Diabetic nephropathy and proximal tubule ROS: Challenging our glomerulocentricity
2007; Elsevier BV; Volume: 71; Issue: 12 Linguagem: Inglês
10.1038/sj.ki.5002286
ISSN1523-1755
Autores Tópico(s)Advanced Glycation End Products research
ResumoDiabetic nephropathy is currently viewed as a predominantly glomerular process with glomerular injury driving secondary tubular loss. Brezniceanu and colleagues apply transgenic methods to support a prominent role for reactive oxygen species as mediators and for the proximal tubule as a major site of early disease activity in diabetes. Results support evidence for early tubular apoptosis and atrophy in human diabetic nephropathy. Diabetic nephropathy is currently viewed as a predominantly glomerular process with glomerular injury driving secondary tubular loss. Brezniceanu and colleagues apply transgenic methods to support a prominent role for reactive oxygen species as mediators and for the proximal tubule as a major site of early disease activity in diabetes. Results support evidence for early tubular apoptosis and atrophy in human diabetic nephropathy. Diabetic nephropathy has long been considered virtually synonymous with glomerulosclerosis, the latter viewed as the cardinal manifestation and primary lesion of this functionally devastating disease. Recent gentle challenges to our collective dogmatism on this topic have emerged, but the paper by Brezniceanu and colleagues1.Brezniceanu M.-L. Liu F. Wei C.-C. et al.Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice.Kidney Int. 2007; 71: 912-923Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar (this issue) takes a giant step toward shifting the glomerulo-tubular balance in the field. These authors have made elegant use of transgenic technology to achieve selective overexpression of catalase in the renal proximal tubular epithelial cell (RPTC). Catalase, an enzyme that breaks down H2O2 to inactive components, is used as a tool to reduce generation of reactive oxygen species (ROS) and thereby probe their function. Using the RPTC-specific, androgen-regulated KAP promoter to drive transgenic catalase expression, Brezniceanu and colleagues1.Brezniceanu M.-L. Liu F. Wei C.-C. et al.Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice.Kidney Int. 2007; 71: 912-923Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar found that adult males exhibited spontaneous overexpression of catalase protein and activity in RPTCs, without the addition of exogenous androgen. The investigators accordingly used the wild-type and transgenic adult male mice to examine the role of ROS in early streptozotocin (STZ)-induced diabetic nephropathy, specifically asking whether limiting ROS generation attenuates selected diabetes-induced abnormalities in the proximal tubule: increased angiotensinogen (Agt), plasminogen activator inhibitor-1, apoptosis, and histologic injury following 2 weeks of diabetes. The emphasis by Brezniceanu and colleagues1.Brezniceanu M.-L. Liu F. Wei C.-C. et al.Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice.Kidney Int. 2007; 71: 912-923Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar on the proximal tubule rather than the glomerulus is the first of several surprises. A handful of recent studies have in fact documented prominent tubular atrophy in both human and experimental diabetes. In eight type 1 diabetics with stage I–II chronic kidney disease, Najafian et al.2.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 found by stereologic methods that atubular glomeruli and glomeruli attached to atrophic tubules made up 68% of all glomeruli, while fractional volumes of atrophic tubules were increased and those of normal tubules were reduced. Also in human diabetic kidneys, Kumar et al.3.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 demonstrated increased apoptosis localized to tubular and interstitial cells but, interestingly, not present in the glomerulus; a similar pattern was found in STZ-induced diabetic nephropathy in rats.4.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 Collectively, these reports point to a previously unrecognized atrophic process in the diabetic tubule and suggest that specifically tubular apoptosis may contribute. The choice by Brezniceanu and colleagues1.Brezniceanu M.-L. Liu F. Wei C.-C. et al.Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice.Kidney Int. 2007; 71: 912-923Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar to monitor expression of Agt in the proximal tubule reflects the recent recognition of an independently regulated intrarenal renin–angiotensin system (RAS) localized to the RPTC. The classic circulating RAS is based on renin secretion by the juxtaglomerular cells of the afferent arteriole; circulating renal renin in turn requires circulating Agt from liver and angiotensin-converting enzyme at the endothelial surface to effect angiotensin II (Ang II) production. The intrarenal RAS, on the other hand, appears to be self-contained, as all components required for Ang II generation can be synthesized within the RPTC.5.Ichihara A. Kobori H. Nishiyama A. Navar L.G. Renal renin-angiotensin system.Contrib Nephrol. 2004; 143: 117-130Crossref PubMed Scopus (56) Google Scholar In contrast to the negative feedback exerted by Ang II on juxtaglomerular-cell renin secretion, the intrarenal RAS operates via a positive-feedback response to Ang II.6.Navar L.G. Nishiyama A. Why are angiotensin concentrations so high in the kidney.Curr Opin Nephrol Hypertens. 2004; 13: 107-115Crossref PubMed Scopus (93) Google Scholar In addition, Ang II in the tubular lumen is also actively taken up into RPTCs, in part by angiotensin receptor type 1 binding/internalization.7.Zhuo J.L. Imig J.D. Hammond T.G. et al.Ang II accumulation in rat renal endosomes during Ang II-induced hypertension: role of AT(1) receptor.Hypertension. 2002; 39: 116-121Crossref PubMed Scopus (121) Google Scholar Once inside, Ang II stimulates increased RPTC expression of both Agt and renin, and thus a feed-forward effect promoting more Ang II formation. By these processes, chronic exogenous Ang II infusion leads to increased Ang II in kidney cortex and specifically in the renal interstitial fluid.8.Nishiyama A. Seth D.M. Navar L.G. Angiotensin II type 1 receptor-mediated augmentation of renal interstitial fluid angiotensin II in angiotensin II-induced hypertension.J Hypertens. 2003; 21: 1897-1903Crossref PubMed Scopus (50) Google Scholar Renal interstitial Ang II levels have been found to be many-fold higher than plasma levels and to be regulated independently of plasma Ang II.9.Nishiyama A. Seth D.M. Navar L.G. Renal interstitial fluid angiotensin I and angiotensin II concentrations during local angiotensin-converting enzyme inhibition.J Am Soc Nephrol. 2002; 13: 2207-2212Crossref PubMed Scopus (86) Google Scholar,10.Nishiyama A. Seth D.M. Navar L.G. Renal interstitial fluid concentrations of angiotensins I and II in anesthetized rats.Hypertension. 2002; 39: 129-134Crossref PubMed Scopus (191) Google Scholar Recent studies also show that transgenic overexpression of Agt confined to the RPTC is alone sufficient to induce systemic hypertension,11.Davisson R.L. Ding Y. Stec D.E. et al.Novel mechanism of hypertension revealed by cell-specific targeting of human angiotensinogen in transgenic mice.Physiol Genomics. 1999; 1: 3-9Crossref PubMed Scopus (102) Google Scholar,12.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 lending support in principle to the concept that intrarenal RAS activation dominates extrarenal regulators of blood pressure. Of particular relevance to the article by Brezniceanu and colleagues,1.Brezniceanu M.-L. Liu F. Wei C.-C. et al.Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice.Kidney Int. 2007; 71: 912-923Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar these Agt overexpression studies demonstrate that Agt excess alone appears capable of initiating intrarenal RAS activation. The intrarenal RAS has generated intense interest in the setting of diabetes, where it was first appreciated that an activated intrarenal RAS can coexist with a suppressed circulating RAS. In experimental diabetes, investigators have subsequently confirmed an increase in intrarenal Ang II levels,13.Awad A.S. Webb R.L. Carey R.M. Siragy H.M. Increased renal production of angiotensin II and thromboxane B2 in conscious diabetic rats.Am J Hypertens. 2005; 18: 544-548Crossref PubMed Scopus (11) Google Scholar together with increased renin14.Zimpelmann J. Kumar D. Levine D.Z. et al.Early diabetes mellitus stimulates proximal tubule renin mRNA expression in the rat.Kidney Int. 2000; 58: 2320-2330Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar and increased Agt15.Hsieh T.J. Zhang S.L. Filep J.G. et al.High glucose stimulates angiotensinogen gene expression via reactive oxygen species generation in rat kidney proximal tubular cells.Endocrinology. 2002; 143: 2975-2985Crossref PubMed Scopus (147) Google Scholar in the early stages of diabetic nephropathy. There is, additionally, solid evidence that both high glucose15.Hsieh T.J. Zhang S.L. Filep J.G. et al.High glucose stimulates angiotensinogen gene expression via reactive oxygen species generation in rat kidney proximal tubular cells.Endocrinology. 2002; 143: 2975-2985Crossref PubMed Scopus (147) Google Scholar and elevated Ang II16.Hannken T. Schroeder R. Zahner G. et al.Reactive oxygen species stimulate p44/42 mitogen-activated protein kinase and induce p27(Kip1): role in angiotensin II-mediated hypertrophy of proximal tubular cells.J Am Soc Nephrol. 2000; 11: 1387-1397Crossref PubMed Google Scholar — considered the two most potent pathophysiologic agonists in diabetic nephropathy — induce increased ROS generation in proximal tubular and mesangial cells in vitro, and that ROS excess may in turn mediate tissue injury and apoptosis. However, we have not had evidence that can assign priority or establish a molecular sequence for how these signaling elements interact in vivo. Brezniceanu and colleagues1.Brezniceanu M.-L. Liu F. Wei C.-C. et al.Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice.Kidney Int. 2007; 71: 912-923Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar designed the transgenic model noted above to test in vivo the importance of ROS in mediating early diabetic nephropathy. The authors first clearly demonstrate that catalase overexpression is confined to the proximal tubule and is functional in situ: isolated renal proximal tubules from the male transgenic mice exhibited virtually no increase in ROS and no increase in Agt mRNA or protein expression in response to either Ang II or high glucose, in contrast to the vigorous responses in wild-type renal proximal tubules. Turning next to the STZ-diabetic state, Brezniceanu and colleagues1.Brezniceanu M.-L. Liu F. Wei C.-C. et al.Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice.Kidney Int. 2007; 71: 912-923Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar show similar outcomes in isolated renal proximal tubules from STZ-diabetic transgenic versus diabetic wild-type mice: catalase overexpression prevented the diabetes-induced increases in Agt expression, in the proinflammatory ROS-dependent plasminogen activator inhibitor-1, in apoptosis-inducing enzyme caspase-3 and p53, and in tubular-cell apoptosis. Finally, by standard renal histologic and immunochemical approaches, the authors confirmed in vivo by TdT-mediated dUTP nick end labeling that catalase overexpression prevented increases in RPTC Agt protein, apoptotic enzymes, and apoptosis in the STZ-diabetic transgenic kidneys. The molecular targets were chosen for their known sensitivity to ROS, underscoring the conclusion that constrained ROS generation in fact mediated the outcomes. Thus, we can reasonably predict that the processes represented by these molecules — specifically, intrarenal RAS activation, inflammation, and apoptosis — are downstream consequences of increased oxidative stress in early diabetic nephropathy. The effects of transgenic catalase overexpression in diabetic RPTCs are equally interesting for what was not blocked. Proteinuria in diabetic transgenic mice persisted at levels comparable to those in diabetic wild-type mice. This is perhaps a reflection of the preserved hyperglycemia with its expected effects at glomerular sites. It is nonetheless a reminder that excess protein continued to bombard the catalase-overexpressing RPTCs in the diabetic transgenic mice. Hyperglycemia enhances tubular uptake of albumin17.Drumm K. Lee E. Stanners S. et al.Albumin and glucose effects on cell growth parameters, albumin uptake and Na(+)/H(+)-exchanger Isoform 3 in OK cells.Cell Physiol Biochem. 2003; 13: 199-206Crossref PubMed Scopus (23) Google Scholar and induces RPTC surface expression of CD36, mediating increased uptake of advanced glycosylation endproducts- modified albumin.18.Susztak K. Ciccone E. McCue P. et al.Multiple metabolic hits converge on CD36 as novel mediator of tubular epithelial apoptosis in diabetic nephropathy.PLoS Med. 2005; 2: e45Crossref PubMed Scopus (137) Google Scholar It is interesting to speculate that the favorable results of limiting ROS in RPTCs might in part reflect prevention of the inflammatory and proapoptotic effects of excess RPTC albumin uptake.19.Abbate M. Zoja C. Remuzzi G. How does proteinuria cause progressive renal damage?.J Am Soc Nephrol. 2006; 17: 2974-2984Crossref PubMed Scopus (544) Google Scholar If true, this would predict an additional contribution of proteinuria-related injury in the early phase of diabetic nephropathy and raises the question of ROS-dependence of protein-induced tubulointerstitial injury. Figure 1 proposes one way of integrating the findings of Brezniceanu and colleagues1.Brezniceanu M.-L. Liu F. Wei C.-C. et al.Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice.Kidney Int. 2007; 71: 912-923Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar and others in the field in a molecular scheme; major weight is given to intrarenal RAS activation and to the feed-forward, autoamplification features of this system in the RPTC. Another intriguing observation by Brezniceanu and colleagues1.Brezniceanu M.-L. Liu F. Wei C.-C. et al.Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice.Kidney Int. 2007; 71: 912-923Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar is the apparent failure of catalase overexpression to block the RPTC hypertrophy in diabetic transgenic mice over the 2 weeks of STZ-diabetes. This also comes as a surprise. Ang II has been reported to induce RPTC hypertrophy via ROS-dependent pathways.20.Hannken T. Schroeder R. Stahl R.A. Wolf G. Angiotensin II-mediated expression of p27Kip1 and induction of cellular hypertrophy in renal tubular cells depend on the generation of oxygen radicals.Kidney Int. 1998; 54: 1923-1933Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar Additionally, in prior work by Brezniceanu and colleagues in RPTC cell culture, hyperglycemia-induced hypertrophy was prevented by Ang II receptor blockade,21.Zhang S.L. To C. Chen X. et al.Effect of renin-angiotensin system blockade on the expression of the angiotensinogen gene and induction of hypertrophy in rat kidney proximal tubular cells.Exp Nephrol. 2001; 9: 109-117Crossref PubMed Scopus (34) Google Scholar suggesting that diabetic hypertrophy is Ang II dependent. On the basis of these observations, catalase overexpression would be expected to reduce diabetes-associated RPTC hypertrophy. The unexpected persistence of RPTC hypertrophy perhaps reflects the greater complexity of the in vivo state, which cannot be fully modeled in the cell-culture setting. One is left to conclude that mechanisms of hyperglycemia-induced hypertrophy remain far from clear. Yet diabetic tubular hypertrophy may well have exceptionally important consequences. Recent studies by Thomson and colleagues have led to an equally tubulocentric proposal: that diabetic tubular hypertrophy is the primary event in diabetic nephropathy and in fact causes glomerular hyperfiltration.22.Thomson S.C. et al.Kidney function in early diabetes: the tubular hypothesis of glomerular filtration.Am J Physiol Renal Physiol. 2004; 286: F8-F15Crossref PubMed Scopus (182) Google Scholar These investigators propose that proximal hypertrophy, via increased proximal sodium reabsorption and reduced sodium delivery to the macula densa, triggers a tubuloglomerular feedback response that initiates and sustains diabetic hyperfiltration.22.Thomson S.C. et al.Kidney function in early diabetes: the tubular hypothesis of glomerular filtration.Am J Physiol Renal Physiol. 2004; 286: F8-F15Crossref PubMed Scopus (182) Google Scholar The findings of Brezniceanu and colleagues1.Brezniceanu M.-L. Liu F. Wei C.-C. et al.Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice.Kidney Int. 2007; 71: 912-923Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar appear to exclude a role for ROS in mediating diabetic RPTC hypertrophy. Studies by Thomson and colleagues provide evidence that ornithine decarboxylase is essential to hyperglycemia-induced RPTC hypertrophy.23.Thomson S.C. Deng A. Bao D. et al.Ornithine decarboxylase, kidney size, and the tubular hypothesis of glomerular hyperfiltration in experimental diabetes.J Clin Invest. 2001; 107: 217-224Crossref PubMed Scopus (187) Google Scholar Taken together, these findings suggest it is likely that neither Ang II nor ROS are etiologic, whereas ornithine decarboxylase remains in the running as a mediator of diabetic tubular hypertrophy. It will be important to learn in the present model whether persistence of diabetic hypertrophy over a longer period leads to adverse consequences despite the catalase-induced protection from excess tubular ROS generation. There are, as always, caveats to ponder. First, the diagnosis of hypertrophy is based primarily on kidney weight–body weight ratio; Brezniceanu and colleagues1.Brezniceanu M.-L. Liu F. Wei C.-C. et al.Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice.Kidney Int. 2007; 71: 912-923Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar did not quantitate RPTC hypertrophy at a cellular level by either cell size or protein–DNA ratio. It thus remains possible that the apparent renal enlargement may reflect edema in diabetic transgenic mice, a histopathologic feature in fact noted by the investigators. If so, resolution of hypertrophy might be masked. Second, endogenous androgens are important, not only postpubertally, but also during fetal development (for example, in sex differentiation); catalase overexpression in fetal male RPTCs during development could induce programming events that permanently change proximal tubular function and confound results in unexpected ways. Thus, as usual, differing approaches to independently confirm these findings will be important. Because these observations cover a very early phase of disease, examination of ROS roles at later stages will also be crucial in developing rational therapeutic interventions. Nonetheless, these remarkable findings provide compelling support for the in vivo participation of ROS in the early phase of diabetic nephropathy and for the proximal tubule as a key site of early ROS-induced disease activity. That tubular processes leading to apoptosis and atrophy in the proximal tubule may drive loss of renal function independently of glomerular disease activity would be, if confirmed, a paradigm shift in our currently glomerulocentric view of diabetic nephropathy.
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