Podocytes as a target of prorenin in diabetes

High plasma prorenin levels predict the onset of microvascular complications, such as albuminuria/proteinuria,in diabetic patients. In diabetic rats with elevated plasma prorenin levels, treatment with HRP, which competitively inhibits the binding of prorenin to the (pro)renin receptor [(P)RR] as a decoy peptide, significantly prevented the development of albuminuria/proteinuria and glomerulosclerosis, suggesting that (P)RR-bound prorenin plays a significant role in the pathogenesis of diabetic nephropathy. Recently, the presence of (P)RR in podocytes, which represent one of the glomerular filtration barriers, has been reported. Although podocytes are subjected to both high glucose levels and mechanical stretching caused by glomerular hyperfiltration under diabetic conditions, the expression of (P)RR is reportedly regulated by high glucose levels in in vitro mesangial cells and the in vivo kidneys of diabetic rats, whereas mechanical stretching is up-regulated by (P)RR expression in human podocytes. In addition, prorenin treatment not only leads to the generation of intracellular angiotensin (Ang)II, but also activates the phosphorylation of ERK via (P)RR in a manner that acts independently of AngII in human podocytes. Thus, the upregulation of prorenin and (P)RR in podocytes as a result of glomerular hyperfiltration might play an important role in the development of albuminuria/proteinuria via the generation of intracellular AngII and the stimulation of (P)RR-dependent intracellular signals. Further inquiry regarding podocyte (P)RR intracellular signal transduction will be needed to develop a new therapeutic approach targeting podocyte (P)RR in patients with diabetic nephropathy.     

Effects of High Glucose on AVP-Induced Hyperplasia, Hypertrophy, and Type IV Collagen Synthesis in Cultured Rat Mesangial Cells

Introduction. Hyperglycemia is a principal characteristic of diabetes and influences many cellular functions. Diabetic nephropathy is characterized by glomerular mesangial expansion which could result from increased mesangial cell extracellular matrix synthesis induced by hyperglycemia. Methods. To investigate whether the physiological functions of mesangial cells are altered in a diabetic environment, we evaluated the effect of high extracellular glucose concentration on thymidine/leucine incorporation, hyperplasia/hypertrophy, and type IV collagen synthesis, induced by vasopressin (AVP), in cultured rat mesangial cells. Results. The exposure of mesangial cells to a high glucose concentration (30 mM) significantly reduced AVP-induced thymidine incorporation and hyperplasia compared with normal glucose (10 mM). By contrast, treatment of mesangial cells with AVP in the presence of high extracellular glucose significantly increased leucine incorporation, hypertrophy, and type IV collagen synthesis compared with those at normal glucose levels. The administration of staurosporine, a protein kinase C inhibitor, reversed these effects of high-glucose conditions. Furthermore, the nonpeptide AVP V(1A) receptor-selective antagonists potently inhibited these AVP-induced physiological responses in mesangial cells cultured in high-glucose conditions. Conclusions. These results demonstrate that high glucose suppresses mesangial cell proliferation but enhances hypertrophy and type IV collagen synthesis induced by AVP. This increased mesangial cell hypertrophy and extracellular matrix synthesis may play a crucial role in the glomerular mesangial expansion common to diabetic nephropathy.     

Cell biology of diabetic nephropathy: Roles of endothelial cells,tubulointerstitial cells and podocytes

Diabetic nephropathy is the major cause of end‐stage renal failure throughout the world in both developed and developing countries. Diabetes affects all cell types of the kidney, including endothelial cells, tubulointerstitial cells, podocytes and mesangial cells. During the past decade, the importance of podocyte injury in the formation and progression of diabetic nephropathy has been established and emphasized. However, recent findings provide additional perspectives on pathogenesis of diabetic nephropathy. Glomerular endothelial damage is already present in the normoalbuminuric stage of the disease when podocyte injury starts. Genetic targeting of mice that cause endothelial injury leads to accelerated diabetic nephropathy. Tubulointerstitial damage, previously considered to be a secondary effect of glomerular protein leakage, was shown to have a primary significance in the progression of diabetic nephropathy. Emerging evidence suggests that the glomerular filtration barrier and tubulointerstitial compartment is a composite, dynamic entity where any injury of one cell type spreads to other cell types, and leads to the dysfunction of the whole apparatus. Accumulation of novel knowledge would provide a better understanding of the pathogenesis of diabetic nephropathy, and might lead to a development of a new therapeutic strategy for the disease.     

Transgenic overexpression of brain natriuretic peptide prevents the progression of diabetic nephropathy in mice

Aims/hypothesis Brain natriuretic peptide (BNP) is a potent vasorelaxing and natriuretic peptide that is secreted from the heart and has cardioprotective properties. We have previously generated hypotensive transgenic mice (BNP-Tg mice) that overproduce BNP in the liver, which is released into the circulation. Using this animal model, we successfully demonstrated the amelioration of renal injury after renal ablation and in proliferative glomerulonephritis. Glomerular hyperfiltration is an early haemodynamic derangement, representing one of the key mechanisms of the pathogenesis of diabetic nephropathy. Based on the suggested involvement of increased endogenous natriuretic peptides, the aim of this study was to investigate their role in the development and progression of diabetic nephropathy. Materials and methods We evaluated the progression of renal injury and fibrogenesis in BNP-Tg mice with diabetes induced by streptozotocin. We also investigated the effect of BNP on high glucose-induced signalling abnormalities in mesangial cells. Results After induction of diabetes, control mice exhibited progressively increased urinary albumin excretion with impaired renal function, whereas these changes were significantly ameliorated in BNP-Tg mice. Notably, diabetic BNP-Tg mice revealed minimal mesangial fibrogenesis with virtually no glomerular hypertrophy. Glomerular upregulation of extracellular signal-regulated kinase, TGF-β and extracellular matrix proteins was also significantly inhibited in diabetic BNP-Tg mice. In cultured mesangial cells, activation of the above cascade under high glucose was abrogated by the addition of BNP. Conclusions/interpretation Chronic excess of BNP prevents glomerular injury in the setting of diabetes, suggesting that renoprotective effects of natriuretic peptides may be therapeutically applicable in preventing the progression of diabetic nephropathy.     

Rosiglitazone enhances glucose uptake in glomerular podocytes using the glucose transporter GLUT1

Aims/hypothesis  Peroxisome proliferator-activated receptor (PPAR) γ agonists are used increasingly in the treatment of type 2 diabetes. In the context of renal disease, PPARγ agonists reduce microalbuminuria in diabetic nephropathy; however, the mechanisms underlying this effect are unknown. Glomerular podocytes are newly characterised insulin-sensitive cells and there is good evidence that they are targeted in diabetic nephropathy. In this study we investigated the functional and molecular effects of the PPARγ agonist rosiglitazone on human podocytes. Methods  Conditionally immortalised human podocytes were cultured with rosiglitazone and functional effects were measured with glucose-uptake assays. The effect of rosiglitazone on glucose uptake was also measured in 3T3-L1 adipocytes, nephrin-deficient podocytes, human glomerular endothelial cells, proximal tubular cells and podocytes treated with the NEFA palmitate. The role of the glucose transporter GLUT1 was investigated with immunofluorescence and small interfering RNA knockdown and the plasma membrane expression of GLUT1 was determined with bis-mannose photolabelling. Results  Rosiglitazone significantly increased glucose uptake in wild-type podocytes and this was associated with translocation of GLUT1 to the plasma membrane. This effect was blocked with GLUT1 small interfering RNA. Nephrin-deficient podocytes, glomerular endothelial cells and proximal tubular cells did not increase glucose uptake in response to either insulin or rosiglitazone. Furthermore, rosiglitazone significantly increased basal and insulin-stimulated glucose uptake when podocytes were treated with the NEFA palmitate. Conclusions/interpretation  In conclusion, rosiglitazone has a direct and protective effect on glucose uptake in wild-type human podocytes. This represents a novel mechanism by which PPARγ agonists may improve podocyte function in diabetic nephropathy.     

Increased glomerular cytosolic phospholipase A2 activity of OLETF rats with early diabetes.

In view of the potential role of prostaglandins (PGs) in development of glomerular hyperfiltration leading to diabetic nephropathy, we studied the temporal relationship of the activity of cytosolic phospholipase A2 (cPLA2), a rate-limiting enzyme for eicosanoid biosynthesis, with hyperfiltration and the histological changes in glomeruli using OLETF rats, a model for non-insulin-dependent diabetes mellitus (NIDDM). Diabetes mellitus and associated histopathological changes, which developed spontaneously by 30-46 weeks after birth of OLETF rats, were accompanied by approximately 65% increase in glomerular cPLA2 activity that showed significant correlations with elevated plasma glucose levels and creatinine clearance. Moreover, mesangial cells cultured for 5 days with high glucose exhibited approximately 2-fold higher cPLA2 activity than those cultured with physiologic level of glucose. These data suggest that increased glomerular cPLA2 activity leads to production of PGs, which may promote the progression of early diabetic glomerular hyperfiltration and subsequent diabetic nephropathy.     

Expression of vascular endothelial growth factor in response to high glucose in rat mesangial cells

Diabetic nephropathy associated with hyperglycemia is characterized by glomerular hyperfiltration and endothelial dysfunction. Vascular endothelial growth factor (VEGF) is known to be primarily involved in neoangiogenesis and increased endothelial permeability. The purpose of this study was to investigate VEGF expression in response to high glucose in rat cultured mesangial cells and to identify its signal pathway via protein kinase C (PKC). Rat mesangial cells were cultured with different concentrations of glucose: normal (5 mM d-glucose), medium (15 mM d-glucose) and high (30 mm d-glucose). Calphostin-C as a PKC inhibitor and phorbol myristate acetate (PMA) as a PKC downregulator were instillated into culture media to evaluate the role of PKC in mediating the glucose-induced increase in VEGF expression. High glucose increased expression of VEGF at the mRNA and protein levels, identified by semi-quantitative RT-PCR and western blotting, within 3 h and in a time- and glucose concentration-dependent manner. Calphostin-C and PMA inhibited glucose-induced increases in VEGF expression at the mRNA and protein levels. In conclusion, high glucose can directly increase VEGF expression in rat mesangial cells via a PKC-dependent mechanism. These results suggest that VEGF could be a potential mediator of glomerular hyperfiltration and proteinuria in diabetic nephropathy.     

Pathogenesis of diabetic nephropathy: the role of oxidative stress and protein kinase C

Hyperglycemia, a well recognized pathogenetic factor of long-term complications in diabetes mellitus, not only generates more reactive oxygen species but also attenuates antioxidative mechanisms through glycation of the scavenging enzymes. Therefore, oxidative stress has been considered to be a common pathogenetic factor of the diabetic complications including nephropathy. A causal relationship between oxidative stress and diabetic nephropathy has been established by observations that (1) lipid peroxides and 8-hydroxydeoxyguanosine, indices of oxidative tissue injury, were increased in the kidneys of diabetic rats with albuminuria; (2) high glucose directly increases oxidative stress in glomerular mesangial cells, a target cell of diabetic nephropathy; (3) oxidative stress induces mRNA expression of TGF-beta1 and fibronectin which are the genes implicated in diabetic glomerular injury, and (4) inhibition of oxidative stress ameliorates all the manifestations associated with diabetic nephropathy. Proposed mechanisms involved in oxidative stress associated with hyperglycemia are glucose autooxidation, the formation of advanced glycosylation end products, and metabolic stress resulting from hyperglycemia. Since the inhibition of protein kinase C (PKC) effectively blocks not only phorbol ester-induced but also high glucose- and H2O2-induced fibronectin production, the activation of PKC under diabetic conditions may also have a modulatory role in oxidative stress-induced renal injury in diabetes mellitus.     

小鼠肾小球系膜细胞葡萄糖转运蛋白的鉴定及其功能研究

目的：研究葡萄糖转运蛋白１（ｇｌｕｃｏｓｅｔｒａｎｓｐｏｒｔｏｒ，ＧＬＵＴ１）在肾小球系膜细胞上的表达与功能，探讨共在糖尿病肾病发病机制中的作用。方法：ＧＬＵＴ１ｍＲＮＡ蛋白质表达及其功能的检测分别采用ＲＴ－ＰＣＲ，细胞免疫荧光染色，流式细胞仪分析技术２－Ｄｅｏｘｙ－（＾３Ｈ）－Ｄ－Ｇｌｕｃｏｓｅ摄入法与根皮素竞争性抑制实验。结果：证实小鼠肾小球系膜细胞ＧＬＵＴ１ｍＲＮＡ和蛋白质的表达并表肯定了系     

Glomerular expression of thrombospondin-1, transforming growth factor beta and connective tissue growth factor at different stages of diabetic nephropathy and their interdependent roles in mesangial response to diabetic stimuli

Aims/hypothesis We quantified the glomerular expression of thrombospondin-1 (THBS1, also known as TSP-1), transforming growth factor beta 1 (TGFB1, also known as TGF-β1) and connective tissue growth factor (CTGF) at each stage of diabetic nephropathy. We also examined the roles of THBS1 and CTGF in mediating high-glucose- and glycated-albumin-induced synthesis of the matrix protein, fibronectin, by mesangial cells. Methods THBS1, latent and active TGFB1, and CTGF, were detected by immunohistochemistry and in situ hybridisation in biopsies from 19 insulin-dependent diabetic patients with incipient, manifest and advanced diabetic nephropathy, and in 11 control kidneys. Findings were quantified by image analysis. Human mesangial cells were cultured with normal or high glucose, albumin or glycated albumin (Amadori product), +/−THBS1 or CTGF antisense oligonucleotides, or with peptide W, an inhibitor of TGFB1 bioactivation by THBS1. Proteins were measured by western blot analysis or ELISA. Results In glomeruli of normal kidneys, mRNA and protein levels for THBS1, latent-TGFB1 and CTGF were low. They were increased in the incipient stage of diabetic nephropathy, predominantly in mesangial areas, with further increases at later stages of the disease. Little or no active TGFB1 immunostaining was detected prior to manifest diabetic nephropathy. In contrast to high-glucose conditions, increases in fibronectin synthesis that were stimulated by glycated albumin were not dependent on THBS1 activation of latent TGFB1. However, increased fibronectin synthesis in both conditions required CTGF. Conclusions/interpretation Increased glomerular expression of all three factors occurs from the earliest stage of diabetic nephropathy. In contrast to THBS1, CTGF is required for mesangial synthesis of fibronectin stimulated by high glucose or glycated albumin, and is thus a potential therapeutic target. N. A. Wahab and L. Schaefer made an equal contribution to the work reported in this paper     

Pathophysiological role of Amadori-glycated proteins in diabetic microangiopathy

Early and advanced nonenzymatic glycation of proteins are increased in diabetes. Although Amadori-glycated proteins are the major glycated modifications, most studies so far have focused on the role of advanced glycation end-products (AGEs) in diabetes-related vascular complications. It was only recently that the role of Amadori-glycated proteins has come under consideration. Here we review data that point to an important role of Amadori-modified glycated serum proteins in diabetic microangiopathy. Amadori-glycated albumin induces the activation of glomerular mesangial and endothelial cells to a phenotype that may be linked to the pathogenesis of diabetic microangiopathy, that is, by the stimulation of protein kinase C, activation of transforming growth factor beta, and the expression of extracellular matrix proteins. In type 1 diabetic patients, levels of Amadori-glycated proteins are independently associated with nephropathy and retinopathy. Reduction of Amadori-glycated albumin levels in diabetic animal models ameliorates the progression of nephropathy and retinopathy, indicating a causal role of Amadori-glycated proteins in the pathogenesis of diabetic nephropathy and retinopathy. Based on these data, inhibition of Amadori-glycated albumin may be a target for reduction of diabetic vascular complications.     

Molecular bases of diabetic nephropathy

The determinant of the diabetic nephropathy is hyperglycemia, but hypertension and other genetic factors are also involved. Glomerulus is the focus of the injury, where mesangial cell proliferation and extracellular matrix occur because of the increase of the intra- and extracellular glucose concentration and overexpression of GLUT1. Sequentially, there are increases in the flow by the poliol pathway, oxidative stress, increased intracellular production of advanced glycation end products (AGEs), activation of the PKC pathway, increase of the activity of the hexosamine pathway, and activation of TGF-beta1. High glucose concentrations also increase angiotensin II (AII) levels. Therefore, glucose and AII exert similar effects in inducing extracellular matrix formation in the mesangial cells, using similar transductional signal, which increases TGF-beta1 levels. In this review we focus in the effect of glucose and AII in the mesangial cells in causing the events related to the genesis of diabetic nephropathy. The alterations in the signal pathways discussed in this review give support to the observational studies and clinical assays, where metabolic and antihypertensive controls obtained with angiotensin-converting inhibitors have shown important and additive effect in the prevention of the beginning and progression of diabetic nephropathy. New therapeutic strategies directed to the described intracellular events may give future additional benefits.     

Pravastatin suppress superoxide and fibronectin production of glomerular mesangial cells induced by oxidized-LDL and high glucose.

Pravastatin is a potent inhibitor of HMG-CoA reductase and is effective in lowering serum lipid levels. Recent studies have shown that pravastatin also reduces oxidative modification of LDL and decreases albuminuria in patients with diabetes. To determine the possible benefit of pravastatin on the diabetic kidney, we have measured the effects of pravastatin on the proliferation and the production of superoxide and fibronectin, and the expression of fibronectin mRNA of glomerular mesangial cells stimulated by oxidized-LDL and high glucose. Our results demonstrated that the [(3)H]-labeled thymidine uptake of mesangial cells decreased after oxidized-LDL stimulation (50 microg/ml, 6 h) and increased after high glucose stimulation (25 mM, 48 h). The production of superoxide and fibronectin and the expression of fibronectin mRNA of glomerular mesangial cells were all significantly increased after stimulation with either oxidized-LDL or high glucose, or the combination of oxidized-LDL and high glucose. Pravastatin (100 microM, 48 h) alone had no effect on unstimulated cells. However, pravastatin significantly reversed thymidine uptake, inhibited the production of superoxide and fibronectin, and inhibited the expression of fibronectin mRNA of glomerular mesangial cells after stimulation with either oxidized-LDL or high glucose. Our results indicate that pravastatin may effect as an antioxidant and may suppress fibronectin synthesis of glomerular mesangial cells in diabetic patients with hyperlipidemia.     

Three-dimensional architecture of glomerular extracellular matrices in diabetic glomerulosclerosis

The three-dimensional ultrastructural changes of the glomerular extracellular matrices in diabetic glomerulosclerosis were studied in acellular rat and human diabetic glomeruli by scanning electron microscopy. The mesangial matrix appeared as fenestrated septa with oval stomata between the glomerular capillaries in normal control specimens. In diabetic glomerulosclerosis, both in humans and rats, expansion of mesangial matrix and narrowing of the mesangial fenestrae were observed. A thin layer of the mesangial matrix extended into the peripheral glomerular basement membrane (GBM) subendothelially. Thickening of the GBM in diabetic nephropathy might be due to expansion of the mesangial matrix into the peripheral GBM.     

Mesangial cells in diabetes

The glomerular mesangium is the site of prominent structural lesions in diabetic nephropathy, including excess deposition of extracellular matrix in a focal, nodular pattern. Increased single-nephron blood flow and filtration are early signs of glomerular involvement in diabetes, and may initiate or contribute to mesangial damage. Vasodilatation results from arteriolar insensitivity to vasoconstrictors. In turn, this may reflect primary metabolic defects of glomerular smooth muscle, including the afferent arteriole and mesangial cells themselves. Parallel alterations in the glomerular basement membrane and related structures, such as mesangial matrix, are likely to result from glycosylation of intrinsic proteins, or accumulation of advanced glycosylation end-products. Structural and haemodynamic changes account for (micro)-albuminuria, with additional possible overloading of the measangium. Mesangial proliferative changes eventually ensue, with excess matrix deposition and progressive fibrosis. Recent evidence that long-standing hyperglycaemia modifies mesangial metabolism, sensitivity to vasoconstrictors and matrix biosynthesis in vitro is reviewed here, in the light of its potential implications for experimental and human diabetic nephropathy.     

亚硒酸钠对大鼠肾小球系膜细胞表达环氧化酶2的影响

分别以高葡萄糖、高胰岛素、过氧化氢和糖基化终产物孵育大鼠肾小球系膜细胞（RGMC），均可使RGMC环氧化酶（COX）-2mRNA和蛋白表达增加（均P〈0．01）；亚硒酸钠预处理后可抑制上述4种因素诱导的COX-2 mRNA和蛋白表达。亚硒酸钠可能通过抑制COX-2在RGMC的表达，从而在防治糖尿病肾病发生发展过程中发挥重要作用。     

Alterations in the expression of the a3b1 integrin in certain membrane domains of the glomerular epithelial cells (podocytes) in diabetes mellitus

Summary In view of the major alterations which take place at the level of the extracellular matrix of the glomerular wall in diabetes mellitus and the key roles played by β ₁ integrins in cell-to-matrix interactions, it is imperative to understand the role played by integrins in the development of diabetic glomerulosclerosis. In the present study, we revealed by immunocytochemistry the ultrastructural distribution of the α₃ β ₁ at the level of the plasma membrane of the different renal glomerular cells from short- and long-term diabetic rats. For the endothelial cells, the labelling present on both the luminal and abluminal plasma membranes was low. For the podocyte epithelial cells, the labelling was present on both the luminal and basal plasma membranes, the former being concentrated at points of contact between podocyte foot processes. The labelling on the basal plasma membrane was more significant and similar in domains facing either the glomerular basement membrane or the mesangial matrix. The plasma membrane of mesangial cells also exhibited α₃ β ₁. The labelling was recorded under diabetic conditions, at the same sites, with similar intensities, alongside that of the basal plasma membrane of podocytes facing the glomerular basement membrane, the density of which decreased significantly. This decrease in labelling was similar in renal tissues from short- and long-term diabetic animals. These results demonstrate that α₃ β ₁ present at the podocyte basal plasma membrane facing the glomerular basement membrane, which undergoes important alterations in diabetes, could be involved in the major dysfunctions of the glomerular wall characteristic of diabetic glomerulosclerosis. Since the changes in integrin were found to occur as early as after 1 month of hyperglycaemia, when morphological alterations of the glomerular basement membrane are not yet established, we propose that they constitute an early event which precedes the onset of diabetic nephropathy. [Diabetologia (1997) 40: 15–22] Received: 20 June 1996 and in revised form: 24 September 1996     

Immunohistochemical characterization of glomerular PDGF B-chain and PDGF beta-receptor expression in diabetic rats

Platelet-derived growth factor (PDGF) was found to contribute to the pathophysiological process in the development and progression of glomerulosclerosis characterized by mesangial cell proliferation and accumulation of extracellular matrix. To examine the role of PDGF in the development of diabetic nephropathy, we conducted immunohistochemical analysis for PDGF B-chain (PDGF-B) and PDGF beta-receptor (PDGFR-beta) in the glomeruli of streptozotocin-induced diabetic rats. At 2, 4, and 12 weeks after the onset of diabetes, the expression of PDGF-B in glomeruli of diabetic rats was increased significantly as compared to control or diabetic rats treated with insulin. Similar changes were observed on PDGFR-beta immunostaining. The immunostaining of mirror sections revealed the existence of PDGF-B or PDGFR-beta not only in mesangial cells but also in visceral epithelial cells. Glomerular volume was significantly increased in diabetes. This early glomerular abnormality was prevented by an inhibition of PDGF system with trapidil as well as by the treatment of insulin. Our results suggest that the activation of the PDGF system in glomerular cells might play an important role in the development of early glomerular lesion in diabetes.