Growth hormone induces transforming growth factor-β1 in podocytes: Implications in podocytopathy and proteinuria

Pituitary growth hormone (GH) is essential for growth, metabolism, and renal function. Overactive GH signaling is associated with impaired kidney function. Glomerular podocytes, a key kidney cell type, play an indispensable role in the renal filtration and express GH receptors (GHR), suggesting the direct action of GH on these cells. However, the precise mechanism and the downstream signaling events by which GH leads to diabetic nephropathy remain to be elucidated. Here we performed proteome analysis of the condition media from human podocytes and confirmed that GH-induces TGF-β1. Inhibition of GH/GHR stimulated-JAK2 signaling abrogates GH-induced TGF-β1 secretion. Mice administered with GH showed glomerular manifestations concomitant with proteinuria. Pharmacological inhibition of TGF-βR1 in mice prevented GH-induced TGF-β dependent SMAD signaling and proteinuria. Conditional deletion of GHR in podocytes protected mice from streptozotocin-induced diabetic nephropathy. GH and TGF-β1 signaling components expression was elevated in the kidneys of human diabetic nephropathy patients. Our study identifies that GH induces TGF-β1 in podocytes, contributing to diabetic nephropathy.     

整合素相关激酶在糖尿病肾病的表达及其意义

目的探讨整合素相关激酶(Integrin-Linked Kinase,ILK)在糖尿病肾病患者肾组织中的表达及其意义.方法对3例正常肾组织,14例糖尿病肾病患者肾穿刺活检标本,应用免疫组织化学方法检测ILK和FN在肾组织的阳性表达强度,并作图像分析处理.结果在正常肾组织,ILK主要表达于肾小球脏层上皮细胞,系膜细胞和小管上皮细胞呈弱表达.在糖尿病肾病,ILK表达于肾小球脏层上皮细胞和系膜细胞,在萎缩变性的肾小管上皮细胞表达增强.在肾小球结节硬化时,ILK表达明显减少.此外,ILK和FN的表达量在糖尿病肾病早、中期成正相关(P<0.001),在糖尿病肾病晚期成负相关(P<0.05).结论 ILK在糖尿病肾病肾组织中表达量显著增加,并与FN的表达有一定的相关性,说明其可能通过促进细胞外基质FN等的积聚,在糖尿病肾小球硬化过程中发挥重要作用.     

Altering expression of alpha3beta1 integrin on podocytes of human and rats with diabetes

The adhesion molecule integrin alpha3beta1 is the major receptor of podocyte to the glomerular capillary basement membrane (GBM). Since progressive alteration of the glomerular extracellular matrix (ECM) compartment leading to GBM thickening is common in diabetic nephropathy, we investigated the cellular distribution of alpha3beta1 integrin in podocytes of patients with diabetic nephropathy and streptozotocin-induced diabetic rats, and we evaluated the effects of high glucose on the cultured rat podocytes. Both human and rat kidneys were stained using the immunoelectron microscopy and immunoperoxidase technique with mouse monoclonal antibodies to human integrin alpha3 subunit. The results showed that both the number of immunogold particles and the staining of integrin alpha3 subunit on podocytes were weaker in patients with diabetic nephropathy than those of control kidneys. The staining of alpha3 on podocytes in the poorly-controlled diabetic rats was also weaker after one and three months of hyperglycemia. However, the staining was identical to controls in rats with only one week of hyperglycemia. High glucose (25 mM) but not streptozotocin in vitro suppressed the alpha3 expression of cultured rat podocytes. Our results demonstrated that the expression of integrin alpha3beta1 on podocytes was suppressed in both human and rats with diabetes, possibly due to the effects of hyperglycemia, and the suppression became more severe with the duration of diabetes.     

Beyond proteinuria: VDR activation reduces renal inflammation in experimental diabetic nephropathy

Local inflammation is thought to contribute to the progression of diabetic nephropathy. The vitamin D receptor (VDR) activator paricalcitol has an antiproteinuric effect in human diabetic nephropathy at high doses. We have explored potential anti-inflammatory effects of VDR activator doses that do not modulate proteinuria in an experimental model of diabetic nephropathy to gain insights into potential benefits of VDR activators in those patients whose proteinuria is not decreased by this therapy. The effect of calcitriol and paricalcitol on renal function, albuminuria, and renal inflammation was explored in a rat experimental model of diabetes induced by streptozotocin. Modulation of the expression of mediators of inflammation by these drugs was explored in cultured podocytes. At the doses used, neither calcitriol nor paricalcitol significantly modified renal function or reduced albuminuria in experimental diabetes. However, both drugs reduced the total kidney mRNA expression of IL-6, monocyte chemoattractant protein (MCP)-1, and IL-18. Immunohistochemistry showed that calcitriol and paricalcitol reduced MCP-1 and IL-6 in podocytes and tubular cells as well as glomerular infiltration by macrophages, glomerular cell NF-κB activation, apoptosis, and extracellular matrix deposition. In cultured podocytes, paricalcitol and calcitriol at concentrations in the physiological and clinically significant range prevented the increase in MCP-1, IL-6, renin, and fibronectin mRNA expression and the secretion of MCP-1 to the culture media induced by high glucose. In conclusion, in experimental diabetic nephropathy VDR activation has local renal anti-inflammatory effects that can be observed even when proteinuria is not decreased. This may be ascribed to decreased inflammatory responses of intrinsic renal cells, including podocytes, to high glucose.     

Morphological changes in the rat kidney following long-term diabetes

The morphological basis of diabetic nephropathy has been studied using light and electron microscopy. Kidneys of streptozotocin-induced diabetic rats were examined on the light microscope at 4 weeks and 8 months after induction of diabetes mellitus. In addition, the 8-month diabetic kidneys were examined with the electron microscope. Renal hypertrophy was evidenced by the increase in the weight of kidneys of diabetic rats. Whilst the diabetic kidneys were approximately twice as large after 4 weeks they were only 30% larger compared to age-matched controls after 8 months of induction of diabetes. After 4 weeks, light microscopy revealed dilated tubules within the cortex of the diabetic kidneys. Light microscopy showed a significant amount of destruction of the distal convoluted tubules while electron microscopy revealed a spectrum of damage that included basement membrane thickening, loss of podocytic foot processes, disruption of tubular basal infoldings and their related mitochondria and fibrosis of the tubules 8 months after induction of diabetes. It is concluded that renal hypertrophy persists after a prolonged occurrence of diabetes but the extensive damage and loss of renal tissue including the loss of the foot processes of podocytes might be partly responsible for the clinical presentation of diabetic nephropathy.     

Growth hormone induces mitotic catastrophe of glomerular podocytes and contributes to proteinuria

Glomerular podocytes are integral members of the glomerular filtration barrier in the kidney and are crucial for glomerular permselectivity. These highly differentiated cells are vulnerable to an array of noxious stimuli that prevail in several glomerular diseases. Elevated circulating growth hormone (GH) levels are associated with podocyte injury and proteinuria in diabetes. However, the precise mechanism(s) by which excess GH elicits podocytopathy remains to be elucidated. Previous studies have shown that podocytes express GH receptor (GHR) and induce Notch signaling when exposed to GH. In the present study, we demonstrated that GH induces TGF-β1 signaling and provokes cell cycle reentry of otherwise quiescent podocytes. Though differentiated podocytes reenter the cell cycle in response to GH and TGF-β1, they cannot accomplish cytokinesis, despite karyokinesis. Owing to this aberrant cell cycle event, GH- or TGF-β1-treated cells remain binucleated and undergo mitotic catastrophe. Importantly, inhibition of JAK2, TGFBR1 (TGF-β receptor 1), or Notch prevented cell cycle reentry of podocytes and protected them from mitotic catastrophe associated with cell death. Inhibition of Notch activation prevents GH-dependent podocyte injury and proteinuria. Similarly, attenuation of GHR expression abated Notch activation in podocytes. Kidney biopsy sections from patients with diabetic nephropathy (DN) show activation of Notch signaling and binucleated podocytes. These data indicate that excess GH induced TGF-β1-dependent Notch1 signaling contributes to the mitotic catastrophe of podocytes. This study highlights the role of aberrant GH signaling in podocytopathy and the potential application of TGF-β1 or Notch inhibitors, as a therapeutic agent for DN.Subject terms: Podocytes, Diabetic nephropathy     

An update: the role of Nephrin inside and outside the kidney

Nephrin is a key molecule in podocytes to maintain normal slit diaphragm structure. Nephin interacts with many other podocyte and slit diaphragm protein and also mediates important cell signaling pathways in podocytes. Loss of nephrin during the development leads to the congenital nephrotic syndrome in children. Reduction of nephrin expression is often observed in adult kidney diseases including diabetic nephropathy and HIV-associated nephropathy. The critical role of nephrin has been confirmed by different animal models with nephrin knockout and knockdown. Recent studies demonstrate that knockdown of nephrin expression in adult mice aggravates the progression of unilateral nephrectomy and Adriamycin-induced kidney disease. In addition to its critical role in maintaining normal glomerular filtration unit in the kidney, nephrin is also expressed in other organs. However, the exact role of nephrin in kidney and extra-renal organs has not been well characterized. Future studies are required to determine whether nephrin could be developed as a drug target to treat patients with kidney disease.     

Glomerular Endothelial Cell Injury and Damage Precedes That of Podocytes in Adriamycin-Induced Nephropathy

The role of podocytes in the development and progression of glomerular disease has been extensively investigated in the past decade. However, the importance of glomerular endothelial cells in the pathogenesis of proteinuria and glomerulosclerosis has been largely ignored. Recent studies have demonstrated that endothelial nitric oxide synthatase (eNOS) deficiency exacerbates renal injury in anti-GBM and remnant kidney models and accelerates diabetic kidney damage. Increasing evidence also demonstrates the importance of the glomerular endothelium in preventing proteinuria. We hypothesize that endothelial dysfunction can initiate and promote the development and progression of glomerulopathy. Administration of adriamycin (ADR) to C57BL/6 mice, normally an ADR resistant strain, with an eNOS deficiency induced overt proteinuria, severe glomerulosclerosis, interstitial fibrosis and inflammation. We also examined glomerular endothelial cell and podocyte injury in ADR-induced nephropathy in Balb/c mice, an ADR susceptible strain, by immunostaining, TUNEL and Western blotting. Interestingly, down-regulation of eNOS and the appearance of apoptotic glomerular endothelial cells occurred as early as 24 hours after ADR injection, whilst synaptopodin, a functional podocyte marker, was reduced 7 days after ADR injection and coincided with a significant increase in the number of apoptotic podocytes. Furthermore, conditioned media from mouse microvascular endothelial cells over-expressing GFP-eNOS protected podocytes from TNF-α-induced loss of synaptopodin. In conclusion, our study demonstrated that endothelial dysfunction and damage precedes podocyte injury in ADR-induced nephropathy. Glomerular endothelial cells may protect podocytes from inflammatory insult. Understanding the role of glomerular endothelial dysfunction in the development of kidney disease will facilitate in the design of novel strategies to treat kidney disease.     

Losartan Affects Glomerular AKT and mTOR Phosphorylation in an Experimental Model of Type 1 Diabetic Nephropathy

The AKT-mTOR pathway is activated in diabetic nephropathy. Renin-angiotensin system modulators exert beneficial effects on the diabetic kidney. We explored the action of losartan on AKT-mTOR phosphorylation in glomeruli and podocytes. Diabetes mellitus was induced to Sprague-Dawley rats by streptozotocin. Five months later, the rats were commenced on losartan and euthanized 2 months later. Kidneys were processed for immunofluorescence studies. Glomeruli were isolated for Western blot analysis. Diabetes increased activated forms of AKT and mTOR both in glomeruli and podocytes. In diabetic rats, losartan decreased phosphorylated/activated forms of AKT (Thr308) and mTOR (Ser2448) in glomeruli but decreased only activated mTOR in podocytes. However, in both glomeruli and podocytes of healthy animals, an inverse pattern was evident. In conclusion, a new body of evidence indicates the differential activation of AKT-mTOR in glomeruli and podocytes of healthy and diabetic animals in response to losartan.     

Co-regulation of Gremlin and Notch signalling in diabetic nephropathy

Diabetic nephropathy is currently the leading cause of end-stage renal disease worldwide, and occurs in approximately one third of all diabetic patients. The molecular pathogenesis of diabetic nephropathy has not been fully characterized and novel mediators and drivers of the disease are still being described. Previous data from our laboratory has identified the developmentally regulated gene Gremlin as a novel target implicated in diabetic nephropathy in vitro and in vivo. We used bioinformatic analysis to examine whether Gremlin gene sequence and structure could be used to identify other genes implicated in diabetic nephropathy. The Notch ligand Jagged1 and its downstream effector, hairy enhancer of split-1 (Hes1), were identified as genes with significant similarity to Gremlin in terms of promoter structure and predicted microRNA binding elements. This led us to discover that transforming growth factor-beta (TGFbeta1), a primary driver of cellular changes in the kidney during nephropathy, increased Gremlin, Jagged1 and Hes1 expression in human kidney epithelial cells. Elevated levels of Gremlin, Jagged1 and Hes1 were also detected in extracts from renal biopsies from diabetic nephropathy patients, but not in control living donors. In situ hybridization identified specific upregulation and co-expression of Gremlin, Jagged1 and Hes1 in the same tubuli of kidneys from diabetic nephropathy patients, but not controls. Finally, Notch pathway gene clustering showed that samples from diabetic nephropathy patients grouped together, distinct from both control living donors and patients with minimal change disease. Together, these data suggest that Notch pathway gene expression is elevated in diabetic nephropathy, co-incident with Gremlin, and may contribute to the pathogenesis of this disease.     

Comparative investigation of kidney mesangial cells from increased oxidative stress-induced diabetic rats by using different microscopy techniques

High glucose and increased oxidative stress levels are the known important mediators of diabetic nephropathy. However, the effects of these mediators on tissue damage basically due to extracellular matrix expansion in mesangial cells have yet to be fully examined within the context of early stage diabetic nephropathy. In this study, we attempted to characterize changes in mesangial cells of streptozotocin-induced diabetic rats with a comparative investigation of kidney tissue by using different microscopy techniques. The serum levels of urea and creatinine of diabetic rats, as biomarkers of kidney degeneration, decreased significantly compared to those of age-matched controls. In diabetic rats, there are increased malondialdehyde and oxidized-glutathione levels as well as reduced-glutathione and glutathione-peroxidase activity levels in renal tissue compared to those of the controls. By using light and electron microscopies, we showed that there were marked thickening in Bowman’s membrane and glomerular capillary wall, increased amount of extracellular matrix often occupying Bowman’s space, degenerations in tubules, an increased number of mesangial cells in the network of glomerular capillary walls, and increased amount of lipid accumulation in proximal tubules in the renal tissue of diabetic rats. Our confocal microscopy data confirmed also the presence of irregularity and widened in glomerular capillaries, their attachment to the Bowman’s capsule, degenerated heterochromatin, thickening in foci of glomerular basement membrane, and marked increase in mesangial cells. These results suggest that a detailed structural investigation of kidney tissue provides further information on the important role of mesangial cells in pathogenesis of diabetic nephropathy.     

Transferrin receptor 2: a new molecule in iron metabolism

Transferrin receptor 1 (TfR1) which mediates uptake of transferrin-bound iron, is essential for life in mammals. Recently, a close homologue of human transferrin receptor 1 was cloned and called transferrin receptor 2 (TfR2). A similar molecule has been identified in the mouse. Human transferrin receptor 2 is 45% identical with transferrin receptor 1 in the extracellular domain, but contains no iron responsive element in its mRNA and is apparently not regulated by intracellular iron concentration nor by interaction with HFE. Transferrin receptor 2, like transferrin receptor 1, binds transferrin in a pH-dependent manner (but with 25 times lower affinity) and delivers iron to cells. However, transferrin receptor 2 distribution differs from transferrin receptor 1, increasing in differentiating hepatocytes and decreasing in differentiating erythroblasts. Expression of both receptors is cell cycle dependent. Mutations in the human transferrin receptor 2 gene cause iron overload disease, suggesting it has a role in iron homeostasis.     

Autophagic flux defect in diabetic kidney disease results in megamitochondria formation in podocytes

Podocyte injury is an important factor in the pathogenesis of diabetic nephropathy. Podocytes are characterized by large numbers of mitochondria. However, mitochondrial dysfunction as it relates to kidney pathology remains poorly understood. The present study found that podocyte mitochondria in different animal models of diabetes mellitus became elongated with the development of albuminuria, suggesting a change in mitochondrial dynamics. We then treated cells with a combination of glucose, fatty acids, and angiotensin II (GFA) to mimic the diabetic milieu. Cultured podocytes exposed to GFA showed megamitochondria formation and decreased autophagosome degradation. We also found that GFA treatment decreased the binding of the autophagosome to the lysosome. Our results suggest that megamitochondria are common in podocytes during diabetic nephropathy and that insufficient autophagy flux may underlie this effect. These findings have expanded our understanding of the pathogenesis of diabetic nephropathy and identified a potential pharmacological target for treatment.     

Lactoferrin and transferrin damage of the gram-negative outer membrane is modulated by Ca2+ and Mg2+

Lactoferrin and transferrin have antimicrobial activity against selected Gram-negative bacteria, but the mechanism of action has not been defined. We studied the ability of lactoferrin and transferrin to damage the Gram-negative outer membrane. Lipopolysaccharide release by the proteins could be blocked by concurrent addition of Ca2+ and Mg2+. Addition of Ca2+ also blocked the ability of lactoferrin to increase the susceptibility of Escherichia coli to rifampicin. Transferrin, but not lactoferrin, increased susceptibility of Gram-negative bacteria to deoxycholate, with reversal of sensitivity occurring with exposure to Ca2+ or Mg2+. In transmission electron microscopy studies polymyxin B caused finger-like membrane projections, but no morphological alterations were seen in cells exposed to EDTA, lactoferrin or transferrin. These data provide further evidence that lactoferrin and transferrin act as membrane-active agents with the effects modulated by Ca2+ and Mg2+.     

MAD2B promotes podocyte injury through regulating Numb-dependent Notch 1 pathway in diabetic nephropathy

Rationale: Recent studies have demonstrated that the loss of podocyte is a critical event in diabetic nephropathy (DN). Previously, our group have found that the mitotic arrest deficient protein MAD2B was involved in high glucose (HG)-induced podocyte injury by regulating APC/C activity. However, the exact mechanism of MAD2B implicated in podocyte injury is still lacking.Methods: The experiments were conducted by using kidney tissues from streptozotocin (STZ) induced diabetic mice with or without podocyte-specific deletion of MAD2B and the cultured podocytes exposed to different treatments. Glomerular pathological injury was evaluated by periodic acid-Schiff staining and transmission electron microscopy. The endogenous interaction between MAD2B and Numb was discovered by yeast two-hybrid analysis and co-immunoprecipitation assay. The expressions of MAD2B, Numb and related pathway were detected by western blot, immunochemistry and immunofluorescence.Results: The present study revealed that MAD2B was upregulated in diabetic glomeruli and cultured podocytes under hyperglycemic conditions. Podocyte-specific deletion of MAD2B alleviated podocyte injury and renal function deterioration in mice of diabetic nephropathy. Afterwards, MAD2B was found to interact with Numb, which was downregulated in diabetic glomeruli and HG-stimulated cultured podocytes. Interestingly, MAD2B genetic deletion could partly reverse the decline of Numb in podocytes exposed to HG and in diabetic mice, and the expressions of Numb downstream molecules such as NICD and Hes-1 were decreased accordingly. In addition, overexpression of Numb ameliorated HG-induced podocyte injury.Conclusions: The present findings suggest that upregulated MAD2B expression contributes to Numb depletion and activation of Notch 1 signaling pathway, which ultimately leads to podocyte injury during DN progression.     

Expression and regulation of a novel identified TNFAIP8 family is associated with diabetic nephropathy

Tumor necrosis factor-α-inducible protein 8 (TNFAIP8) family are very recently identified proteins which share considerable sequence homology to regulate cellular and immune homeostasis. However, it is unknown whether TNFAIP8 family is expressed in the kidney and contributes to the regulation of renal functions. Therefore, the present study was designed to characterize the members of TNFAIP8 family in the kidney and to explore their possible roles in the development and progression of diabetic nephropathy. By RT-PCR and Western blot analyses, we found that all members of TNFAIP8 family were detected in the kidney. TNFAIP8 and TIPE2 expression was significantly increased in glomeruli from streptozotocin (STZ)-induced diabetic rats, and this upregulation was further confirmed in renal biopsies of diabetic patients. In in vitro study, TNFAIP8 was upregulated in response to high glucose in mesangial cells rather than podocytes. Moreover, a direct correlation was observed between expression of TNFAIP8 and mesangial cell proliferation and this regulation was associated with NADPH oxidase-mediated signaling pathway. However, we failed to observe the upregulation of TIPE2 in both mesangial cells and podocytes in response to high glucose. In conclusion, the present study addressed the role of TNFAIP8 family in diabetic nephropathy. These findings for the first time demonstrate that TNFAIP8 is one of critical components of a signal transduction pathway that links mesangial cell proliferation to diabetic renal injury.     

Nicorandil as a novel therapy for advanced diabetic nephropathy in the eNOS-deficient mouse

Nicorandil is an orally available drug that can act as a nitric oxide donor, an antioxidant, and an ATP-dependent K channel activator. We hypothesized that it may have a beneficial role in treating diabetic nephropathy. We administered nicorandil to a model of advanced diabetic nephropathy (the streptozotocin-induced diabetes in mice lacking endothelial nitric oxide synthase, eNOSKO); controls included diabetic eNOS KO mice without nicorandil and nondiabetic eNOS KO mice treated with either nicorandil or vehicle. Mice were treated for 8 wk. Histology, blood pressure, and renal function were determined. Additional studies involved examining the effects of nicorandil on cultured human podocytes. Here, we found that nicorandil did not affect blood glucose levels, blood pressure, or systemic endothelial function, but significantly reduced proteinuria and glomerular injury (mesangiolysis and glomerulosclerosis). Nicorandil protected against podocyte loss and podocyte oxidative stress. Studies in cultured podocytes showed that nicorandil likely protects against glucose-mediated oxidant stress via the ATP-dependent K channel as opposed to its NO-stimulating effects. In conclusion, nicorandil may be beneficial in diabetic nephropathy by preserving podocyte function. We recommend clinical trials to determine whether nicorandil may benefit diabetic nephropathy or other conditions associated with podocyte dysfunction.     

Advances in pathogenetic mechanisms of diabetic nephropathy.

The diabetic epidemic that is being experienced around the world has many ramifications. Since diabetes is the most common cause of end stage renal disease in the United States and the Western world, we can expect the increase in prevalence to continue. Minority individuals with diabetes suffer a disproportionately high incidence of diabetic nephropathy leading to end stage renal disease. The data suggest that aggressive medical management should be their mainstay of therapy. In the past five years, our knowledge of the mechanisms involved in the pathology of diabetic glomerulosclerosis has greatly expanded. Transforming growth factor (TGF)-beta still maintains a key role in the pathogenesis. However, many of the signaling mechanisms have now been described. Furthermore, TGF-beta may also function to damage glomerular epithelial cells or podocytes, resulting in podocyteuria and proteinuria that worsen with progressive diabetic nephropathy. Additionally, TGF-beta upregulation in diabetes may cause injury or transformation of tubular epithelial cells that contribute to interstitial fibrosis. The use of thiazolidinediones in type 2 diabetes is associated with improvement in insulin sensitivity, as well as improvement in albuminuria. The mechanisms by which these ligands function remain unclear, but there may be several targets that could include mesangial cells and podocytes.