimbra T.M. Araujo M. et al.N-acetylcysteine attenuates the progression of chronic renal failure.Kidney Int. 2005; 68: 2208-2217Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 22.Kurata H. Takaoka M. Kubo Y. et al.Protective effect of nitric oxide in ischemia/reperfusion-induced renal injury and endothelin-1 overproduction.Eur J Pharmacol. 2005; 517: 232-239Crossref PubMed Scopus (40) Google Scholar, 23.Sun K. Kiss E. Bedke J. et al.Role of xanthine oxireductase in experimental acute renal-allograft rejection.Transplantation. 2004; 77: 1683-1692Crossref PubMed Scopus (25) Google Scholar including diabetic nephropathy.24.Bhatti F. Mankhey R.W. Asico L. et al.Mechanisms of antioxidant and pro-oxidant effects of α-lipoic acid in the diabetic and non-diabetic kidney.Kidney Int. 2005; 67: 1371-1380Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar In vitro, ROS contribute to apoptosis of podocytes, mesangial and tubular cells exposed to high glucose.25.Verzola D. Bertolotto M.B. Villaggio B. et al.Taurine prevents apoptosis induced by high ambient glucose in human tubule renal cells.J Invest Med. 2002; 50: 443-451Crossref PubMed Google Scholar, 26.Kang B.P. Frencher S. Reddy V. et al.High glucose promotes mesangial cell apoptosis by oxidant-dependent mechanism.Am J Physiol Renal Physiol. 2003; 284: F455-F466Crossref PubMed Scopus (137) Google Scholar, 27.Susztak K. Raff A.C. Schiffer M. et al.Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy.Diabetes. 2006; 55: 225-233Crossref PubMed Scopus (810) Google Scholar Angiotensin II (Ang II) is a potent stimulator of ROS generation by heightened NADPH oxidase activity in mesangial cells and RPTCs, and antioxidants appear to provide renal protection, in part, by ameliorating oxidative stress induced by Ang II.28.Haugen E.N. Croatt A.J. Nath K.A. Angiotensin II induces renal oxidant stress in vivo and heme oxygenase-1 in vivo and in vitro.Kidney Int. 2000; 58: 144-152Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 29.James E.A. Galceran J.M. Rajp L. Angiotensin II induces superoxide anion production by mesangial cells.Kidney Int. 1998; 54: 775-784Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 30.Aigava T. Ishizaka N. Taguchi J. et al.Heme oxygenase-1 is upregulated in the kidney of angiotensin II-induced hypertensive rats: possible role in renoprotection.Hypertension. 2000; 35: 800-806Crossref PubMed Scopus (136) Google Scholar Such observations strongly indicate a causal link between Ang II, ROS, and apoptosis within the kidney. We previously documented that transgenic (Tg) mice overexpressing rat angiotensinogen (Agt), the sole precursor of angiotensins, in their RPTCs are prone to develop hypertension, albuminuria, and renal injury.31.Sachetelli S. Liu Q. Zhang S.-L. et al.Ras blockade decreases blood pressure and proteinuria in transgenic mice overexpressing rat angiotensinogen gene in the kidney.Kidney Int. 2006; 69: 1016-1023Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar This study investigated whether ROS generation mediates the proapoptotic and profibrotic effects engendered by upregulation of the intrarenal renin-angiotensin system (RAS) in proximal tubules in vivo, and whether inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase with apocynin (an inhibitor of NAPDH oxidase) could prevent RPTC apoptosis, independently of systemic hypertension. Tg mice exhibited significantly augmented in ROS generation in their RPTCs (Figure 1a) and increased albumin/creatinine ratios as compared to non-Tgs (Figure 1b), though their kidney/body weight ratios were similar (Figure 1c). Treatment with either apocynin or perindopril, but not with hydralazine, normalized ROS generation and the urinary albumin/creatinine ratio, but had no effect on the kidney/body weight ratio (Figure 1a–c). Mean systolic blood pressure (SBP) in male Tg mice increased from week 11 of life, becoming statistically significant at week 12 and thereafter (Figure 2a). Perindopril and hydralazine (commenced at week 13) normalized the SBP of Tg mice after 1 week of treatment (Figure 2a). In contrast, apocynin did not significantly reduce SBP in Tg mice (Figure 2a and b). Renal damage was evident in Tg mice (Figure 3b) as compared to non-Tg mice (Figure 3a). Histologic findings included vacuoles and loss of the brush border in RPTCs, a large number of detached cells and accumulation of cellular debris in the tubular lumen in Tg kidneys, indicating RPTC damage. Some RPTCs were flattened or atrophied. Treatment with apocynin (Figure 3c) or perindopril (Figure 3d) markedly attenuated these abnormalities in Tg mice, whereas hydralazine (Figure 3e) had minimal effect. RPTC volume was significantly higher in untreated Tg mice than in non-Tg littermates (Figure 3f). Treatment with apocynin, perindopril, or hydralazine effectively attenuated this change. Glomerular volume did not differ significantly between Tg and non-Tg mice, and was unaffected by apocynin, perindopril or hydralazine treatment (Figure 3g). Apoptotic RPTCs were more abundant in Tg (Figure 4b) than non-Tg mice (Figure 4a). Apocynin (Figure 4c) and perindopril (Figure 4d) effectively attenuated RPTC apoptosis, whereas hydralazine was without effect (Figure 4e). Semiquantitative estimation of the number of apoptotic RPTCs confirmed these findings (Figure 4f). Apoptotic RPTCs were detected in at least 16 and 3% of tubules at the glomerulo-tubular (G-T) junction in Tg and non-Tg kidneys, respectively (Figure 4g). Treatment with apocynin or perindopril significantly reduced the number of apoptotic RPTCs at the G-T junction to 4 and 3%, respectively, whereas hydralazine had little effect (14%). Immunohistochemistry revealed increased staining for active caspase-3 (Figure 5b) and Bax (Figure 5g) in RPTCs of Tg mice compared to non-Tg mice (Figure 5a and f). Treatment with apocynin (Figure 5c and h) or perindopril (Figure 5d and i) effectively attenuated these changes, whereas hydralazine had no effect (Figure 5e and j). Similar trends were observed in caspase-3 activity assays (Figure 5k) and western blotting of Bax (Figure 5l) from isolated renal proximal tubules (RPTs). Taken together, these data demonstrate that apocynin and the RAS blocker perindopril effectively prevented RAS-induced RPTC apoptosis, whereas hydralazine had minimal effect. Tg kidneys exhibited enhanced expression of extracellular matrix proteins (Figure 6b), collagenous components (Figure 6g), and immunoreactive collagen type IV (Figure 6l) as compared to the kidneys of non-Tg mice (Figure 6a, f and k). These changes were attenuated by apocynin (Figure 6c, h and m), perindopril (Figure 6d, i and n), and hydralazine (Figure 6e, j and o) as quantitated in sections stained with Masson's trichrome (Figure 6p) or immunoreactive collagen type IV (Figure 6q). Similarly, Tg kidneys also showed higher expression of transforming growth factor-β1 (TGF-β1) and plasminogen activator inhibitor-1 (PAI-1) (Figure 7b and g) than non-Tg kidneys (Figure 7a and f). Treatment with apocynin (Figure 7c and h), perindopril (Figure 7d and i), or hydralazine (Figure 7e and j) attenuated these changes. TGF-β1 and PAI-1 expression was quantitated by western blotting (Figure 7k and l). Bax mRNA expression was significantly higher (Figure 8a), whereas Bcl-xL mRNA expression was significantly lower (Figure 8b) in Tg than in non-Tg RPTs. Treatment with apocynin or perinodpril markedly reversed these changes, whereas hydralazine had no detectable effects. In contrast, all agents attenuated the increases in TGF-β1 mRNA (Figure 8c) and PAI-1 mRNA (Figure 8d) expression in Tg RPTs. ROS generation and NADPH oxidase activity were significantly higher in Tg than in non-Tg RPTs (Figure 9a and b). Treatment with apocynin effectively inhibited ROS generation and NADPH activity in Tg RPTs, but not in non-Tg RPTs. Furthermore, phosphorylated (p)-p47phox and p47phox expression in membrane (Figure 9c) and cytosolic (Figure 9d) fractions of Tg RPTs was significantly higher in Tg RPTs than in non-Tg RPTs. Apocynin effectively attenuated p-p47phox and p47phox expression in membrane fractions of Tg RPTs without affecting non-Tg RPTs (Figure 9c). In contrast, apocynin did not effect p-p47phox and p47phox expression in cytosolic fractions of Tg and non-Tg RPTs (Figure 9d). This study demonstrates that apocynin treatment prevents RPTC apoptosis in Agt-Tg mice independently of systemic hypertension. These findings indicate that ROS generation by intrarenal RAS activation is likely important in the induction of RPTC apoptosis. Our Tg model specifically expresses Agt in RPTCs but not in other tissues.31.Sachetelli S. Liu Q. Zhang S.-L. et al.Ras blockade decreases blood pressure and proteinuria in transgenic mice overexpressing rat angiotensinogen gene in the kidney.Kidney Int. 2006; 69: 1016-1023Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar Our data significantly demonstrate elevated ROS generation in RPTs of these Tg mice compared to non-Tg mice, consistent with previous reports on Ang II induction of ROS generation in rat32.Hsieh T.-J. Fustier P. Wei C.-C. et al.Reactive oxygen species blockade and action of insulin on expression of angiotensinogen gene in proximal tubular cells.J Endocrinol. 2004; 183: 535-550Crossref PubMed Scopus (41) Google Scholar and human RPTCs.33.Bhaskaran M. Reddy K. Radhakrishanan N. et al.Angiotensin II induces apoptosis in renal proximal tubular cells.Am J Physiol Renal Physiol. 2003; 284: F955-F965Crossref PubMed Scopus (133) Google Scholar The observation that apocynin and perindopril blocked ROS generation in RPTs of Tg mice, whereas hydralazine had no effect, indicates that enhanced ROS generation and albuminuria in Tg mice may be attributed to augmented intrarenal RAS activation per se rather than to increased SBP. Unlike hydralazine, both apocynin and perindopril reversed albuminuria in Tg mice. Baseline SBP was significantly higher in Tg than in non-Tg mice. Between weeks 12 and 20, the SBP in Tg mice rose on average by 20 mm Hg (P<0.05) as compared to non-Tg mice (mean SBP was 100 and 120 mm Hg in non-Tg and Tg mice, respectively). Hydralazine and perindopril effectively attenuated SBP after 1 week of treatment. Surprisingly, apocynin did not lower SBP in Agt-Tg mice. Previous studies have reported that apocynin decreased blood pressure in Dahl salt-sensitive hypertensive rats,34.Taylor N.E. Glocka P. Liang M. et al.NADPH oxidase in the renal medulla causes oxidative stress and contributes to salt-sensitive hypertension in Dahl S rats.Hypertension. 2006; 47: 692-698Crossref PubMed Scopus (151) Google Scholar aldosterone-induced hypertensive rats35.Park Y.M. Park M.Y. Suh Y.L. et al.NADPH oxidase inhibitor prevents blood pressure elevation and cardiovascular hypertrophy in aldosterone-infused rats.Biochem Biophys Res Commun. 2004; 313: 812-817Crossref PubMed Scopus (116) Google Scholar and Ang II-induced hypertensive rats.36.Virdis A. Neves M.F. Amiri F. et al.Role of NADPH oxidase on vascular alternations in angiotensin II-infused mice.J Hypertens. 2004; 22: 535-542Crossref PubMed Scopus (198) Google Scholar The reasons for this apparent discrepancy are unknown. One possible explanation is that Ang II derived from Agt in RPTCs (that is, Ang II generated by RPTC renin and angiotensin-converting enzyme in RPTCs37.Tang S.S. Jung F. Diamant D. et al.Temperature-sensitive SV 40 immortalized rat proximal tubule cell line has functional renin-angiotensin system.Am J Physiol. 1995; 268: F435-F446PubMed Google Scholar) might affect SBP through direct vasoconstriction of glomerular arterioles by the Ang II AT1-receptor independently of ROS generation. This possibility is further supported by the observation that hydralazine prevented hypertension in Tg mice without ameliorating intrarenal abnormalities. Another possibility is that apocynin is not effective in blocking the membrane translocation of p-p47phox in glomerular arteriole smooth muscle cells, although it does block the translocation of p-p47phox in RPTs, as seen in our study. Indeed, Heumuller et al.38.Heumuller S. Wind S. Barbosa-Sicard E. et al.Apocynin is not an inhibitor of vascular NADPH oxidase but an antioxidant.Hypertension. 2008; 51: 211-217Crossref PubMed Scopus (590) Google Scholar recently reported that apocynin is not an effective inhibitor of vascular NADPH oxidase due to the absence of myeloperoxidase in vascular smooth muscle cells to convert apocynin into active apocynin dimer. In contrast, myeloperoxidase protein has been detected in RPTs of patients with membranous glomerulopathy,39.Porubsky S. Schmid H. Bonrouhi M. et al.Influence of native and hypochloride-modified low density lipoprotein on gene expression in human proximal tubular epithelium.Am J Pathol. 2004; 164: 2175-2187Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar consistent with formation of active apocynin. Although oxidative stress is associated with hypertension, it remains unclear whether ROS could initiate the development of hypertension. In clinical studies, treatment with antioxidants failed to decrease high blood pressure.40.Heart Protection Study Collaborative Group MRC/BHF Heart protection study of antioxidant vitamin supplementation in 20,536 high-risk individuals: a randomized placebo-controlled trial.Lancet. 2002; 360: 23-33Abstract Full Text Full Text PDF PubMed Scopus (1278) Google Scholar, 41.Kim M.K. Sasaki S. Sasazuki S. et al.Lack of long-term effect of vitamin C supplementation on blood pressure.Hypertension. 2002; 40: 797-803Crossref PubMed Scopus (96) Google Scholar Likewise, apocynin did not affect hypertension in this study but yet attenuated renal ROS generation. Thus, it is conceivable that oxidative stress is not the cause but rather a consequence of hypertension, such that blocking ROS ameliorates the renal lesions from a downstream point. Our data highlight the importance of intrarenal Agt gene expression and ROS generation in mediating RPTC hypertrophy and tubular apoptosis in the mouse kidney. Although the relationship between RPTC hypertrophy and apoptosis is not well understood, it is speculated that low cellular ROS levels induce cell hypertrophy whereas high ROS levels evoke cell apoptosis. Indeed, Griendling's group and others have shown that ROS stimulates both cellular hypertrophy and apoptosis, depending on cellular ROS levels and their molecular species.42.Li P.F. Dietz R. von Harsdorf R. Differential effect of hydrogen peroxide and superoxide anion on apoptosis and proliferation of vascular smooth muscle cells.Circulation. 1997; 96: 3602-3609Crossref PubMed Scopus (260) Google Scholar, 43.Lee M.Y. Griendling K.K. Redox signalling, vascular function, and hypertension.Antioxid Redox Signal. 2008; 10 (review): 1045-1060Crossref PubMed Scopus (186) Google Scholar The numbers of terminal transferase-mediated deoxyuridine triphosphate nick-end-labeling (TUNEL)-positive RPTCs were significantly higher in Tg than in non-Tg mice. These data are consistent with previous reports concerning Ang II induction of RPTC apoptosis both in vitro and in vivo.33.Bhaskaran M. Reddy K. Radhakrishanan N. et al.Angiotensin II induces apoptosis in renal proximal tubular cells.Am J Physiol Renal Physiol. 2003; 284: F955-F965Crossref PubMed Scopus (133) Google Scholar, 44.Aizawa T. Ishizaka N. Kurokawa K. et al.Different effects of angiotensin II and catecholamine on renal cell apoptosis and proliferation in rats.Kidney Int. 2001; 59: 645-653Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar Consistent with the TUNEL assay, RPTs of Tg mice exhibited enhanced active caspase-3 and Bax expression. These changes were attenuated by apocynin and perindopril but not by hydralazine. Heightened Bax expression occurred with concomitant downregulation of the Bcl-xL gene. Apocynin and perindopril but not hydralazine reversed the Bax/Bcl-xL ratio. An increased Bax/Bcl-xL ratio is consistent with promotion of apoptosis, and is a likely mechanism by which intrarenal RAS activation and ROS enhance tubular apoptosis in Tg mice. The precise mechanism(s) by which apocynin prevents Ang II-induced ROS generation and subsequent tubular injury (albuminuria, interstitial fibrosis, and RPTC apoptosis) in Tg mice is far from being fully understood. It has been postulated that apocynin blocks NADPH oxidase assembly by interfering with p-p47phox binding to gp91phox,45.Ximens V.F. Kanegae M.P. Rissato S.R. et al.The oxidation of apocynin catalyzed by myeloperoxidase: proposal for NADPH oxidase inhibition.Arch Biochem Biophys. 2007; 457: 134-141Crossref PubMed Scopus (118) Google Scholar as Ang II activates protein kinase C and subsequently phosphorylates p47phox.46.Mollnau H. Wendt M. Szocs K. et al.Effects of angiotensin II infusion on the expression and function of NADPH oxidase and components of nitric oxide/cGMP signaling.Circ Res. 2002; 90: E58-E65Crossref PubMed Google Scholar Indeed, our data support this notion. Likewise, the mechanism(s) by which ROS induce RPTC apoptosis remains undefined. One possibility is that ROS, through activating p38 mitogen-activated protein kinase signaling, stimulate p53 phosphorylation.47.Perfettini J.L. Castedo M. Nardacci R. et al.Essentail role of p53 phosphorylation by p38 MAPK in apoptosis induction by the HIV-1 envelope.J Exp Med. 2005; 201: 279-289Crossref PubMed Scopus (120) Google Scholar Phosphorylated p53 then translocates to the nucleus and enhances Bax gene transcription.48.Schuler M. Green D.R. Mechanisms of p53-dependent apoptosis.Biochem Soc Trans. 2001; 29: 684-688Crossref PubMed Google Scholar, 49.Punj A. Chakrabarty A.M. Redox proteins in mammalian cell death: an evolutionary conserved function in mitochondria and prokaryotes.Cell Microbiol. 2003; 5: 225-231Crossref PubMed Scopus (40) Google Scholar Bax translocates to the mitochondria and binds the anti-apoptotic proteins Bcl-2 and Bcl-xL, inhibiting their protective actions on gate-keeping and, ultimately, leading to mitochondrial dysfunction and caspase-3 activation. Our data on increased Bax and caspase-3 expression, combined with decreased Bcl-xL expression in RPTs of diabetic Tg mice, lend support to this notion. Furthermore, our data indicate that enhanced intrarenal RAS activation stimulates Bax mRNA and protein expression in Tg mice. The present results may have clinical implications. As tubular apoptosis is detectable in various renal diseases7.Marcussen N. Olsen T.S. Atubular glomeruli in patients with chronic pyelonephritis.Lab Invest. 1990; 62: 467-473PubMed Google Scholar, 8.Markowitz G.S. Radhakrishnan J. Kambham N. et al.Lithium nephrotoxicity: a progressive combined glomerular and tubulointerstitial nephropathy.J Am Soc Nephrol. 2000; 11: 1439-1448PubMed Google Scholar, 9.Benigni A. Gagliardini E. Remuzzi A. et al.Angiotensin-converting enzyme inhibition prevents glomerular-tubule disconnection and atrophy in passive Heymann nephritis, an effect not observed with a calcium antagonist.Am J Pathol. 2001; 159: 1743-1750Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 10.Cosio F.G. Grande J.P. Larson T.S. et al.Kidney allograft fibrosis and atrophy early after living donor transplantation.Am J Transplant. 2005; 5: 1130-1136Crossref PubMed Scopus (106) Google Scholar, 11.Horiguchi H. Oguma E. Kayama F. Cadmium and cisplatin damage erythropoietin-producing proximal renal tubular cells.Arch Toxicol. 2006; 80: 680-686Crossref PubMed Scopus (29) Google Scholar, 12.Kimura M. Asano M. Abe K. et al.Role of atrophic changes in proximal tubular cells in the peritubular deposition of type IV collagen in a rat renal ablation model.Nephrol Dial Transplant. 2005; 20: 1559-1565Crossref PubMed Scopus (35) Google Scholar, 13.Najafian B. Kim Y. Crosson J.T. et al.Atubular glomeruli and glomerulotubular junction abnormalities in diabetic nephropathy.J Am Soc Nephrol. 2003; 14: 908-917Crossref PubMed Scopus (89) Google Scholar, 14.Najafian B. Crosson J.T. Kim Y. et al.Glomerulotubular junction abnormalities are associated with proteinuria in type 1 diabetes.J Am Soc Nephrol. 2006; 17: S53-S60Crossref PubMed Scopus (49) Google Scholar and tubular atrophy appears to be a better indicator of disease progression than glomerular pathology,4.Risdon R.A. Sloper J.C. De Wardener H.E. Relationship between renal function and histological changes found in renal-biopsy specimens from patients with persistent glomerular nephritis.Lancet. 1968; 2: 363-366Abstract PubMed Google Scholar, 19.Gilbert R.E. Cooper M.E. The tubulointerstitium in progressive diabetic kidney disease: more than an aftermath of glomerular injury?.Kidney Int. 1999; 56: 1627-1637Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 50.Marcussen N. Tubulointerstitial damage leads to atubular glomeruli: significance and possible role in progression.Nephrol Dial Transplant. 2000; 15: 74-75Crossref PubMed Scopus (36) Google Scholar, 51.Lindop G.B.M. Gibson I.W. Downie T.T. et al.The glomerulo-tubular junction: a target in renal disease.J Pathol. 2002; 197 (review): 1-3Crossref PubMed Scopus (16) Google Scholar we postulate that RPTC apoptosis may be an initial step leading to tubular atrophy and that ROS is one of the key mediators of this process. In summary, our study indicates an important role for ROS in albuminuria, interstitial fibrosis and RPTC apoptosis in Agt-Tg mice independently of systemic hypertension in
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