Claire E. Hills

TGF-β1-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-β 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-β and associated sub-cellular proteins in EMT. Methods: To understand the pathology of EMT and the role of TGF-β, we reviewed the literature using PubMed for English language articles that contained key words related to EMT, TGF-β 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-β1 is pivotal in mediating these phenotypic changes. Conclusion: TGF-β-induced EMT is a key contributor to fibrotic scar formation as seen in DN, and novel routes for future therapeutic intervention are discussed.

The role of TGF-β and epithelial-to mesenchymal transition in diabetic nephropathy

Transforming Growth Factor-beta (TGF-β) is a pro-sclerotic cytokine widely associated with the development of fibrosis in diabetic nephropathy. Central to the underlying pathology of tubulointerstitial fibrosis is epithelial-to-mesenchymal transition (EMT), or the trans-differentiation of tubular epithelial cells into myofibroblasts. This process is accompanied by a number of key morphological and phenotypic changes culminating in detachment of cells from the tubular basement membrane and migration into the interstitium. Ultimately these cells reside as activated myofibroblasts and further exacerbate the state of fibrosis. A large body of evidence supports a role for TGF-β and downstream Smad signalling in the development and progression of renal fibrosis. Here we discuss a role for TGF-β as the principle effector in the development of renal fibrosis in diabetic nephropathy, focusing on the role of the TGF-β1 isoform and its downstream signalling intermediates, the Smad proteins. Specifically we review evidence for TGF-β1 induced EMT in both the proximal and distal regions of the nephron and describe potential therapeutic strategies that may target TGF-β1 activity.

Cellular and physiological effects of C-peptide.

In recent years, accumulating evidence indicates a biological function for proinsulin C-peptide. These results challenge the traditional view that C-peptide is essentially inert and only useful as a surrogate marker of insulin release. Accordingly, it is now clear that C-peptide binds with high affinity to cell membranes, probably to a pertussis-toxin-sensitive G-protein-coupled receptor. Subsequently, multiple signalling pathways are potently and dose-dependently activated in multiple cell types by C-peptide with the resulting activation of gene transcription and altered cell phenotype. In diabetic animals and Type 1 diabetic patients, short-term studies indicate that C-peptide also enhances glucose disposal and metabolic control. Furthermore, results derived from animal models and clinical studies in Type 1 diabetic patients suggest a salutary effect of C-peptide in the prevention and amelioration of diabetic nephropathy and neuropathy. Therefore a picture of Type 1 diabetes as a dual-hormone-deficiency disease is developing, suggesting that the replacement of C-peptide alongside insulin should be considered in its management.

C-peptide reverses TGF-β1-induced changes in renal proximal tubular cells: implications for treatment of diabetic nephropathy

The crucial pathology underlying progressive chronic kidney disease in diabetes is tubulointerstitial fibrosis. Central to this process is epithelial-mesenchymal transformation (EMT) of proximal tubular epithelial cells driven by maladaptive transforming growth factor-beta1 (TGF-beta1) signaling. Novel signaling roles for C-peptide have recently been discovered with evidence emerging that C-peptide may mitigate microvascular complications of diabetes. We studied the potential for C-peptide to interrupt injurious TGF-beta1 signaling pathways and thus block development of EMT in HK2 human kidney proximal tubular cells. Cells were incubated with TGF-beta1 either alone or with C-peptide in low or high glucose. Changes in cell morphology, TGF-beta1 receptor expression, vimentin, E-cadherin, and phosphorylated Smads were assessed. Luciferase reporters were used to assess Smad activity. The cytoskeleton was visualized by TRITC-phalloidin staining. The typical TGF-beta1-stimulated, EMT-associated morphological alterations of proximal tubular cells, including increased vimentin expression, decreased E-cadherin expression, and cytoskeletal rearrangements, were prevented by C-peptide treatment. C-peptide also blocked TGF-beta1-induced upregulation of expression of both type I and type II TGF-beta1 receptors and attenuated TGF-beta1-mediated Smad phosphorylation and Smad transcriptional activity. These effects of C-peptide were inhibited by pertussis toxin. The results demonstrate that C-peptide almost completely reversed the morphological changes in PT cells induced by TGF-beta1 and suggest a role or C-peptide as a renoprotective agent in diabetic nephropathy.

C-Peptide as a Therapeutic Tool in Diabetic Nephropathy

Background/Aims: Insulin is synthesised as a pro-hormone with an interconnecting C-peptide, cleaved during post-translational modification. This review discusses growing evidence which indicates that C-peptide is biologically active, benefiting microvascular complications associated with diabetes. Methods: To explore the renoprotective role of C-peptide in diabetic nephropathy (DN), we reviewed the literature using PubMed for English language articles that contained key words related to C-peptide, kidney and DN. Results: Numerous studies have demonstrated that C-peptide ameliorates a number of the structural and functional renal disturbances associated with uncontrolled hyperglycaemia in human and animal models of type 1 diabetes mellitus that lead to the development and progression of nephropathy, including abrogation of glomerular hyperfiltration, reduced microalbuminuria, decreased mesangial expansion and increased endothelial nitric oxide synthase levels. The in vitro exposure of kidney proximal tubular cells to physiological concentrations of C-peptide activates extracellular signal-regulated kinase, phosphatidylinositol 3-kinase, protein kinase C, elevates intracellular calcium, and stimulates transcription factors NF-ĸB and peroxisome proliferator-activated receptor-γ. Conclusion: Burgeoning studies suggest that C-peptide is more than merely a link between the A and B chains of the proinsulin molecule and represents a future therapeutic tool in reducing complications of DN.

High glucose up-regulates ENaC and SGK1 expression in HCD-cells

Background/Aim: Diabetic nephropathy is associated with progressive renal damage, leading to impaired function and end-stage renal failure. Secondary hypertension stems from a deranged ability of cells within the kidney to resolve and appropriately regulate sodium resorption in response to hyperglycaemia. However, the mechanisms by which glucose alters sodium re-uptake have not been fully characterised. Methods: Here we present RT-PCR, western blot and immunocytochemistry data confirming mRNA and protein expression of the serum and glucocorticoid inducible kinase (SGK1) and the α conducting subunit of the epithelial sodium channel (ENaC) in a model in vitro system of the human cortical collecting duct (HCD). We examined changes in expression of these elements in response to glucose challenge, designed to mimic hyperglycaemia associated with type 2 diabetes mellitus. Changes in Na+ concentration were assessed using single-cell microfluorimetry. Results: Incubation with glucose, the Ca2+-ionophore ionomycin and the cytokine TGF-β1 were all found to evoke significant and time-dependent increases in both SGK1 and αENaC protein expression. These molecular changes were correlated to an increase in Na+-uptake at the single-cell level. Conclusion: Together these data offer a potential explanation for glucose-evoked Na+-resorption and a potential contributory role of SGK1 and ENaCs in development of secondary hypertension, commonly linked to diabetic nephropathy.

Intracellular signalling by C-peptide.

C-peptide, a cleavage product of the proinsulin molecule, has long been regarded as biologically inert, serving merely as a surrogate marker for insulin release. Recent findings demonstrate both a physiological and protective role of C-peptide when administered to individuals with type I diabetes. Data indicate that C-peptide appears to bind in nanomolar concentrations to a cell surface receptor which is most likely to be G-protein coupled. Binding of C-peptide initiates multiple cellular effects, evoking a rise in intracellular calcium, increased PI-3-kinase activity, stimulation of the Na+/K+ ATPase, increased eNOS transcription, and activation of the MAPK signalling pathway. These cell signalling effects have been studied in multiple cell types from multiple tissues. Overall these observations raise the possibility that C-peptide may serve as a potential therapeutic agent for the treatment or prevention of long-term complications associated with diabetes.

Proinsulin C-Peptide Antagonizes the Profibrotic Effects of TGF-β1 via Up-Regulation of Retinoic Acid and HGF-Related Signaling Pathways

Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameliorate complications of type 1 diabetes. Here we sought to identify new pathways regulated by C-peptide of relevance to the pathophysiology of diabetic nephropathy. Microarray analysis was performed to identify genes regulated by either C-peptide and/or TGF-beta1 in a human proximal tubular cell line, HK-2. Expression of retinoic acid receptor beta (RARbeta), hepatocyte growth factor (HGF), cellular retinoic acid-binding protein II (CRABPII), vimentin, E-cadherin, Snail, and beta-catenin was assessed by immunoblotting. The cellular localization of vimentin and beta-catenin was determined by immunocytochemistry. Changes in cell morphology were assessed by phase contrast microscopy. Gene expression profiling demonstrated differential expression of 953 and 1458 genes after C-peptide exposure for 18 h or 48 h, respectively. From these, members of the antifibrotic retinoic acid (RA)- and HGF-signaling pathways were selected. Immunoblotting demonstrated that C-peptide increased RARbeta, CRABPII, and HGF. We confirmed a role for RA in reversal of TGF-beta1-induced changes associated with epithelial-mesenchymal transition, including expression changes in Snail, E-cadherin, vimetin, and redistribution of beta-catenin. Importantly, these TGF-beta1-induced changes were inhibited by C-peptide. Further, effects of TGF-beta1 on Snail and E-cadherin expression were blocked by HGF, and inhibitory effects of C-peptide were removed by blockade of HGF activity. This study identifies a novel role for HGF as an effector of C-peptide, possibly via an RA-signaling pathway, highlighting C-peptide as a potential therapy for diabetic nephropathy.

'Special K' and a Loss of Cell-To-Cell Adhesion in Proximal Tubule-Derived Epithelial Cells: Modulation of the Adherens Junction Complex by Ketamine

Ketamine, a mild hallucinogenic class C drug, is the fastest growing ‘party drug’ used by 16–24 year olds in the UK. As the recreational use of Ketamine increases we are beginning to see the signs of major renal and bladder complications. To date however, we know nothing of a role for Ketamine in modulating both structure and function of the human renal proximal tubule. In the current study we have used an established model cell line for human epithelial cells of the proximal tubule (HK2) to demonstrate that Ketamine evokes early changes in expression of proteins central to the adherens junction complex. Furthermore we use AFM single-cell force spectroscopy to assess if these changes functionally uncouple cells of the proximal tubule ahead of any overt loss in epithelial cell function. Our data suggests that Ketamine (24–48 hrs) produces gross changes in cell morphology and cytoskeletal architecture towards a fibrotic phenotype. These physical changes matched the concentration-dependent (0.1–1 mg/mL) cytotoxic effect of Ketamine and reflect a loss in expression of the key adherens junction proteins epithelial (E)- and neural (N)-cadherin and β-catenin. Down-regulation of protein expression does not involve the pro-fibrotic cytokine TGFβ, nor is it regulated by the usual increase in expression of Slug or Snail, the transcriptional regulators for E-cadherin. However, the loss in E-cadherin can be partially rescued pharmacologically by blocking p38 MAPK using SB203580. These data provide compelling evidence that Ketamine alters epithelial cell-to-cell adhesion and cell-coupling in the proximal kidney via a non-classical pro-fibrotic mechanism and the data provides the first indication that this illicit substance can have major implications on renal function. Understanding Ketamine-induced renal pathology may identify targets for future therapeutic intervention.

Calcium-sensing receptor activation increases cell-cell adhesion and ß-cell function

Background/Aims: The extracellular calcium-sensing receptor (CaR) is expressed in pancreatic β-cells where it is thought to facilitate cell-to-cell communication and augment insulin secretion. However, it is unknown how CaR activation improves β-cell function. Methods: Immunocytochemistry and western blotting confirmed the expression of CaR in MIN6 β-cell line. The calcimimetic R568 (1µM) was used to increase the affinity of the CaR and specifically activate the receptor at a physiologically appropriate extracellular calcium concentration. Incorporation of 5-bromo-2’-deoxyuridine (BrdU) was used to measure cell proliferation, whilst changes in non-nutrient-evoked cytosolic calcium were assessed using fura-2-microfluorimetry. AFM-single-cell-force spectroscopy related CaR-evoked changes in epithelial (E)-cadherin expression to improved functional tethering between coupled cells. Results: Activation of the CaR over 48hr doubled the expression of E-cadherin (206±41%) and increased L-type voltage-dependent calcium channel expression by 70% compared to control. These changes produced a 30% increase in cell-cell tethering and elevated the basal-to-peak amplitude of ATP (50µM) and tolbutamide (100µM)-evoked changes in cytosolic calcium. Activation of the receptor also increased PD98059 (1-100µM) and SU1498 (1-100µM)-dependent β-cell proliferation. Conclusion: Our data suggest that activation of the CaR increases E-cadherin mediated functional tethering between β-cells and increases expression of L-type VDCC and secretagogue-evoked changes in [Ca2+]i. These findings could explain how local changes in calcium, co-released with insulin, activate the CaR on neighbouring cells to help ensure efficient and appropriate secretory function.