TGF-beta 1-induced epithelial-to-mesenchymal transition and therapeutic intervention in diabetic nephropathy

Background/Aims: Epithelial-to-mesenchymal cell transformation (EMT) is the trans-differentiation of tubular epithelial cells into myofibroblasts, an event underlying progressive chronic kidney disease in diabetes, resulting in fibrosis. Mainly reported in proximal regions of the kidney, EMT is now recognized as a key contributor to the loss of renal function throughout the nephron in diabetic nephropathy (DN). Concomitant upregulation of TGF-beta in diabetes makes this pro-fibrotic cytokine an obvious candidate in the development of these fibrotic complications. This article reviews recent findings clarifying our understanding of the role of TGF-beta and associated sub-cellular proteins in EMT. Methods: To understand the pathology of EMT and the role of TGF-beta, we reviewed the literature using PubMed for English language articles that contained key words related to EMT, TGF-beta and DN. Results: EMT and phenotypic plasticity of epithelial cells throughout the nephron involves cytoskeletal reorganization and de novo acquisition of classic mesenchymal markers. Concurrent downregulation of epithelial adhesion molecules results in a loss of function and decreased cell coupling, contributing to a loss of epithelial integrity. TGF-beta 1 is pivotal in mediating these phenotypic changes. Conclusion: TGF-beta-induced EMT is a key contributor to fibrotic scar formation as seen in DN, and novel routes for future therapeutic intervention are discussed

SGK1 in the kidney: disrupted sodium transport in diabetes and beyond

Renal complications of diabetes can be severe; however, the mechanisms that underlie the development and progression of diabetic nephropathy are poorly understood. Recent evidence suggests that the serum and glucocorticoid induced kinase-1 (SGK1) may be key to this process. SGK1 expression and function are increased in models of diabetes and polymorphisms of the SGK1 gene are associated with type 2 diabetes mellitus. A key regulator of sodium transport within the renal epithelium of the distal nephron, SGK1 was originally isolated as a glucocorticoid-sensitive gene that regulated the epithelial sodium channel (ENaC; known also as the sodium channel, nonvoltage-gated 1, SCNN1). It is now apparent that SGK1 modulates sodium re-absorption by a number of sodium transporters/channels throughout the length of the nephron including; the Na+/H+ exchange isoform 3 (NHE3), the Na+Cl- co-transporter (NCC) and the Na+/K+-ATPase. In addition, SGK1 is regulated by a diverse range of factors including; insulin, glucose, intracellular calcium, transforming growth factor-beta1, flow rate and osmolality. This brief review examines the evidence supporting an involvement of SGK1 in diabetic nephropathy and discusses how dysregulated sodium transport may account for the development of secondary hypertension associated with the condition. Furthermore, the article examines how aberrant SGK1 expression and activity may be responsible for the cellular changes seen in the damaged nephron

Functional expression of TRPV4 channels in human collecting duct cells: implications for secondary hypertension in diabetic nephropathy

Background. The Vanilloid subfamily of transient receptor potential (TRPV) ion channels has been widely implicated in detecting osmotic and mechanical stress. In the current study, we examine the functional expression of TRPV4 channels in cell volume regulation in cells of the human collecting duct. Methods. Western blot analysis, siRNA knockdown, and microfluorimetry were used to assess the expression and function of TRPV4 in mediating Ca2+-dependent mechanical stimulation within a novel system of the human collecting duct (HCD). Results. Native and siRNA knockdown of TRPV4 protein expression was confirmed by western blot analysis. Touch was used as a cell-directed surrogate for osmotic stress. Mechanical stimulation of HCD cells evoked a transient increase in [Ca2+]i that was dependent upon thapsigargin-sensitive store release and Ca2+ influx. At 48 hrs, high glucose and mannitol (25 mM) reduced TRPV4 expression by 54% and 24%, respectively. Similar treatment doubled SGK1 expression. Touch-evoked changes were negated following TRPV4 knockdown. Conclusion. Our data confirm expression of Ca2+-dependent TRPV4 channels in HCD cells and suggest that a loss of expression in response to high glucose attenuates the ability of the collecting duct to exhibit regulatory volume decreases, an effect that may contribute to the pathology of fluid and electrolyte imbalance as observed in diabetic nephropathy

Visfatin reduces gap junction mediated cell-to-cell communication in proximal tubule-derived epithelial cells

Background/Aims: In the current study we examined if the adipocytokine, visfatin, alters connexin-mediated intercellular communication in proximal tubule-derived epithelial cells. Methods: The effects of visfatin (10-200ng/mL) on cell viability and cytotoxicity in HK2-cells were assessed by MTT, crystal violet and lactate dehydrogenase assays. Western blot analysis was used to confirm expression of Cx26, Cx40 and Cx43. The effect of visfatin (10-200ng/mL) on TGF-β1 secretion was confirmed by ELISA, and the effects of both TGF-β1 (2-10ng/mL) and visfatin (10-200ng/mL) on connexin expression were assessed by western blot. Functional intercellular communication was determined using transfer of Lucifer Yellow and paired-whole cell patch clamp electrophysiology. Results: In low glucose (5mM), visfatin (10-200ng/mL) did not affect membrane integrity, cytotoxicity or cell viability at 48hrs, but did evoke a concentration-dependent reduction in Cx26 and Cx43 expression. The expression of Cx40 was unaffected. At 48hrs, visfatin (10-200ng/mL) increased the secretion of TGF-β1 and the visfatin-evoked changes in connexin expression were mimicked by exogenous application of the pro-fibrotic cytokine (2-10ng/ml). Visfatin reduced dye transfer between coupled cells and decreased functional conductance, with levels falling by 63% as compared to control. Although input resistance was increased following visfatin treatment by 166%, the change was not significant as compared to control. The effects of visfatin on Cx-expression and cell-coupling were blocked in the presence of a TGF-β1 specific neutralizing antibody. Conclusions: The adipocytokine visfatin selectively evoked a non-toxic reduction in connexin expression in HK2-cells. The loss in gap-junction associated proteins was mirrored by a loss in functional conductance between coupled cells. Visfatin increased TGF-β secretion and the pattern of change for connexins expression was mimicked by exogenous application of TGF-β1. The effect of visfatin on Cx-expression and dye transfer were negated in the presence of a TGF-β1 neutralising antibody. These data suggest that visfatin reduces connexin-mediated intercellular communication in proximal tubule-derived epithelial cells via a TGF-β dependent pathway. © 2013 S. Karger AG, Base

A role for the extracellular calcium-sensing receptor in cell-cell communication in pancreatic islets of Langerhans

Background: The extracellular calcium-sensing receptor (CaR) is expressed in many tissues that are not associated with Ca2+ homeostasis, including the endocrine cells in pancreatic islets of Langerhans. We have demonstrated previously that pharmacological activation of the CaR stimulates insulin secretion from islet -cells and insulin-secreting MIN6 cells. Methods: In the present study we have investigated the effects of CaR activation on MIN6 cell proliferation and have used shRNA-mediated CaR knockdown to determine whether the CaR is involved in the regulation of insulin secretion via cell-cell communication. Results: CaR activation caused the phosphorylation and activation of the p42/44 MAPK signalling cascade, and this activation was prevented by the shRNA-induced down-regulation of CaR mRNA expression. CaR activation also resulted in increased proliferation of MIN6 cells, consistent with the known role of the p42/44 MAPK system in the regulation of -cell proliferation. Down-regulation of CaR expression had no detectable effects on glucose-induced insulin secretion from MIN6 cells maintained as monolayers, but blocked the increases in insulin secretion that were observed when the cells were configured as three-dimensional islet-like structures (pseudoislets), consistent with a role for the CaR in cell-cell communication in pseudoislets. Conclusion: It is well established that islet function is dependent on communication between islet cells and the results of this study suggest that the CaR is required for -cell to -cell interactions within islet-like structures