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* Department of Pathology, Northwestern University School of Medicine, Chicago, Illinois 60611; Department of Medicine, Okayama University School of Medicine, Okayama, 700-8558 Japan; and Department of Medicine, Northwestern University School of Medicine, Chicago, Illinois 60611
1 To whom requests for reprints should be addressed at Department of Pathology, Northwestern University Medical School, Chicago, IL 60611. E-mail: y-kanwar{at}northwestern.edu
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Key Words: hyperglycemia • advance glycation products • protein kinase C • GTP binding proteins • reactive oxygen species • hypertension
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The activity of transporters in various renal cell types has not been clearly defined (4); nevertheless, upon glucose entry the intracellular signaling events may be similar in most of them. However, one may expect differential handling of glucose intermediaries depending on a given molecule expressed in a particular cell, e.g., aldose reductase and myo-inositol oxidase in the tubular cells and PKC-
and -β isoforms in the glomeruli. In general, the cellular events include increased flux of polyols and hexosamines, generation of advanced glycation end products (AGEs), increased activity of protein kinase C (PKC), TGF-β-Smad-MAPK and G-proteins, altered expression of cyclin kinases and their inhibitors, and of matrix degrading enzymes and their inhibitors with a conceivable common signaling denominator as ROS and a final outcome of increased synthesis and deposition of extracellular matrix (ECM) (5–7). The ROS, whether mitochondrial or cell membrane–derived, may also be responsible for the activation of "renin-angiotensin system" (RAS), contributing further compromise in the renal functions. As suggested by recent studies it is conceivable that there may be a crosstalk between the common metabolic denominator, i.e., ROS and RAS, and both in synchrony may amplify signaling events (8, 9) (Figure 1
), and this review briefly summarizes various pathogenetic mechanisms and pertinent molecules involved in the progression of diabetic nephropathy.
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Relevance of AGEs in the Pathogenesis of Diabetic Nephropathy.
AGEs are heterogeneous groups of macromolecules that are normally formed non-enzymatically by the interaction of reducing sugars with free amino groups of proteins, lipids, and nucleic acids, but their formation increases under high glucose ambience. Initially a labile Schiff base is formed that undergoes a series of chain reactions, i.e., Amadori rearrangement, dehydration, polymerization, and ultimately AGEs’ formation. The AGEs are formed both extracellularly from glucose and intracellularly from various dicarbonyls. The extracellular AGEs interact with their receptor, RAGE, and other binding proteins, i.e., OST-48, 80K-H, galectin-3, and induce various intracellular events (7). The intracellular AGEs also initiate several signaling events by activating PKC, MAP kinase, and transcription factors such as NF-
B. This would increase the activity of various growth factors, such as TGF-β, and thereby alter expression of ECM proteins (15). The latter include increased synthesis of type I and IV collagens, decreased expression of proteoglycans, and anomalous ECM polymerization and expansion. Intriguingly, the AGEs themselves can covalently bind with proteins, thus compounding their deleterious effects in various tissues (15). In addition, there are perturbed cell:matrix interactions, altered adhesiveness, and capillary permeability. These ECM-vascular abnormalities are partially reversed by interrupting AGE:RAGE interactions (16). Another important AGEs-induced cellular event includes formation of reactive oxygen species (ROS) that as well modulates the activity of various kinases and transcription factors that ultimately contribute to ECM pathology (7, 17).
Activation of PKC and Ensuing Various Cellular Signaling Events.
Among various signaling kinases, PKC seems to be a centerpiece in the pathogenesis of diabetic nephropathy (17). Under high glucose ambience it is activated by DAG formed during glycolytic intermediary steps and by ROS generated following AGE:RAGE interactions (7, 18). Such interactions at the cell membrane activate PKC by increasing the activity of phospholipase C with an increase in intracellular Ca2+ and DAG. This cyclic generation of DAG would suggest an intimate "level and activity" relationship between DAG and PKC, and such a parallelism in their expression has been observed in various tissues in diabetes (18). The PKC activation also leads to endothelial dysfunction with decreased nitric oxide production, increased expression of endothelin-1, and vascular endothelial growth factor. This would alter blood flow and capillary permeability that are compounded by increased TGF-β1–induced synthesis of integral ECM proteins of the vasculature (18). At the same time, increased expression of NF-B and PAI-1 would induce a local tissue inflammatory response and thrombotic microangiopathy, thus accentuating the vascular injury, which is further augmented by additional ROS generated by plasmalemmal NADPH oxidoreductase (7). The fact that PKC activation occurs by multiple routes and that the administration of PKC inhibitor, ruboxistaurin mesylate, reduces renal abnormalities in db/db mice suggests its central signaling role in hyperglycemia-induced vascular injury (7, 18).
Cellular Pathways Induced by TGF-β.
The AGEs, ROS, DAG, PKC, and hexosamines are the potential candidates that activate TGF-β signaling (19), while others compounding the hyperglycemic injury include vasoactive substances, i.e, angiotensin II, endothelin, and thromboxane, and cyclical stretch and relaxation of mesangial cells mimicking intraglomerular hypertension (20). The final outcome would be aberrant accumulation of ECM proteins, which is further facilitated by an inhibition of matrix proteases (MMPs) and activation of the corresponding inhibitors (TIMPs). At present, TGF-β is recognized as the major cytokine responsible for ECM pathobiology seen in diabetic nephropathy (21). Initially, TGF-β binds to a type II receptor, which trans-phosphorylates type I serine/threonine kinase receptor. The latter interacts with Smad2 and 3, which then forms a complex with Co-Smad4 (22). Following nuclear translocation of this complex, it binds with promoters of TGF-β target genes, e.g., collagen 1(I), PAI-1, Jun B, c-Jun, and fibronectin and regulates their transcription. These events are negatively regulated by Smad7. Besides Smads, the mitogen activated protein kinases (MAPKs) involved in hyperglycemia-induced TGF-β signaling include extracellular signal regulated kinases 1 and 2 (ERK1 and 2, p44/p42 MAPKs), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38 MAP kinase (22). The MAPKs also modulate transcriptional regulation of ECM genes conceivably via activated protein-1 (AP-1), a heterodimer of c-Fos and c-Jun. Since AP-1, regulated by ERK and JNK, can bind to Smad3 while Smad3:Smad4 complex to AP-1 consensus sequences in various promoters of TGF-β target genes, this would suggest a cross-talk between Smad and MAPK pathways activated by high glucose ambience (22). Another cytokine relevant to TGF-β signaling is connective tissue growth factor (CTGF) that is induced by TGF-β via consensus Smad and transcription enhancer factor (TEF) elements localized within the CTGF promoter (23). The above alluded signaling has been worked out in mesangial cell culture systems, and its in vivo relevance is also well-described in mice models of diabetes, where heightened TGF-β bioactivity has been observed. Moreover, administration of neutralizing anti–TGF-β antibodies prevents renal hypertrophy, mesangial matrix expansion, increased collagen and fibronectin mRNA expression, and deterioration of renal functions in db/db or STZ-induced diabetes in mice (21). Likewise, its upregulated tissue expression and increased urinary excretion of TGF-β in patients with diabetic nephropathy has been observed. Interestingly, angiotensin-converting enzyme inhibitors that ameliorate renal damage concomitantly also lower the TGF-β production, suggesting a relationship of hypertension and ECM pathobiology in diabetes mellitus (20).
Mechanisms Related to Generation of ROS.
The ROS seem to be the common denominators and amplifiers of the above-described signaling cellular pathways activated by hyperglycemia (5, 7, 24). They are continuously produced and degraded normally to maintain homeostasis, but when generated at high concentrations, such as in hyperglycemia, they are capable of inducing injury in various organs (25). The ROS that can induce renal injury include superoxide anion (O2·–), H2O2, hydroxyl radical, and peroxynitrite (26). Their redox balance is maintained by various enzymes, including cytoplasmic Cu/Zn superoxide dismutase (Cu/ZnSOD) and mitochondrial manganese SOD (MnSOD), the most important being heme oxygenase-1(HO-1) that undergoes remarkable induction by hyperglycemia (27). Most of the ROS are generated during mitochondrial oxidative phosphorylation and small amounts via NADPH-oxidase system (28–30).
During oxidative phosphorylation the electron donors generate a high membrane potential by pumping protons across the mitochondrial inner membrane (5, 31). As a consequence, the electron transport is inhibited and the half life of free-radical intermediates of ubiquinone increases, which reduces O2 to O2· – with ensuing oxidant stress. Rotenone, an inhibitor of electron transport, blocks the DCF-sensitive ROS generation, and the fact that a similar effect is observed by overexpression of mitochondrial MnSOD or uncoupling protein-1 (UCP-1) supports this notion (5, 32). Conceivably, the MnSOD or UCP-1 reverses the oxidant stress generated by the high glucose-induced cellular events, including the polyol and hexosamine fluxes, AGE formation, and PKC activation. Whereas, O2· – generated initially (vide supra) inhibits glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that in turn amplifies these cellular events with repetitious generation of ROS (24). Similarly, since gene disruption of GADPH also amplifies the above cellular event it would support the role of mitochondrial ROS in hyperglycemia-induced injury and ECM accumulation. Along these lines, it is interesting to note that the overexpression of extra-mitochondrial cytoplasmic Cu/ZnSOD reduces the glomerular pathophysiologic changes in db/db mice and STZ-induced diabetes (33, 34).
The ROS are generated via NADPH-oxidase system by the interaction of membrane-bound flavocytochrome b558 (heterodimer of gp91phox and p22phox) with various cytosolic proteins (p47phox, p67phos, p40phox, and a GTP-binding protein, p21rac) (29, 35); as a result O2· – is generated, which gets dismutated to H2O2. The relevance of this system in renal pathobiology lies in the fact that Nox 4, a homologue of neutrophil gp91phox, is expressed in the kidney (29). Besides AGEs, PKC, DAG, IP3 (inositol 1, 4, 5-trophosphate), and TGF-β the metabolites of cyclooxygenase (COX) pathway and can also activate NADPH oxidase under high glucose ambience (25, 32). Moreover, decreased fibronectin expression by the inhibitors of oxidase, apocynin, and diphenylene iodonium (DPI) suggests a potential role of NADPH oxidase in hyperglycemic injury and relevance in redox-sensitive processes, i.e., cell growth, apoptosis, migration, and extracellular matrix (ECM) modeling that are modulated by various signaling pathways and transcription and growth factors such as TGF-β. The latter adversely affects the biology of tubular cells with induction of epithelial–mesenchymal transition, and loss of epithelial cell adhesion, -smooth muscle actin expression, cytoskeletal organization, and cell migration through the basement membrane and ensuing of tubulointerstitial fibrosis (36). Other systems in which ROS are generated include nitrosoredox balance, where reactive nitrogen species (RNS), e.g., derivatives of NO, are produced (37). Briefly, NO synthesis is modulated by a cofactor of nitric oxide synthase (NOS), known as tetrahydrobiopterin (BH4) (38). BH4 levels are reduced in high glucose ambience with reduced synthesis of NO by the endothelium, leading to an altered ratio of BH4 and its oxidized form, BH2, with increased generation of superoxide (39). Further support for the role of RNS in pathogenesis of hyperglycemic injury is derived from the studies elucidating an amelioration of endothelial/smooth muscle dysfunction in diabetic rat aortic rings exposed to BH4 (39).
Amplification of Hyperglycemic Injury by Glomerular Capillary Hypertension.
Like the ROS that amplify the hyperglycemic injury, hypertension, or, more importantly, the increased glomerular capillary pressure also significantly contributes to the acceleration of diabetes-related complications, perhaps via a cross-talk between metabolic and hemodynamic factors being operative under high glucose ambience—a concept that has emerged in recent years and deserves much attention (40). The evidence for intraglomerular hypertension was derived from micro-puncture studies in experimental diabetes, and interestingly the systemic blood pressure was found to be normal in these animals (41). This implied that hyperglycemia impairs "autoregulation" of local glomerular microcirculation with dilatation of arterioles, more so of the afferent arteriole, thus affecting the transcapillary hydraulic pressure difference and plasma flow (41). Conceivably, hyperglycemia sensitizes the target organs to blood pressure–induced damage, most likely by activation of renin-angiotensin system (RAS) with local production of angiotensin II (Ang II) in the kidney (9). The support for this notion comes from studies in which lowering of the blood pressure had comparable or even more beneficial effects, e.g., on microalbuminuria, than controlling hyperglycemia (42). The cells that may be involved in the activation of local RAS include proximal tubular epithelia, glomerular mesangial cells, and podocytes; the latter apparently also produce Ang II and express AT1 receptors (43, 44). Hence, it seems that due to some error of evolution the stage is set in which the kidney would be a bidirectional target of hyperglycemia-induced as well as pressure-induced injury in a diabetic state, although one may be a consequence of the other; meaning thereby that inhibition of angiotensin converting enzyme (ACE) would be an integral part of the therapy for amelioration of diabetic nephropathy. In this regard, it is worth mentioning that ACE gene insertion/deletion (I/D) polymorphism studies indicate that individuals with ACE D/D genotype respond poorly to ACE inhibition, suggesting that in this quandary of metabolically induced altered hemodynamics the genetics also have a considerable influence in the ultimate outcome of this disease (45). Along these lines, ACE2 knockout mice have been reported to develop glomerulosclerosis that is reminiscent of diabetic nephropathy (46). Similarly, deficiency of ACE2, a negative regulator of ACE, is associated with a local increase of tubular Ang II and tubulointerstitial fibrosis in long-term experimental diabetes (47).
With respect to the pathogenesis of diabetic nephropathy, the relevant question would be as to what are the mechanism(s) by which glucose sensitizes the kidney to pressure-induced damage via the generation of Ang II, which is central to early hyperplasia and late hypertrophy of the renal cells with upregulation of various cytokines, e.g., TGF-β, CTGF, IL-6, monocyte chemotactic protein-1 (MCP-1), and vascular endothelial growth factor (VEGF), that is followed by accumulation of the ECMs (8, 9, 41). To begin with high glucose has been shown to increase the expression of renin and angiotensinogen (AGT) in mesangial and tubular cells, which could increase intrarenal concentration of Ang II and then via various autocrine and paracrine pathways lead to generation of various cytokines and ECM accumulation (48). Secondly, in review of the metabolic events discussed earlier (vide supra) it is conceivable that glucose-mediated generation of ROS via AGE:RAGE interaction could also upregulate the expression of AGT and Ang II in tubular cells (7, 49). Similarly, infusion of AGEs in vivo results in a remarkable increase in the expression of various components of the RAS (49). Interestingly, the infusion of Ang II increases both serum and renal accumulation of AGEs, thus highlighting autocrinicity and complexity of the events in a diabetic milieu (49). In terms of autocrinicity the subject matter of discussion would be to address the mechanisms by which altered intraglomerular hemodynamics amplify hyperglycemia-induced renal injury. An expected effect of increased capillary pressure would be a stretch-stress on the glomerular cells that parallels to changes in glomerular volume and activation of various signaling pathways.
The mechanical stretch has a profound effect on the pathobiology of mesangial cells. First of all, in vitro studies indicate that such a stress induces expression of GLUT-1, which certainly would lead to an increased cellular concentration of glucose and activation of various pathways (50). Secondly, repetitive cyclic stretch/relaxation of mesangial cells has been shown to enhance their proliferation and increase synthesis of ECM while decreasing expression of ECM-degrading enzymes (51). Increased ECM synthesis may in part be due to the direct mechanical stretch-induced expression of TGF-β and its receptors as well. The TGF-β activation in this scenario could also be due to stretch-induced activation of PKC and p38 MAPK with consequential increased synthesis of fibronectin (51). Ang II mediated stretch-stress in mesangial cells also induces the expression of an inflammatory cytokine, MCP-1 (52). As a consequence an upregulation of intercellular adhesion molecule-1 (ICAM-1) and generation of ROS would be anticipated with amplification of the inflammatory response and renal injury. In support of such anticipation are renal biopsy studies, which at times show an influx of monocytes in patients with diabetic nephropathy (53). The stretch-stress also modulates the biology of glomerular podocytes, conceivably via elaboration of VEGF (54). These podocytes undergo reversible reorganization of the cytoskeletal elements and reduced 3β1 integrin; as a result the cells detach from the underlying GBM and undergo apoptosis (54). Another interesting aspect of the podocyte biology is that the expression of nephrin, a slit membrane protein, is reduced, conceivably via VEGF signaling, with expected increased urinary excretion of proteins in diabetic state (54). Intriguingly enough, decreased expression of nephrin and relative loss of podocytes is also observed in offsprings in states of maternal hyperglycemia, and it has been postulated that this underdosing of nephrons or podocytes during fetal development may be a susceptibility factor to develop hypertension in later adult life (55). Overall, it appears that the metabolic and hemodynamic events are interlinked in states of hyperglycemia with a certain degree of influence of genetic background.
Pathogenetic Mechanisms Related to GTP-Binding Proteins and Cell Cycle Proteins.
The role of GTP binding protein is not well-appreciated, but certainly deserves a brief discussion. Major GTP proteins activated in hyperglycemic state belong to the Ras and Rho family of small GTPases (56). They exert pleiotropic effects, i.e., regulate growth, morphogenesis, cell motility, axonal guidance, cytokinesis, and intracellular trafficking by cycling between inactive (GDP-bound) and active (GTP-bound) state. The Ras and Rho are of special significance since they transduce signals from extracellular stimuli to intracellular signal transduction pathways relevant to the pathobiology of diabetes. For instance, in Ras/Raf/MEK signaling, the Ras acts as an intermediary between the phosphorylated growth factor receptor and MEK (MAP kinase/ERK) activation. Whereas, Rho and Rho-related Rac can directly activate SAPK/JNK and p38 MAPK pathways, respectively (57). The activation of Ras-related Rap1 occurs upon binding with Raf1, which itself is activated by a phosphorylated form of growth factor receptor. The Rap1 can also be induced by intracellular second messengers, e.g., DAG, which can directly or via PKC also activate the Rap/Raf/MAPK pathway under high glucose ambience (58, 59). The activated Rho-induced ECM fibronectin synthesis in renal cells most likely is modulated by the TGF-β–mediated upregulation of CTGF and also by other known profibrogenic molecules, including angiotensin II, PDGF, and endothelin-1 (60). The Rho GTPases–induced SAPK/JNK kinases also activate a number of transcription factors, including cAMP-responsive element binding protein (CREB). Since cis-acting cAMP responsive elements TGACGTCA are present in the fibronectin promoter, the CREB may be the key transcription factor that regulates ECM protein expression in diabetic milieu. Finally, post-translational modifications, such as farnesylation or geranylgeranylation of GTPases, may be responsible for their activation with increased synthesis of ECM proteins (61).
Other cellular processes modulated by GTPases include cellular proliferation by activating SAPK/JNK and p38 MAPK pathways like TGF-β under high glucose ambience (56, 62). The proliferation of mesangial cells, as seen in initial stages of diabetic nephropathy, is modulated by cell cycle proteins, cyclin and cyclin-dependent kinases (CDK), and their inhibitors, CDIs (61, 63, 64). The latter include 2 major groups, i.e., Cip/Kip (p21Cip1and p27Kip1) and 1NK4/ARF (p161NK4 and p14ARF). The expression of p27Kip1 of Cip/Kip is increased in the kidneys of db/db and streptozotocin-induced diabetes in mice and mesangial cells exposed to high glucose ambience. Conceivably, this increased expression is related to p27Kip1 phosphorylation since MAP kinase and PKC are activated in diabetic milieu. Interestingly, gene disruption of p27Kip1 results in decreased protein synthesis, suggesting conversion from hypertrophic to a hyperplastic state of cells exiting the G1 phase of the cell cycle. Similarly, induction of diabetes in p27Kip1–/– or p21Cip1 –/– mice does not exhibit any hypertrophic response, but hyperplasia of renal tubular cells. Typically, the p27Kip1–/– mice do not develop diabetic nephropathy as no renal or glomerular hypertrophy develops (65). With respect to p21Cip1, its decreased expression is associated with reduced CDK-2 and -4 activities and increased expression of Ras and Rho in proliferating cells exposure to high glucose, thus establishing a link between activated GTPases and cell cycle proteins (61).
In summary, mechanisms that are relevant to the pathogenesis of diabetic nephropathy are discussed. They involve various signaling pathways modulated by large number molecules regulating the bioactivity of one another and at times amplifying via autocrine loops with a final outcome of amassing of ECM in various compartments of the kidney as characteristically seen in diabetic nephropathy. A more recent interesting concept that seems to enjoy a universal consensus has emerged which implicates that both the metabolic and hemodynamic events are interwoven together in the pathogenesis of diabetic nephropathy. Still, further advancements are necessary to delineate other cellular pathways modulated by known or unknown genes to gain new insights into its pathogenesis. In this regard, mitochondrial genome needs to be searched for mutations in the genes that could be relevant to ROS and oxidative phosphorylation and perturb the biology of target cells. Similarly, the understudied subject of the role of GTP-binding proteins in the hyperglycemia-induced cellular changes needs further attention as well.
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-dependent pathway. Diabetes 53:2921–2930, 2004.B-dependent MCP-1-mediated pathway in human mesangial cells: inhibition by rosiglitazone. J Am Soc Nephrol 16:688–696, 2005.This article has been cited by other articles:
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  D. Schlondorff and B. Banas The Mesangial Cell Revisited: No Cell Is an Island J. Am. Soc. Nephrol., June 1, 2009; 20(6): 1179 - 1187. [Abstract] [Full Text] [PDF]
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 C. E. Hills, N. Al-Rasheed, N. Al-Rasheed, G. B. Willars, and N. J. Brunskill C-peptide reverses TGF-{beta}1-induced changes in renal proximal tubular cells: implications for treatment of diabetic nephropathy Am J Physiol Renal Physiol, March 1, 2009; 296(3): F614 - F621. [Abstract] [Full Text] [PDF]
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J. H. Garman, S. Mulroney, M. Manigrasso, E. Flynn, and C. Maric Omega-3 fatty acid rich diet prevents diabetic renal disease Am J Physiol Renal Physiol, February 1, 2009; 296(2): F306 - F316. [Abstract] [Full Text] [PDF]
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 B. Laczy, B. G. Hill, K. Wang, A. J. Paterson, C. R. White, D. Xing, Y.-F. Chen, V. Darley-Usmar, S. Oparil, and J. C. Chatham Protein O-GlcNAcylation: a new signaling paradigm for the cardiovascular system Am J Physiol Heart Circ Physiol, January 1, 2009; 296(1): H13 - H28. [Abstract] [Full Text] [PDF]
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R. V. Durvasula and S. J. Shankland Activation of a local renin angiotensin system in podocytes by glucose Am J Physiol Renal Physiol, April 1, 2008; 294(4): F830 - F839. [Abstract] [Full Text] [PDF]
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