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 METHODS: Using the Nephroseq database, analysis of unpublished transcriptomic data, examined gene expression and correlation in human CKD.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.

Quantification of bush-cricket acoustic trachea mechanics using Atomic Force Microscopy nanoindentation.

Derived from the respiratory tracheae, bush-crickets' acoustic tracheae (or ear canals) are hollow tubes evolved to transmit sounds from the external environment to the interior ear. Due to the location of the ears in the forelegs, the acoustic trachea serves as a structural element that can withstand large stresses during locomotion. In this study, we report a new Atomic Force Microscopy Force Spectroscopy (AFM-FS) approach to quantify the mechanics of taenidia in the bush-cricket Mecopoda elongata. Mechanical properties were examined over the longitudinal axis of hydrated taenidia, by indenting single fibres using precision hyperbolic tips. Analysis of the force-displacement (F-d) extension curves at low strains using the Hertzian contact model showed an Elastic modulus distribution between 13.9 MPa to 26.5 GPa, with a mean of 5.2 ± 7 GPa and median 1.03 GPa. Although chitin is the primary component of stiffness, variation of elasticity in the nanoscale suggests that resilin significantly affects the mechanical properties of single taenidia fibres (38% of total data). For indentations up to 400 nm, an intricate chitin-resilin response was observed, suggesting structural optimization between compliance and rigidity. Finite-element analysis on composite materials demonstrated that the Elastic modulus is sensitive to the percentage of resilin and chitin content, their location and structural configuration. Based on our results, we propose that the distinct moduli of taenidia fibres indicate sophisticated evolution with elasticity playing a key role in optimization. STATEMENT OF SIGNIFICANCE: In crickets and bush-crickets, the foreleg tracheae have evolved into acoustic canals, which transport sound to the ears located on the tibia of each leg. Tracheae are held open by spiral cuticular micro-fibres called taenidia, which are the primary elements of mechanical reinforcement. We developed an AFM-based method to indent individual taenidia at the nanometre level, to quantify local mechanical properties of the interior acoustic canal of the bush-cricket Mecopoda elongata, a model species in hearing research. Taenidia fibres were immobilized on a hard substrate and the indenter directly approached the epicuticle surface. This is the first characterization of the nano-structure of unfixed tracheal taenidia, and should pave the way for further in vivo mechanical investigations of auditory structures.

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.

Carboxyfluorescein Dye Uptake to Measure Connexin-mediated Hemichannel Activity in Cultured Cells.

Connexins are membrane bound proteins that facilitate direct and local paracrine mediated cell-to-cell communication through their ability to oligomerise into hexameric hemichannels. When neighbouring channels align, they form gap-junctions that provide a direct route for information transfer between cells. In contrast to intact gap junctions, which typically open under physiological conditions, undocked hemichannels have a low open probability and mainly open in response to injury. Hemichannels permit the release of small molecules and ions (approximately 1kDa) into the local intercellular environment, and excessive expression/activity has been linked to a number of disease conditions. Carboxyfluorescein dye uptake measures functional expression of hemichannels, where increased hemichannel activity/function reflects increased loading. The technique relies on the uptake of a membrane-impermeable fluorescent tracer through open hemichannels, and can be used to compare channel activity between cell monolayers cultured under different conditions, e.g. control versus disease. Other techniques, such as biotinylation and electrophysiology can measure cell surface expression and hemichannel open probability respectively, however, carboxyfluorescein uptake provides a simple, rapid and cost-effective method to determine hemichannel activity in vitro in multiple cell types. Graphic abstract: Using dye uptake to measure hemichannel activity.

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.

Connexin-mediated cell communication in the kidney: A potential therapeutic target for future intervention of diabetic kidney disease?: Joan Mott Prize Lecture.

The ability of cells to communicate and synchronise their activity is essential for the maintenance of tissue structure, integrity and function. A family of membrane-bound proteins called connexins are largely responsible for mediating the local transfer of information between cells. Assembled in the cell membrane as a hexameric connexon, they either function as a conduit for paracrine signalling, forming a transmembrane hemi-channel, or, if aligned with connexons on neighbouring cells, form a continuous aqueous pore or gap junction, which allows for the direct transmission of metabolic and electrical signals. Regulation of connexin synthesis and activity is critical to cellular function, and a number of diseases are attributed to changes in the expression and/or function of these important proteins. A link between hyperglycaemia, connexin expression, altered nucleotide concentrations and impaired function highlights a potential role for connexin-mediated cell communication in complications of diabetes. In the diabetic kidney, glycaemic injury is the leading cause of end-stage renal failure, reflecting multiple aetiologies including glomerular hyperfiltration, albuminuria, increased deposition of extracellular matrix and tubulointerstitial fibrosis. Loss of connexin-mediated cell-to-cell communication in diabetic nephropathy may represent an early sign of disease progression, but our understanding of the process remains severely limited. This review focuses on recent evidence demonstrating that glucose-evoked changes in connexin-mediated cell communication and associated purinergic signalling may contribute to the pathogenesis of kidney disease in diabetes, highlighting the tantalising potential of targeting these proteins as a novel therapeutic intervention.

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.

Quantifying cellular mechanics and adhesion in renal tubular injury using single cell force spectroscopy.

Tubulointerstitial fibrosis represents the major underlying pathology of diabetic nephropathy where loss of cell-to-cell adhesion is a critical step. To date, research has predominantly focussed on the loss of cell surface molecular binding events that include altered protein ligation. In the current study, atomic force microscopy single cell force spectroscopy (AFM-SCFS) was used to quantify changes in cellular stiffness and cell adhesion in TGF-ß1 treated kidney cells of the human proximal tubule (HK2). AFM indentation of TGF-ß1 treated HK2 cells showed a significant increase (42%) in the elastic modulus (stiffness) compared to control. Fluorescence microscopy confirmed that increased cell stiffness is accompanied by reorganization of the cytoskeleton. The corresponding changes in stiffness, due to F-actin rearrangement, affected the work of detachment by changing the separation distance between two adherent cells. Overall, our novel data quantitatively demonstrate a correlation between cellular elasticity, adhesion and early morphologic/phenotypic changes associated with tubular injury. FROM THE CLINICAL EDITOR: Diabetes affects many patients worldwide. One of the long term problems is diabetic nephropathy. Here, the authors utilized atomic force microscopy single cell force spectroscopy (AFM- SCFS) to study cellular stiffness and cell adhesion after TGF1 treatment in human proximal tubule kidney cells. The findings would help further understand the overall disease mechanism in diabetic patients.

Mind the gap: connexins and cell-cell communication in the diabetic kidney.

Connexins, assembled as a hexameric connexon, form a transmembrane hemichannel that provides a conduit for paracrine signalling of small molecules and ions to regulate the activity and function of adjacent cells. When hemichannels align and associate with similar channels on opposing cells, they form a continuous aqueous pore or gap junction, allowing the direct transmission of metabolic and electrical signals between coupled cells. Regulation of gap junction synthesis and channel activity is critical for cell function, and a number of diseases can be attributed to changes in the expression/function of these important proteins. Diabetic nephropathy is associated with several complex metabolic and inflammatory responses characterised by defects at the molecular, cellular and tissue level. In both type 1 and type 2 diabetes, glycaemic injury of the kidney is the leading cause of end-stage renal failure, a consequence of multiple aetiologies, including increased deposition of extracellular matrix, glomerular hyperfiltration, albuminuria and tubulointerstitial fibrosis. In diabetic nephropathy, loss of connexin mediated cell-cell communication within the nephron may represent an early sign of disease; however, our current knowledge of the role of connexins in the diabetic kidney is sparse. This review highlights recent evidence demonstrating that maintenance of connexin-mediated cell-cell communication could benefit region-specific renal function in diabetic nephropathy and suggests that these proteins should be viewed as a tantalising novel target for therapeutic intervention.

Quantitative investigation of calcimimetic R568 on beta cell adhesion and mechanics using AFM single-cell force spectroscopy.

In this study we use a novel approach to quantitatively investigate mechanical and interfacial properties of clonal ß-cells using AFM-Single Cell Force Spectroscopy (SCFS). MIN6 cells were incubated for 48 h with 0.5 mM Ca(2+) ± the calcimimetic R568 (1 µM). AFM-SCFS adhesion and indentation experiments were performed by using modified tipless cantilevers. Hertz contact model was applied to analyse force-displacement (F-d) curves for determining elastic or Young's modulus (E). Our results show CaSR-evoked increases in cell-to-cell adhesion parameters and E modulus of single cells, demonstrating that cytomechanics have profound effects on cell adhesion characterization.

The calcium-sensing receptor and ß-cell function.

In addition to its central role controlling systemic calcium homeostasis, the extracellular calcium-sensing receptor (CaSR) can be found on multiple cell types not associated with controlling plasma calcium. The endocrine pancreas is one such tissue, and it is apparent that the receptor plays an important role in regulating ß-cell function. During exocytosis, divalent cations are coreleased with insulin and their concentration within the restricted intercellular compartments of the pancreatic islet increases sufficiently to activate the CaSR on neighboring cells. Acute and chronic activation of the receptor has multiple effects on the ß-cell, from increasing cadherin-based cell-cell adhesion to directly altering the expression and function of various potassium and voltage-dependent calcium channels. The promiscuous activation of multiple binding partners improves cell adhesion, cell coupling, and cell-to-cell communication within the islet and is the basis for the effect of the CaSR on ß-cell function and improved glucose responsiveness.

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.

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 Ca²âº-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 [Ca²âº](i) that was dependent upon thapsigargin-sensitive store release and Ca²âº 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 Ca²âº-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.

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.