Connexin-43 hemichannels orchestrate NOD-like receptor protein-3 (NLRP3) inflammasome activation and sterile inflammation in tubular injury.

BACKGROUND: Without a viable cure, chronic kidney disease is a global health concern. Inflammatory damage in and around the renal tubules dictates disease severity and is contributed to by multiple cell types. Activated in response to danger associated molecular patterns (DAMPs) including ATP, the NOD-like receptor protein-3 (NLRP3) inflammasome is integral to this inflammation. In vivo, we have previously observed that increased expression of Connexin 43 (Cx43) is linked to inflammation in chronic kidney disease (CKD) whilst in vitro studies in human proximal tubule cells highlight a role for aberrant Cx43 hemichannel mediated ATP release in tubule injury. A role for Cx43 hemichannels in priming and activation of the NLRP3 inflammasome in tubule epithelial cells remains to be determined. METHODS: Using the Nephroseq database, analysis of unpublished transcriptomic data, examined gene expression and correlation in human CKD. The unilateral ureteral obstruction (UUO) mouse model was combined with genetic (tubule-specific Cx43 knockout) and specific pharmacological blockade of Cx43 (Peptide5), to explore a role for Cx43-hemichannels in tubule damage. Human primary tubule epithelial cells were used as an in vitro model of CKD. RESULTS: Increased Cx43 and NLRP3 expression correlates with declining glomerular filtration rate and increased proteinuria in biopsies isolated from patients with CKD. Connexin 43-tubule deletion prior to UUO protected against tubular injury, increased expression of proinflammatory molecules, and significantly reduced NLRP3 expression and downstream signalling mediators. Accompanied by a reduction in F4/80 macrophages and fibroblast specific protein (FSP1+) fibroblasts, Cx43 specific hemichannel blocker Peptide5 conferred similar protection in UUO mice. In vitro, Peptide5 determined that increased Cx43-hemichannel activity primes and activates the NLRP3 inflammasome via ATP-P2X7 receptor signalling culminating in increased secretion of chemokines and cytokines, each of which are elevated in individuals with CKD. Inhibition of NLRP3 and caspase 1 similarly decreased markers of tubular injury, whilst preventing the perpetual increase in Cx43-hemichannel activity. CONCLUSION: Aberrant Cx43-hemichannel activity in kidney tubule cells contributes to tubule inflammation via activation of the NLRP3 inflammasome and downstream paracrine mediated cell signalling. Use of hemichannel blockers in targeting Cx43-hemichannels is an attractive future therapeutic target to slow or prevent disease progression in CKD. Video Abstract.

Blocking connexin 43 hemichannel-mediated ATP release reduces communication within and between tubular epithelial cells and medullary fibroblasts in a model of diabetic nephropathy.

INTRODUCTION: Fibrosis of renal tubules is the final common pathway in diabetic nephropathy and develops in the face of tubular injury and fibroblast activation. Aberrant connexin 43 (Cx43) hemichannel activity has been linked to this damage under euglycaemic conditions, however, its role in glycaemic injury is unknown. This study investigated the effect of a Cx43 blocker (Tonabersat) on hemichannel activity and cell-cell interactions within and between tubular epithelial cells and fibroblasts in an in vitro model of diabetic nephropathy. METHODS: Human kidney (HK2) proximal tubule epithelial cells and medullary fibroblasts (TK173) were treated in low (5 mM) or high (25 mM) glucose ± transforming growth factor beta-1 (TGFß1) ± Tonabersat in high glucose. Carboxyfluorescein dye uptake and ATPlite luminescence assessed changes in hemichannel-mediated ATP release, while immunoblotting determined protein expression. Co-incubation with the ATP-diphosphohydrolase apyrase or a P2X7R inhibitor (A438079) assessed ATP-P2X7R signalling. Indirect co-culture with conditioned media from the alternate cell type evaluated paracrine-mediated heterotypic interactions. RESULTS: Tonabersat partially negated glucose/TGFß1-induced increases in Cx43 hemichannel-mediated ATP release and downstream changes in adherens junction and extracellular matrix (ECM) protein expression in HK2 and TK173 cells. Apyrase and A438079 highlighted the role for ATP-P2X7R in driving changes in protein expression in TK173 fibroblasts. Indirect co-culture studies suggest that epithelial cell secretome increases Tonabersat-sensitive hemichannel-mediated dye uptake in fibroblasts and downstream protein expression. CONCLUSION: Tonabersat-sensitive hemichannel-mediated ATP release enhances TGFß1-driven heterotypic cell-cell interaction and favours myofibroblast activation. The data supports the potential benefit of Cx43 inhibition in reducing tubulointerstitial fibrosis in late-stage diabetic nephropathy.

Connexin 43: A Target for the Treatment of Inflammation in Secondary Complications of the Kidney and Eye in Diabetes.

Of increasing prevalence, diabetes is characterised by elevated blood glucose and chronic inflammation that precedes the onset of multiple secondary complications, including those of the kidney and the eye. As the leading cause of end stage renal disease and blindness in the working population, more than ever is there a demand to develop clinical interventions which can both delay and prevent disease progression. Connexins are membrane bound proteins that can form pores (hemichannels) in the cell membrane. Gated by cellular stress and injury, they open under pathophysiological conditions and in doing so release 'danger signals' including adenosine triphosphate into the extracellular environment. Linked to sterile inflammation via activation of the nod-like receptor protein 3 inflammasome, targeting aberrant hemichannel activity and the release of these danger signals has met with favourable outcomes in multiple models of disease, including secondary complications of diabetes. In this review, we provide a comprehensive update on those studies which document a role for aberrant connexin hemichannel activity in the pathogenesis of both diabetic eye and kidney disease, ahead of evaluating the efficacy of blocking connexin-43 specific hemichannels in these target tissues on tissue health and function.

Danegaptide Prevents TGFß1-Induced Damage in Human Proximal Tubule Epithelial Cells of the Kidney.

Chronic kidney disease (CKD) is a global health problem associated with a number of comorbidities. Recent evidence implicates increased hemichannel-mediated release of adenosine triphosphate (ATP) in the progression of tubulointerstitial fibrosis, the main underlying pathology of CKD. Here, we evaluate the effect of danegaptide on blocking hemichannel-mediated changes in the expression and function of proteins associated with disease progression in tubular epithelial kidney cells. Primary human proximal tubule epithelial cells (hPTECs) were treated with the beta1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFß1) ± danegaptide. qRT-PCR and immunoblotting confirmed mRNA and protein expression, whilst a cytokine antibody array assessed the expression/secretion of proinflammatory and profibrotic cytokines. Carboxyfluorescein dye uptake and ATP biosensing measured hemichannel activity and ATP release, whilst transepithelial electrical resistance was used to assess paracellular permeability. Danegaptide negated carboxyfluorescein dye uptake and ATP release and protected against protein changes associated with tubular injury. Blocking Cx43-mediated ATP release was paralleled by partial restoration of the expression of cell cycle inhibitors, adherens and tight junction proteins and decreased paracellular permeability. Furthermore, danegaptide inhibited TGFß1-induced changes in the expression and secretion of key adipokines, cytokines, chemokines, growth factors and interleukins. The data suggest that as a gap junction modulator and hemichannel blocker, danegaptide has potential in the future treatment of CKD.

Collagen I Modifies Connexin-43 Hemichannel Activity via Integrin α2ß1 Binding in TGFß1-Evoked Renal Tubular Epithelial Cells.

Chronic Kidney Disease (CKD) is associated with sustained inflammation and progressive fibrosis, changes that have been linked to altered connexin hemichannel-mediated release of adenosine triphosphate (ATP). Kidney fibrosis develops in response to increased deposition of extracellular matrix (ECM), and up-regulation of collagen I is an early marker of renal disease. With ECM remodeling known to promote a loss of epithelial stability, in the current study we used a clonal human kidney (HK2) model of proximal tubular epithelial cells to determine if collagen I modulates changes in cell function, via connexin-43 (Cx43) hemichannel ATP release. HK2 cells were cultured on collagen I and treated with the beta 1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFß1) ± the Cx43 mimetic Peptide 5 and/or an anti-integrin α2ß1 neutralizing antibody. Phase microscopy and immunocytochemistry observed changes in cell morphology and cytoskeletal reorganization, whilst immunoblotting and ELISA identified changes in protein expression and secretion. Carboxyfluorescein dye uptake and biosensing measured hemichannel activity and ATP release. A Cytoselect extracellular matrix adhesion assay assessed changes in cell-substrate interactions. Collagen I and TGFß1 synergistically evoked increased hemichannel activity and ATP release. This was paralleled by changes to markers of tubular injury, partly mediated by integrin α2ß1/integrin-like kinase signaling. The co-incubation of the hemichannel blocker Peptide 5, reduced collagen I/TGFß1 induced alterations and inhibited a positive feedforward loop between Cx43/ATP release/collagen I. This study highlights a role for collagen I in regulating connexin-mediated hemichannel activity through integrin α2ß1 signaling, ahead of establishing Peptide 5 as a potential intervention.

Examining Local Cell-to-Cell Signalling in the Kidney Using ATP Biosensing.

Cell-to-cell communication is an essential process for the efficient function of cells and tissues. Central to this is the purinergic transmission of purines, with ligands such as adenosine triphosphate (ATP). Altered cell-to-cell communication, and in particular changes in the paracrine release of extracellular ATP, plays crucial roles in pathophysiological conditions, such as diabetes. ATP biosensing provides a reliable, real-time measurement of local extracellular ATP concentrations. This allows the detection of altered ATP release, which underlies the progression of inflammation and fibrosis and is a potential therapeutic target. Here we describe in a step-by-step basis how to utilize sensitive microelectrode biosensors to detect low, real-time concentrations of ATP, in vitro.

Blocking Connexin-43 mediated hemichannel activity protects against early tubular injury in experimental chronic kidney disease.

BACKGROUND: Tubulointerstitial fibrosis represents the key underlying pathology of Chronic Kidney Disease (CKD), yet treatment options remain limited. In this study, we investigated the role of connexin43 (Cx43) hemichannel-mediated adenosine triphosphate (ATP) release in purinergic-mediated disassembly of adherens and tight junction complexes in early tubular injury. METHODS: Human primary proximal tubule epithelial cells (hPTECs) and clonal tubular epithelial cells (HK2) were treated with Transforming Growth Factor Beta1 (TGF-ß1) ± apyrase, or ATPγS for 48 h. For inhibitor studies, cells were co-incubated with Cx43 mimetic Peptide 5, or purinergic receptor antagonists Suramin, A438079 or A804598. Immunoblotting, single-cell force spectroscopy and trans-epithelial electrical resistance assessed protein expression, cell-cell adhesion and paracellular permeability. Carboxyfluorescein uptake and biosensing measured hemichannel activity and real-time ATP release, whilst a heterozygous Cx43+/- mouse model with unilateral ureteral obstruction (UUO) assessed the role of Cx43 in vivo. RESULTS: Immunohistochemistry of biopsy material from patients with diabetic nephropathy confirmed increased expression of purinergic receptor P2X7. TGF-ß1 increased Cx43 mediated hemichannel activity and ATP release in hPTECs and HK2 cells. The cytokine reduced maximum unbinding forces and reduced cell-cell adhesion, which translated to increased paracellular permeability. Changes were reversed when cells were co-incubated with either Peptide 5 or P2-purinoceptor inhibitors. Cx43+/- mice did not exhibit protein changes associated with early tubular injury in a UUO model of fibrosis. CONCLUSION: Data suggest that Cx43 mediated ATP release represents an initial trigger in early tubular injury via its actions on the adherens and tight junction complex. Since Cx43 is highly expressed in nephropathy, it represents a novel target for intervention of tubulointerstitial fibrosis in CKD. Video Abstract In proximal tubular epithelial cells (PTECs), tight junction proteins, including zona occuludens-1 (ZO-1), contribute to epithelial integrity, whilst the adherens junction protein epithelial (E)-cadherin (ECAD) maintains cell-cell coupling, facilitating connexin 43 (Cx43) gap junction-mediated intercellular communication (GJIC) and the direct transfer of small molecules and ions between cells. In disease, such as diabetic nephropathy, the pro-fibrotic cytokine transforming growth factor beta1 (TGF-ß1) binds to its receptor and recruits SMAD2/3 signalling ahead of changes in gene transcription and up-regulation of Cx43-mediated hemichannels (HC). Uncoupled hemichannels permit the release of adenosine triphosphate (ATP) in to the extracellular space (↑[ATP]e), where ATP binds to the P2X7 purinoreceptor and activates the nucleotide-binding domain and leucine-rich repeat containing (NLR) protein-3 (NLRP3) inflammasome. Inflammation results in epithelial-to-mesenchymal transition (EMT), fibrosis and tubular injury. A major consequence is further loss of ECAD and reduced stickiness between cells, which can be functionally measured as a decrease in the maximum unbinding force needed to uncouple two adherent cells (Fmax). Loss of ECAD feeds forward to further lessen cell-cell coupling exacerbating the switch from GJIC to HC-mediated release of ATP. Reduction in ZO-1 impedes tight junction effectiveness and decreases trans-epithelial resistance (↓TER), resulting in increased paracellular permeability.

Examining Cell-Cell Interactions in the Kidney Using AFM Single-Cell Force Spectroscopy.

The ability of individual cells to synchronize activity is a basic feature of efficient and appropriate tissue function. Central to this is the physicochemical binding between cells through multiprotein complexes that functionally mediate adhesion. Importantly, the direct connection of physical properties and intercellular signaling is of great importance to certain pathologies including diabetes. Atomic force microscopy (AFM) single-cell force spectroscopy (SCFS) is a high-resolution technique that provides a statistically reliable measurement of the minute forces involved in cell tethering and membrane dynamics. Detection of altered nanoscale forces underlying the loss of adhesion in early tubular injury is pivotal for the development of novel therapeutic strategies for diabetic nephropathy. Here we describe the step-by-step use of an integrated AFM-SCFS system designed to measure functional force-displacement in separating renal tubular epithelial cells. Parameters such as unbinding forces, detachment energy, and distance to complete separation can be obtained from force-displacement (F-d) curves and are critical in assessing how physical changes of cellular adhesion contribute to cell contact, coupling, and communication in the diabetic kidney.

Purinergic receptor (P2X7) activation reduces cell-cell adhesion between tubular epithelial cells of the proximal kidney.

Loss of epithelial (E)-cadherin mediated cell-cell adhesion impairs gap junction formation and facilitates hemichannel-mediated ATP release in the diabetic kidney. Linked to inflammation and fibrosis, we hypothesized that local increases in inter-cellular ATP activate P2X7 receptors on neighboring epithelial cells of the proximal tubule, to further impair cell-cell adhesion and ultimately exacerbate tubular injury. Immunoblotting confirmed changes in E-cadherin expression in human kidney cells treated with non-hydrolysable ATPγS ± the P2X7 antagonist, A438079. Atomic force microscopy based single-cell force spectroscopy quantified maximum unbinding force, tether rupture events, and work of detachment. Confocal microscopy assessed cytoskeletal reorganization. Our studies confirmed that ATPγS downregulated E-cadherin expression in proximal kidney cells, loss of which was paralleled by a reduction in intercellular ligation forces, decreased tether rupture events and cytoskeletal remodeling. Co-incubation with A438079 restored loss of adhesion, suggesting that elevated extracellular ATP mediates tubular injury through P2X7 induced loss of E-cadherin mediated adhesion.

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-ß1 secretion was confirmed by ELISA, and the effects of both TGF-ß1 (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-ß1 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-ß1 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-ß secretion and the pattern of change for connexins expression was mimicked by exogenous application of TGF-ß1. The effect of visfatin on Cx-expression and dye transfer were negated in the presence of a TGF-ß1 neutralising antibody. These data suggest that visfatin reduces connexin-mediated intercellular communication in proximal tubule-derived epithelial cells via a TGF-ß dependent pathway.

'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 ß-catenin. Down-regulation of protein expression does not involve the pro-fibrotic cytokine TGFß, 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 ß-cells where it is thought to facilitate cell-to-cell communication and augment insulin secretion. However, it is unknown how CaR activation improves ß-cell function. METHODS: Immunocytochemistry and western blotting confirmed the expression of CaR in MIN6 ß-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 ß-cell proliferation. CONCLUSION: Our data suggest that activation of the CaR increases E-cadherin mediated functional tethering between ß-cells and increases expression of L-type VDCC and secretagogue-evoked changes in [Ca(2+)](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.

Glucose decreases extracellular adenosine levels in isolated mouse and rat pancreatic islets.

The pancreatic islets of Langerhans are responsible for the regulated release of the endocrine hormones insulin and glucagon that participate in the control of glucose homeostasis. Abnormal regulation of these hormones can result in glucose intolerance and lead to the development of diabetes. Numerous efforts have been made to better understand the physiological regulators of insulin and glucagon secretion. One of these regulators is the purine nucleoside, adenosine. Though exogenous application of adenosine has been demonstrated to stimulate glucagon release and inhibit insulin release, the physiological significance of this pathway has been unclear. We used a novel 7 µm enzyme-coated electrode biosensor to measure adenosine levels in isolated rodent islets. In the mouse islets, basal adenosine levels in the presence of 3 mM glucose were estimated to be 5.7 ± 0.6 µM. As glucose was increased, extracellular adenosine diminished. A 10-fold increase of extracellular KCl increased adenosine levels to 16.4 ± 2.0 µM. This release required extracellular Ca (2+) suggesting that it occurred via an exocytosis-dependent mechanism. We also found that while rat islets were able to convert exogenous ATP into adenosine, mouse islets were unable to do this. Our study demonstrates for the first time the basal levels of adenosine and its inverse relationship to extracellular glucose in pancreatic islets.

17Beta-estradiol elevates cGMP and, via plasma membrane recruitment of protein kinase GIalpha, stimulates Ca2+ efflux from rat hepatocytes.

Rapid non-genomic effects of 17beta-estradiol, the principal circulating estrogen, have been observed in a wide variety of cell types. Here we investigate rapid signaling effects of 17beta-estradiol in rat hepatocytes. We show that, above a threshold concentration of 1 nm, 17beta-estradiol, but not 17alpha-estradiol, stimulates particulate guanylyl cyclase to elevate cGMP, which through activation and plasma membrane recruitment of protein kinase G isoform Ialpha, stimulates plasma membrane Ca(2+)-ATPase-mediated Ca(2+) efflux from rat hepatocytes. These effects are extremely rapid in onset and are mimicked by a membrane-impermeant 17beta-estradiol-BSA conjugate, suggesting that 17beta-estradiol acts at the extracellular face of the plasma membrane. We also show that 17beta-estradiol binds specifically to the intact hepatocyte plasma membrane through an interaction that is competed by an excess of atrial natriuretic peptide but also shows many similarities to the pharmacological characteristics of the putative gamma-adrenergic receptor. We, therefore, propose that the observed rapid signaling effects of 17beta-estradiol are mediated either through the guanylyl cyclase A receptor for atrial natriuretic peptide or through the gamma-adrenergic receptor, which is either itself a transmembrane guanylyl cyclase or activates a transmembrane guanylyl cyclase through cross-talk signaling.

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-kappaB and peroxisome proliferator-activated receptor-gamma. 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.

TGF-beta1-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-beta1 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.

TGF-beta1 mediates glucose-evoked up-regulation of connexin-43 cell-to-cell communication in HCD-cells.

BACKGROUND/AIMS: In the current study we examined if the multifunctional cytokine TGF-beta1 mediated glucose-evoked increases in connexin-43(Cx43)-mediated intercellular communication in cells of the human collecting duct (HCD). METHODS: RT-PCR and western blot analysis were used to confirm mRNA and protein expression of TGF-beta1 and Cx43 in HCD-cells. The effect of TGF-beta1 and high glucose (25 mM) on Cx43 protein expression, cytoskeletal organisation and cell-cell communication was determined in the presence/absence of TGF-beta1 specific immuno-neutralising antibodies. Functional cell-cell communication was determined using Ca2+-microfluorimetry. RESULTS: At 24 hrs, high glucose (25 mM) significantly increased Cx43 mRNA and protein expression. Changes were mimicked by TGF-beta1 (2 ng/ml) at low glucose (5 mM). Both high glucose and TGF-beta1 mediated changes were completely reversed by a pan-specific immuno-neutralising antibody to TGF-beta. Furthermore, high glucose-evoked changes were inhibited by a TGF-beta1-specific monoclonal antibody. Mannitol (25 mM), an osmotic control for high glucose, failed to alter Cx43 expression. TGF-beta1 evoked changes in Cx43 expression were biphasic. An early (4-8 hr) transient decrease in expression was followed by an increase in protein expression (12-24 hr). The decrease in Cx43 expression was paralleled by a transient reorganisation of the actin cytoskeleton, whilst increased Cx43 expression at 24 hrs coincided with a TGF-beta1 specific increase in touch-evoked transmission of Ca2+-signals between coupled cells. CONCLUSIONS: High glucose evoked a TGF-beta1 mediated increase in Cx43 expression and gap-junction mediated cell-cell communication in HCD-cells. These changes may maintain epithelial integrity of the collecting duct following hyperglycaemic assault as observed in diabetes.

Phorbol ester-stimulated NF-kappaB-dependent transcription: roles for isoforms of novel protein kinase C.

Since protein kinase C (PKC) isoforms are variously implicated in the activation of NF-kappaB, we have investigated the role of PKC in the activation of NF-kappaB-dependent transcription by the diacyl glycerol (DAG) mimetic, phorbol 12-myristate 13-acetate (PMA), and by tumour necrosis factor (TNF) alpha in pulmonary A549 cells. The PKC selective inhibitors, Ro31-8220, Gö6976, GF109203X and Gö6983, revealed no effect on TNFalpha-induced NF-kappaB DNA binding and a similar lack of effect on serine 32/36 phosphorylated IkappaBalpha and the loss of total IkappaBalpha indicates that activation of the core IKK-IkappaBalpha-NF-kappaB cascade by TNFalpha does not involve PKC. In contrast, differential sensitivity of an NF-kappaB-dependent reporter to Ro31-8220, Gö6976, GF109203X and Gö6983 (EC(50)s 0.46 microM, 0.34 microM, >10 microM and >10 microM respectively) suggests a role for protein kinase D in transcriptional activation by TNFalpha. Compared with TNFalpha, PMA weakly induces NF-kappaB DNA binding and this effect was not associated with serine 32/36 phosphorylation of IkappaBalpha. However, PMA-stimulated NF-kappaB DNA binding was inhibited by Ro31-8220 (10 microM), GF109203X (10 microM) and Gö6983 (10 microM), but not by Gö6976 (10 microM), suggesting a role for novel PKC isoforms. Furthermore, a lack of positive effect of calcium mobilising agents on both NF-kappaB DNA binding and on transcriptional activation argues against major roles for classical PKCs. This, combined with the ability of both GF109203X and Gö6983 to prevent enhancement of TNFalpha-induced NF-kappaB-dependent transcription by PMA, further indicates a role for novel PKCs in NF-kappaB transactivation. Finally, siRNA-mediated knockdown of PKCdelta and epsilon expression did not affect TNFalpha-induced NF-kappaB-dependent transcription. However, knockdown of PKCdelta expression significantly inhibited PMA-stimulated luciferase activity, whereas knockdown of PKCepsilon was without effect. Furthermore, combined knockdown of PKCdelta and epsilon revealed an increased inhibitory effect on PMA-stimulated NF-kappaB-dependent transcription suggesting that PMA-induced NF-kappaB-dependent transcription is driven by novel PKC isoforms, particularly PKCdelta and epsilon.

Function and expression of melatonin receptors on human pancreatic islets.

Melatonin is known to inhibit insulin secretion from rodent beta-cells through interactions with cell-surface MT1 and/or MT2 receptors, but the function of this hormone in human islets of Langerhans is not known. In the current study, melatonin receptor expression by human islets was examined by reverse transcription-polymerase chain reaction (RT-PCR) and the effects of exogenous melatonin on intracellular calcium ([Ca2+]i) levels and islet hormone secretion were determined by single cell microfluorimetry and radioimmunoassay, respectively. RT-PCR amplifications indicated that human islets express mRNAs coding for MT1 and MT2 melatonin receptors, although MT2 mRNA expression was very low. Analysis of MT1 receptor mRNA expression at the single cell level indicated that it was expressed by human islet alpha-cells, but not by beta-cells. Exogenous melatonin stimulated increases in intracellular calcium ([Ca2+]i) in dissociated human islet cells, and stimulated glucagon secretion from perifused human islets. It also stimulated insulin secretion and this was most probably a consequence of glucagon acting in a paracrine fashion to stimulate beta-cells as the MT1 receptor was absent in beta-cells. Melatonin did not decrease 3', 5'-cyclic adenosine monophosphate (cyclic AMP) levels in human islets, but it inhibited cyclic AMP in the mouse insulinoma (MIN6) beta-cell line and it also inhibited glucose-stimulated insulin secretion from MIN6 cells. These data suggest that melatonin has direct stimulatory effects at human islet alpha-cells and that it stimulates insulin secretion as a consequence of elevated glucagon release. This study also indicates that the effects of melatonin are species-specific with primarily an inhibitory role in rodent beta-cells and a stimulatory effect in human islets.

Atrial natriuretic peptide attenuates elevations in Ca2+ and protects hepatocytes by stimulating net plasma membrane Ca2+ efflux.

Elevations in intracellular Ca(2+) concentration and calpain activity are common early events in cellular injury, including that of hepatocytes. Atrial natriuretic peptide is a circulating hormone that has been shown to be hepatoprotective. The aim of this study was to examine the effects of atrial natriuretic peptide on potentially harmful elevations in cytosolic free Ca(2+) and calpain activity induced by extracellular ATP in rat hepatocytes. We show that atrial natriuretic peptide, through protein kinase G, attenuated both the amplitude and duration of ATP-induced cytosolic Ca(2+) rises in single hepatocytes. Atrial natriuretic peptide also prevented stimulation of calpain activity by ATP, taurolithocholate, or Ca(2+) mobilization by thapsigargin and ionomycin. We therefore investigated the cellular Ca(2+) handling mechanisms through which ANP attenuates this sustained elevation in cytosolic Ca(2+). We show that atrial natriuretic peptide does not modulate the release from or re-uptake of Ca(2+) into intracellular stores but, through protein kinase G, both stimulates plasma membrane Ca(2+) efflux from and inhibits ATP-stimulated Ca(2+) influx into hepatocytes. These findings suggest that stimulation of net plasma membrane Ca(2+) efflux (to which both Ca(2+) efflux stimulation and Ca(2+) influx inhibition contribute) is the key process through which atrial natriuretic peptide attenuates elevations in cytosolic Ca(2+) and calpain activity. Moreover we propose that plasma membrane Ca(2+) efflux is a valuable, previously undiscovered, mechanism through which atrial natriuretic peptide protects rat hepatocytes, and perhaps other cell types, against Ca(2+)-dependent injury.