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Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice

2007; Elsevier BV; Volume: 71; Issue: 9 Linguagem: Inglês

10.1038/sj.ki.5002188

1523-1755

Brezniceanu, F. Liu, Wei Wei, Stella Tran, Sébastien Sachetelli, Zhang, Deng‐Fu Guo, János G. Filep, Julie R. Ingelfinger, John S.D. Chan,

Chronic Kidney Disease and Diabetes

Increased generation of reactive oxygen species (ROS) leads to oxidative stress in diabetes. Catalase is a highly conserved heme-containing protein that reduces hydrogen peroxide to water and oxygen and is an important factor decreasing cellular injury owing to oxidative stress. Hyperglycemic conditions increase oxidative stress and angiotensinogen gene expression. Angiotensinogen conversion to angiotensin II leads to a furtherance in oxidative stress through increased generation of reactive oxygen species. In this study, we utilized mice transgenically overexpressing rat catalase in a kidney-specific manner to determine the impact on ROS, angiotensinogen and apoptotic gene expression in proximal tubule cells of diabetic animals. Proximal tubules isolated from wild-type and transgenic animals without or with streptozotocin-induced diabetes were incubated in low glucose media in the absence or presence of angiotensin II or in a high-glucose media. Our results show that the overexpression of catalase prevents the stimulation of ROS and angiotensinogen mRNA in tubules owing to elevated glucose or angiotensin II in vitro. Additionally, overexpression of catalase attenuated ROS generation, angiotensinogen and proapoptotic gene expression and apoptosis in the kidneys of diabetic mice in vivo. Our studies point to an important role of ROS in the pathophysiology of diabetic nephropathy. Increased generation of reactive oxygen species (ROS) leads to oxidative stress in diabetes. Catalase is a highly conserved heme-containing protein that reduces hydrogen peroxide to water and oxygen and is an important factor decreasing cellular injury owing to oxidative stress. Hyperglycemic conditions increase oxidative stress and angiotensinogen gene expression. Angiotensinogen conversion to angiotensin II leads to a furtherance in oxidative stress through increased generation of reactive oxygen species. In this study, we utilized mice transgenically overexpressing rat catalase in a kidney-specific manner to determine the impact on ROS, angiotensinogen and apoptotic gene expression in proximal tubule cells of diabetic animals. Proximal tubules isolated from wild-type and transgenic animals without or with streptozotocin-induced diabetes were incubated in low glucose media in the absence or presence of angiotensin II or in a high-glucose media. Our results show that the overexpression of catalase prevents the stimulation of ROS and angiotensinogen mRNA in tubules owing to elevated glucose or angiotensin II in vitro. Additionally, overexpression of catalase attenuated ROS generation, angiotensinogen and proapoptotic gene expression and apoptosis in the kidneys of diabetic mice in vivo. Our studies point to an important role of ROS in the pathophysiology of diabetic nephropathy. Oxidative stress is defined as tissue injury induced by increased reactive oxygen species (ROS) generation. The key initial step in ROS formation is the conversion of molecular oxygen (O2) to superoxide (O2•-). Several enzymatic pathways can generate O2•-, but in quantitative terms, the electron transport chain in mitochondria and nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase in membrane are the most important sources in both physiological and pathological conditions (reviewed in references1.Thannikal V. Fanburg B. Reactive oxygen species in cell signalling.Am J Physiol Lung Cell Mol Physiol. 2000; 279: L1005-L1028PubMed Google Scholar, 2.England K. Cotter T.G. Direct oxidative modifications of signalling proteins in mammalian cells and their effects on apoptosis.Redox Rep. 2005; 10: 237-245Crossref PubMed Scopus (91) Google Scholar, 3.Wardle E.N. Cellullar oxidative processes in relation to renal disease.Am J Nephrol. 2005; 25: 13-22Crossref PubMed Scopus (35) Google Scholar, 4.Nohl H. Gille L. Staniek K. 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Biochemistry of peroxisomes.Annu Rev Biochem. 1992; 61: 157-197Crossref PubMed Scopus (723) Google Scholar In the kidney, CAT is localized predominantly in the cytoplasm of proximal tubules of the juxtamedullary cortex, with a lesser amount in proximal tubules of the superficial cortex. It is not detected in the glomeruli, distal tubules, loop of Henle, and collecting ducts.12.Zhou Z. Kang Y.J. Cellular and subcellular localization of catalase in the heart of transgenic mice.J Histochem Cytochem. 2000; 48: 585-594Crossref PubMed Scopus (67) Google Scholar The role of CAT in defending against cell and tissue injury by oxidative stress has been studied extensively. CAT overexpression renders cells more resistant to H2O2 toxicity and oxidant-mediated injury.13.Yoo J.H. Erzurum S.C. Hay J.G. et al.Vulnerability of the human airway epithelium to hyperoxia. Constitutive expression of the catalase gene in human bronchial cells despite oxidant stress.J Clin Invest. 1994; 93: 297-302Crossref PubMed Google Scholar,14.Brown M.R. Miller Jr, F.J. Li W.G. et al.Overexpression of human catalase inhibits proliferation and promotes apoptosis in vascular smooth muscle cells.Circ Res. 1999; 85: 524-533Crossref PubMed Scopus (182) Google Scholar Transgenic (Tg) mice overexpressing CAT are protected against myocardial injury in hypertension.15.Kang Y.J. Chen Y. Epstein P.N. Suppression of doxorubicin cardiotoxicity by overexpression of catalase in the heart of transgenic mice.J Biol Chem. 1996; 271: 12610-12616Crossref PubMed Scopus (266) Google Scholar,16.Yang H. Roberts L.J. Shi M.J. et al.Retardation of atherosclerosis by overexpression of catalase or both Cu/Zn-superoxide dismustase and catalase in mice lacking apolipoprotein E.Circ Res. 2004; 95: 1075-1081Crossref PubMed Scopus (169) Google Scholar In contrast, CAT knockout or null mice are phenotypically normal,17.Ho Y.S. Xiong Y. Ma W. et al.Mice lacking catalase develop normally but show differential sensitivity to oxidant tissue injury.J Biol Chem. 2004; 279: 32804-32812Crossref PubMed Scopus (277) Google Scholar without those clinical manifestations of acatalasemia as seen in humans including oral ulcers and periodic hemolytic episodes.18.Ogata M. Acatalasemia (review).Hum Genet. 1991; 86: 331-340Crossref PubMed Scopus (81) Google Scholar However, CAT-null mice show differential sensitivity to oxidant-mediated tissue injury.17.Ho Y.S. Xiong Y. Ma W. et al.Mice lacking catalase develop normally but show differential sensitivity to oxidant tissue injury.J Biol Chem. 2004; 279: 32804-32812Crossref PubMed Scopus (277) Google Scholar H2O2 is also known to control cell proliferation via modulation of cell signalling.19.Rhee S.G. Chang T.S. Bae Y.S. et al.Cellular regulation by hydrogen peroxide.J Am Soc Nephrol. 2003; 14: S211-S215Crossref PubMed Google Scholar It is postulated that CAT overexpression might modulate H2O2 levels and thereby alter specific gene expression and cellular function in a tissue-specific manner. Glomerular damage is a hallmark of renal injury in diabetes.20.Drummond K. Mauer M. The early natural history of nephropathy in type 1 diabetes. II. Early renal structure changes in type 1 diabetes.Diabetes. 2002; 51: 1580-1587Crossref PubMed Scopus (232) Google Scholar However, a number of studies reported that tubular atrophy might be a better predictor of renal disease progression than glomerular pathology.21.Schainuck L.I. Striker G.E. Cutler R.E. Benditt E.P. Structural-functional correlations in renal disease.Hum Pathol. 1970; 1: 631-641Abstract Full Text PDF PubMed Scopus (424) Google Scholar, 22.Bohle A. MacKensen-Haen S. Von Gise H. Significance of tubulointerstitial changes in the renal cortex for the excretory function and concentration ability of the kidney: a morphometric contribution.Am J Nephrol. 1988; 7: 421-433Crossref Scopus (261) Google Scholar, 23.Bohle A. Muller G.A. Wehrmann M. et al.Pathogenesis of chronic renal failure in the primary glomerulopathies, renal vasculopathies, and chronic interstitial nephritis.Am J Kidney Dis. 1996; 49: S2-S9Google Scholar Both tubular atrophy and interstitial fibrosis are closely associated with loss of renal function.24.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,25.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 Furthermore, 71% of glomeruli from proteinuric type I diabetic patients have glomerulo-tubular junction abnormalities, including 8–17% atubular glomeruli,26.Najafian B. Kim Y. Crosson J.T. Mauer M. Atubular glomeruli and glomerulotubular junction abnormalities in diabetic nephropathy.J Am Soc Nephrol. 2003; 14: 908-917Crossref PubMed Scopus (89) Google Scholar,27.Najafian B. Crosson J.T. Kim Y. Mauer M. Glomerulotubular junction abnormalities are associated with proteinuria in type 1 diabetes.J Am Soc Nephrol. 2006; 17: S53-S60Crossref PubMed Scopus (49) Google Scholar indicating that proximal tubular atrophy contributes significantly to renal failure in diabetes. The mechanisms underlying tubular atrophy are not well understood. One attractive mechanism is apoptosis, which has been demonstrated to mediate cell death in a variety of renal diseases including diabetes.28.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, 29.Kang B.P. Frencher S. Reddy V. et al.High glucose promotes mesangial cell apoptosis by oxidant-dependent mechanism.Am J Physiol. 2003; 284: F455-F466Crossref PubMed Scopus (137) Google Scholar, 30.Allen D.A. Harwood S. Varagunam M. et al.High glucose-induced oxidative stress causes apoptosis in proximal tubular epithelial cells and is mediated by multiple caspases.FASEB J. 2003; 17: 908-910Crossref PubMed Scopus (187) Google Scholar, 31.Kumar D. Zimpelmann J. Robertson S. Burns K.D. Tubular and interstitial cell apoptosis in the streptozotocin-diabetic rat kidney.Nephron Exp Nephrol. 2004; 96: e77-e88Crossref PubMed Scopus (70) Google Scholar, 32.Kumar D. Robertson S. Burns K.D. Evidence of apoptosis in human diabetic kidney.Mol Cell Biochem. 2004; 259: 67-70Crossref PubMed Scopus (123) Google Scholar, 33.Mishra R. Emancipator S.N. Kern T. Simonson M.S. High glucose evokes an intrinsic proapoptotic signalling pathway in mesangial cells.Kidney Int. 2005; 67: 82-93Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar We reported previously that high glucose evokes ROS generation and enhances angiotensinogen (Agt, the sole substrate of the renin-angiotensin system (RAS)) gene expression in rat renal proximal tubular cells (RPTCs).34.Hsieh T.-J. Zhang S.-L. Filep J.G. et al.High glucose stimulates angiotensinogen gene expression via reactive oxygen species (ROS) generation in rat kidney proximal tubular cells.Endocrinology. 2002; 143: 2975-2985Crossref PubMed Scopus (147) Google Scholar,35.Hsieh T.-J. Fustier P. Wei C.-C. et al.Reactive oxygen species blockade and insulin action on angiotensinogen gene expression in proximal tubular cells.J Endocrinol. 2004; 183: 535-550Crossref PubMed Scopus (41) Google Scholar The objective of this study was to define whether overexpressing CAT in RPTCs could attenuate ROS generation by high glucose and Ang II that lead to increased Agt and proapoptotic gene expression as well as to the induction of RPTC apoptosis in diabetic mice in vivo. For this purpose, we generated Tg mice overexpressing rat CAT (rCAT) in their RPTCs using the kidney-specific androgen-regulated protein (KAP)-gene promoter linked to rCAT cDNA. This promoter targets the gene of interest to proximal tubules, where the transgene will respond to androgen.36.Ding Y. Sigmund C.D. Androgen-dependent regulation of human angiotensinogen expression in KAP-hAGT transgenic mice.Am J Physiol Renal Physiol. 2001; 280: F54-F60PubMed Google Scholar Tg mice were generated to produce specific and inducible expression of rCAT in RPTCs using KAP2-rCAT construct (Figure 1a). Southern blot analysis confirmed the presence of the transgene in heterozygote and homozygote animals (Figure 1b). Figure 1c displays the specific reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of rCAT-HA transgene expression in various tissues of Tg line 688. Male Tg mice express the transgene in the kidney but not in other tissues, and exogenous testosterone further enhances renal transgene expression. An increase in CAT protein and CAT activity in the renal proximal tubules (RPTs) of male Tg mice without exogenous testosterone induction was also apparent in comparison with male wild type (WT) mice (Figure 1d and e). Testosterone administration evoked a three-fold increase in CAT activity in RPTs in female Tg mice (Figure 1e). Taken together, these results confirm that KAP2 directs rCAT transgene expression in RPTs of Tg mice. Thus, in subsequent experiments, we used male Tg mice without exogenous testosterone induction, as endogenous testosterone was sufficient to stimulate rCAT expression and activity in RPTs. To demonstrate rCAT transgene functionality, freshly isolated RPTs were incubated ex vivo for 5 h in normal glucose medium (5 mM D-glucose plus 20 mM D-mannitol) in the absence or presence of Ang II (10−9 mM) or in high glucose medium (25 mM D-glucose). ROS generation was augmented in RPTs of WT mice incubated in normal glucose medium plus (Ang II) or in high glucose medium (Figure 2a). No apparent increment of ROS generation, however, was observed in RPTs from Tg mice stimulated by Ang II or high-glucose. Similarly, Agt mRNA levels were also increased significantly in RPTs of WT mice incubated in high glucose or in normal glucose plus Ang II for 24 h (Figure 2b). The stimulatory effect of high glucose and Ang II on Agt mRNA expression was abolished in RPTs of Tg mice. Western blotting confirmed the effect of high glucose and Ang II on Agt protein expression in RPTs of WT and Tg mice (Figure 2c). These results indicate that ROS may mediate, at least in part, the effect of high glucose and Ang II on Agt mRNA and protein expression in RPTs in vivo. Figure 3 presents the physical and biochemical data on both non-diabetic and diabetic WT and Tg mice. Diabetes in both WT and Tg mice as induced by streptozotocin (STZ) led to blood glucose elevation (Figure 3a), increases in 24-h urinary albumin excretion (Figure 3b), and higher kidney weight/body weight ratios (Figure 3c) compared with non-diabetic animals. Normalization of blood glucose by insulin reversed these parameters to non-diabetic levels. Next, we investigated STZ-induced structural damage to the kidney by standard light microscopy. Glomeruli and RPTs appeared to be hypertrophic in both diabetic WT and Tg animals compared with their non-diabetic controls (Figure 4). Insulin treatment reversed the glomerular and RPT hypertrophy in diabetic WT and Tg mice. Furthermore, an increase of cellular edema and detachment and loss of RPTC brush-border were evident in diabetic WT kidneys. These pathological changes appeared to be attenuated in diabetic Tg kidneys. Insulin treatment ameliorated these pathological changes in both diabetic WT and Tg kidneys. RT-quantitative PCR (RT-qPCR) analysis revealed that Agt mRNA expression is significantly increased (at least four- to five-fold) in RPTs of diabetic WT mice compared with non-diabetic WT mice (Figure 5a). Insulin significantly inhibited RPT Agt mRNA expression in diabetic WT mice but could not normalize to levels as in non-diabetic WT mice. Similarly, Agt protein expression (immunostaining) was increased in diabetic WT mouse kidneys (Figure 5b–g). Insulin treatment attenuated but could not completely prevented RPT Agt expression in diabetic WT mice. In contrast, RPT Agt expression was not increased in diabetic Tg mouse kidneys with or without insulin treatment (Figure 5b–g). Non-immune normal rabbit serum displayed no immunostaining for ANG in both non-Tg and Tg mouse kidneys (data not shown). Semiquantitative estimation of the number of Agt-positive stained tubules confirmed a three-fold increase of Agt-positive tubules in diabetic WT mice compared with non-diabetic WT mice (Figure 5h). Treatment with insulin attenuated but could not completely normalize the number of Agt-positive tubules to levels as in non-diabetics. In contrast, there was no apparent increase of Agt-positive stained tubules in diabetic Tg mice with or without insulin treatment compared with non-diabetic Tg mice. Conventional RT-PCR analysis also showed that plasminogen activator inhibition-1 (PAI-1) and Agt mRNA (Figure 6a and b, left panel) levels in RPTs were significantly increased in diabetic WT mice compared with non-diabetic WT mice. rCAT overexpression prevented the increment of PAI-1 and Agt mRNA in RPTs of diabetic Tg mice (Figure 6a and b, right panel). Insulin treatment was not effective, however, in normalizing PAI-1 and Agt mRNA levels in diabetic WT mouse RPTs (Figure 6a and b, left panel). These studies confirm that hyperglycemia evokes ROS generation (as implied by increased PAI-1 mRNA expression) and subsequently upregulates Agt mRNA expression in diabetic mice RPTs. Figure 7 shows that the expression of p53 and Bax mRNA was significantly elevated in RPTs of diabetic WT compared with non-diabetic WT mice (Figure 7a and b, left panel). Increases in p53 and Bax mRNA levels were prevented in Tg mice overexpressing rCAT in their RPTs (Figure 7a and b, right panel). Insulin treatment was effective, however, in diminishing Bax mRNA but not p53 mRNA levels in diabetic WT mouse RPTs to controls. To investigate whether the caspase-3 cascade was activated in diabetic RPTs, caspase-3 mRNA and activity were analyzed by RT-PCR and activity assay, respectively. Figure 8a reveals that caspase-3 mRNA expression was significantly increased in RPTs of diabetic WT compared with non-diabetic WT mice. Insulin treatment normalized the caspase-3 mRNA level. Furthermore, a significant increase in caspase-3 activity was observed in RPTs of diabetic WT mice (Figure 8b). In contrast, there were no apparent increases in caspase-3 mRNA levels and activity in diabetic Tg mice (Figure 8a and b). Active caspase-3 and apoptosis in mouse kidneys were examined by immunohistochemistry and TUNEL assay, respectively. Active caspase-3 was present in RPTCs as well as in distal tubules (DT) of diabetic mice (Figure 9b) but not in non-diabetic WT and Tg mice (Figure 9a and c) and diabetic Tg mice (Figure 9d). Furthermore, TUNEL assay revealed positively stained nuclei in RPTCs as well as in distal tubules of diabetic WT mice (Figure 10b and c) but not in RPTCs of non-diabetic WT and Tg mice (Figure 10a and d) and diabetic Tg mice (Figure 10e and f). These data demonstrate that apoptosis is clearly induced in RPTs and DT of diabetic mice and that CAT overexpression prevents RPTC apoptosis in diabetes. We report here that rCAT overexpression in RPTs of Tg mice prevents ROS generation, Agt and proapoptotic gene expression as well as RPT apoptosis in diabetes, indicating occurrence of oxidative stress in mouse RPTs during early phases of diabetes. Consistently, upregulation of CAT reverses oxidative stress and RPT apoptosis in vivo. To demonstrate that the antioxidant enzyme CAT can prevent ROS generation and attenuate kidney injury in diabetic mouse RPTs, Tg mice overexpressing rCAT specifically in their RPTs were generated. Tg line 688 showed that the transgene was highly expressed in male kidneys with or without exogenous testosterone induction but was not expressed in other tissues. Protein expression and activity of the transgene was confirmed by respective Western blotting and activity assay in isolated mouse RPTs. CAT activity was at least three- to four-fold higher in RPTs of Tg compared WT. Thus, these findings are consistent with our previous report as well as others that KAP2 directs the transgene expression in RPTCs.36.Ding Y. Sigmund C.D. Androgen-dependent regulation of human angiotensinogen expression in KAP-hAGT transgenic mice.Am J Physiol Renal Physiol. 2001; 280: F54-F60PubMed Google Scholar,37.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 High glucose levels evoke ROS generation and enhance Agt gene expression in rat and mouse RPTC lines in vitro.34.Hsieh T.-J. Zhang S.-L. Filep J.G. et al.High glucose stimulates angiotensinogen gene expression via reactive oxygen species (ROS) generation in rat kidney proximal tubular cells.Endocrinology. 2002; 143: 2975-2985Crossref PubMed Scopus (147) Google Scholar, 35.Hsieh T.-J. Fustier P. Wei C.-C. et al.Reactive oxygen species blockade and insulin action on angiotensinogen gene expression in proximal tubular cells.J Endocrinol. 2004; 183: 535-550Crossref PubMed Scopus (41) Google Scholar, 38.Brezniceanu M.-L. Wei C.-C. Zhang S.-L. et al.Transforming growth factor-beta 1 stimulates angitoensinogen gene expression in kidney proximal tubular cells.Kidney Int. 2006; 69: 1977-1985Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 39.Hannken T. Schroeder R. Zahner G. et al.Reactive oxygen species stimulate p44/42 mitogen-activated protein kinase and induce p27Kip1: role in angiotensin II-mediated hypertrophy of proximal tubular cells.J Am Soc Nephrol. 2000; 11: 1387-1397Crossref PubMed Google Scholar Consistently, high glucose and Ang II evoked ROS generation in RPTs of WT mice ex vivo. Most importantly, there was no apparent increase of ROS generation in RPTs of Tg male mice when stimulated with high glucose or Ang II. Furthermore, Agt mRNA levels were significantly elevated in WT RPTs incubated in high glucose or stimulated with Ang II, compared with RPTs incubated in normal glucose medium. rCAT overexpression completely abolished the increment of Agt mRNA in Tg RPTs stimulated by high glucose and Ang II. Western blotting confirmed the high glucose and Ang II upregulation of Agt protein expression in RPTs of WT but not Tg. Taken together, these observations suggest that rCAT overexpression in Tg RPTs is effective in preventing ROS generation stimulated by high glucose or Ang II and consequently abrogates Agt gene expression. STZ is effective in inducing diabetes, with renal injury including renal hyperperfusion, albuminuria, glomerular and RPT hypertrophy, glomerular basement thickening and extracellular matrix protein gene expression in the mouse.40.Tay Y.C. Wang Y. Kairaitis L. et al.Can murine diabetic nephropathy be separated from superimposed acute renal failure.Kidney Int. 2005; 68: 391-398Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar,41.Breyer M.D. Bottinger E. Brosius F.C. et al.Mouse models of diabetic nephropathy.J Am Soc Nephrol. 2005; 16: 27-45Crossref PubMed Scopus (412) Google Scholar After 2 weeks of induction with STZ, all mice exhibited increased blood glucose, increased kidney to body weight ratio and proteinuria (a marker of kidney injury42.Johnson C.A. Levey A.S. Coresh J. et al.Clinical practice guidelines for chronic kidney disease in adults: Part II. Glomerular filtration rate, proteinuria, and other markers.Am Fam Physician. 2004; 70: 1091-1097PubMed Google Scholar). These parameters were not ameliorated by rCAT overexpression in RPTs of Tg mice, but these were completely abrogated by insulin treatment. Thus, our data indicate that rCAT overexpression in RPTCs alone was not sufficient to prevent the physiological changes seen in the diabetic kidney. We would suggest that the increased kidney/body weight ratio and albuminuria were because of the diabetic state per se and not to a nephrotoxic effect of STZ, as normalization of blood glucose by insulin reversed these parameters. Indeed, periodic acid schiff (PAS) staining of kidney sections from non-diabetic and diabetic mice (both WT and Tg mice) revealed structural damage to the kidney in STZ-treated animals, including glomerular and tubular hypertrophy, loss of brush border, cellular edema and detachment. These pathological changes were ameliorated with insulin treatment in both WT and Tg kidneys. PAI-1 is overexpressed in pathological conditions associated with renal fibrosis, including diabetic nephropathy.43.Mottonen J. Strand A. Symersky J. et al.Structural basis of latency in plasminogen activator inhibitor-1.Nature. 1992; 355: 270-273Crossref PubMed Scopus (516) Google Scholar, 44.Niedbala M.J. Picarella M.S. Tumor necrosis factor induction of endothelial cell urokinase-type plasminogen activator mediated proteolysis of extracellular matrix and its antagonism by gamma-interferon.Blood. 1992; 79: 678-687PubMed Google Scholar, 45.Sawdey M.S. Loskutoff D.J. Regulation of murine type 1 plasminogen activator inhibitor gene expression in vivo. Tissue specificity and induction by lipopolysaccharide, tumor necrosis factor-alpha, and transforming growth factor-alpha.J Clin Invest. 1991; 88: 1346-1353Crossref PubMed Scopus (360) Google Scholar ROS mediate high glucose-induced up-regulation of PAI-1 expression in cultured mesangial cells and diabetic glomeruli.46.Jiang Z. Seo J.Y. Ha H. et al.Reactive oxygen species mediate TGF-beta 1-induced plasminogen activator inhibitor-1 upregulation in mesangial cells.Biochem Biophys Res Commun. 2003; 309: 961-966Crossref PubMed Scopus (96) Google Scholar,47.Lee H.B. Ha H. Plasminogen activator inhibitor-1 and diabetic nephropathy.Nephrology. 2005; 10: S11-S13Crossref PubMed Scopus (51) Google Scholar Thus, PAI-1 is a useful marker of ROS-inducible gene. PAI-1 and Agt mRNA expression were increased in RPTs of diabetic WT mice. Furthermore, the increase of PAI-1 and Agt mRNA was abolished in RPTs of male Tg mice, providing further evidence that ROS mediate the effect of high glucose-induced expression of Agt gene and RAS activation. It is noteworthy that insulin treatment did not completely normalize Agt protein as well as PAI-1, ANG and p53 mRNA levels in diabetic WT mice. These data are consistent with our previous observations that prolonged exposure to high glucose evokes insulin resistance on inhibition of Agt mRNA expression in RPTCs,35.Hsieh T.-J. Fustier P. Wei C.-C. et al.Reactive oxygen species blockade and insulin action on angiotensinogen gene expression in proximal tubular cells.J Endocrinol. 2004; 183: 535-550Crossref PubMed Scopus (41) Google Scholar and hyperglycemia induces insulin resistance on Agt gene expression in diabetic rat RPTCs.48.Zhang S.-L. Chen X. Hsieh T.-J. et al.Hyperglycemia induces insulin resistance on angiotensinogen gene expression in diabetic rat kidney proximal tubular cells.J Endocrinol. 2002; 172: 333-344Crossref PubMed Scopus (32) Google Scholar Although the exact molecular mechanism(s) of high-glucose induction of insulin resistance remains uncertain, persistent ROS generation might be involved.35.Hsieh T.-J. Fustier P. Wei C.-C. et al.Reactive oxygen species blockade and insulin action on angiotensinogen gene expression in proximal tubular cells.J Endocrinol. 2004; 183: 535-550Crossref PubMed Scopus (41) Google Scholar High glucose is a potent inducer of apoptosis in RPTCs via ROS generation and activation of multiple caspases.28.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, 29.Kang B.P. Frencher S. Reddy V. et al.High glucose promotes mesangial cell apoptosis by oxidant-dependent mechanism.Am J Physiol. 2003; 284: F455-F466Crossref PubMed Scopus (137) Google Scholar, 30.Allen D.A. Harwood S. Varagunam M. et al.High glucose-induced oxidative stress causes apoptosis in proximal tubular epithelial cells and is mediated by multiple caspases.FASEB J. 2003; 17: 908-910Crossref PubMed Scopus (187) Google Scholar, 31.Kumar D. Zimpelmann J. Robertson S. Burns K.D. Tubular and interstitial cell apoptosis in the streptozotocin-diabetic rat kidney.Nephron Exp Nephrol. 2004; 96: e77-e88Crossref PubMed Scopus (70) Google Scholar, 32.Kumar D. Robertson S. Burns K.D. Evidence of apoptosis in