Connexin-43 hemichannel mediated ATP release stimulates fibroblast activation in an in vitro model of diabetic kidney disease

Aims: Tubulointerstitial fibrosis is the underlying pathology of diabetic nephropathy and develops in response to aberrant activation of multiple cell types within and around the proximal tubule of the kidney, including extracellular matrix (ECM) producing fibroblasts. Whilst we previously reported a role for connexin-43 (Cx43) hemichannel activity in tubule inflammation, the function and extent to which fibroblast hemichannels contribute to this damage, remains to be determined. Methods: Human kidney fibroblasts (TK173) were cultured in the glucose-evoked cytokine transforming growth factor-beta1 (TGFb1) ± Cx43 hemichannel blocker Tonabersat, for 48hrs. Immunoblotting determined protein expression, whilst carboxyfluorescein dye uptake and an ATP lite assay assessed hemichannel-mediated ATP release. Results: TGFb1 significantly increased hemichannel-mediated dye uptake by 73.6±3.9%, (P < 0.001, n = 4) in TK173 cells compared to control, an effect reduced when co-incubated with Tonabersat (P < 0.01, n = 4). The profibrotic cytokine TGFb1 increased ATP release by 92.8±13.9%, with Tonabersat decreasing ATP release by 90.8±25.8% (P < 0.05, n = 4). Immunoblotting deter- mined that TGFb1 increased expression of the ECM pro-teins, fibronectin (330.8±16.4%, P < 0.001, n = 5) and collagen I (42.9±4.6%, P < 0.001, n = 5), and the prin-cipal Wnt signalling mediator b-catenin (91.8±6.6%, P < 0.001, n = 5) compared to control. Tonabersat restored expression of fibronectin, collagen I and b-catenin by 98±29.6%, (P < 0.01, n = 5), 20±6.8%, (P < 0.05, n = 5), and 56.9±26.7%, (P < 0.05, n = 5) respectively. Conclusion: These data suggest that glucose-evoked changes in TGFb1, increase hemichannel-mediated ATP release and downstream expression of fibrotic candidates in human renal fibroblasts. The study indicates that Cx43 hemichannels may represent a future therapeutic target for alleviating tubulointerstitial fibrosis in people with diabetic kidney disease

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 medul- lary fibroblasts (TK173) were treated in low (5mM) or high (25mM) glucose ± transforming growth factor beta-1 (TGFβ1)±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 het- erotypic interactions. Results: Tonabersat partially negated glucose/TGFβ1-induced increases in Cx43 hemichannel-mediated ATP release and downstream changes in adherens junc- tion 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 en- hances TGFβ1-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

The Role of the NLRP3 Inflammasome in Mediating Glomerular and Tubular Injury in Diabetic Nephropathy

The NOD-like receptor protein 3 (NLRP3) inflammasome is a multi-protein signalling complex integral to the chronic inflammatory response, activated in response to sterile and non-sterile cellular damage. The assembly and activation of the NLRP3 inflammasome comprise a two-step process involving nuclear factor kappa B (NFkB)-mediated priming, followed by canonical, non-canonical or alternative signalling pathways. These result in the maturation and release of inflammatory cytokines interleukin 1 beta (IL1ß) and interleukin- 18 (IL18), which are associated with chronic inflammatory conditions including diabetic kidney disease. Diabetic nephropathy is a condition affecting ~40% of people with diabetes, the key underlying pathology of which is tubulointerstitial inflammation and fibrosis. There is growing evidence to suggest the involvement of the NLRP3 inflammasome in this chronic inflammation. Early deterioration of kidney function begins in the glomerulus, with tubular inflammation dictating the progression of late- stage disease. Priming and activation of the NLRP3 inflammasome have been linked to several clinical markers of nephropathy including proteinuria and albuminuria, in addition to morphological changes including mesangial expansion. Treatment options for diabetic nephropathy are limited, and research that examines the impact of directly targeting the NLRP3 inflammasome, or associated downstream components are beginning to gain favour, with several agents currently in clinical trials. This review will explore a role for NLRP3 inflammasome activation and signalling in mediating inflammation in diabetic nephropathy, specifically in the glomerulus and proximal tubule, before briefly describing the current position of therapeutic research in this field

Collagen I Modifies Connexin‐43 Hemichannel Activity via Integrin α2β1 Binding in TGFβ1‐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 (TGFβ1) ± the Cx43 mimetic Peptide 5 and/or an anti‐integrin α2β1 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 TGFβ1 synergistically evoked increased hemichannel activity and ATP release. This was paralleled by changes to markers of tubular injury, partly mediated by integrin α2β1/integrin‐like kinase signaling. The co‐incubation of the hemichannel blocker Peptide 5, reduced collagen I/TGFβ1 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 α2β1 signaling, ahead of establishing Peptide 5 as a potential intervention

Connexin mediated cell communication in the kidney, a potential therapeutic target for future intervention of diabetic kidney disease?

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 function either as a conduit for paracrine signalling, forming a trans-membrane 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, however, our understanding of the process remains severely limited. This review focusses 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

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 patho- physiological 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

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 &lsquo;danger signals&rsquo; 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

A role for connexin-43 hemichannel-mediated adenosine triphosphate triphosphate (ATP) release in inflammation of the diabetic proximal tubule

Aim: Glucose-evoked transforming growth factor beta1 (TGF-b1) dysregulates connexin (Cx)-mediated gap-junction intercellular communication and increases hemichannel adenosine triphosphate (ATP) release. Linked with inflammation in diabetes, we investigated a role for ATP in activation of the NLRP3 inflammasome and production of inflammatory mediators in models of diabetic kidney disease. Methods: Human proximal tubule epithelial cells (HK2) were treated for 48 h with ATPyS (100micoM) or TGF-b1 (10ng/ ml) +/- NLRP3 inhibitor CY09 (10microM), +/- caspase1 inhibitor ACYVAD-CHO (10microM), +/-P2X7 inhibitor A438079 (50microM). Primary proximal tubule cells (hPTECs) were treated with TGF-b1 (10ng/ml) +/- Cx43 inhibitor peptide5 (25microM). Protein expression was determined by immunoblotting, whilst a GLO assay assessed caspase1 activity. Results: TGF-b1 upregulated NLRP3, caspase1, IL1b and IL18 to 285.1 +/- 59.62%, 211.2 +/- 17.06%, 261.6 +/- 33.8% and 163.3 +/- 17.75% respectively (n=3, p < 0.001); an effect replicated by ATPyS, with expression at 340.7 +/- 23.75%, 193.7 +/- 13.47%, 220.6 +/- 17.46% and 192.5 +/- 7.5% (n=3, p < 0.001). Caspase1 activity increased to 601.8 +/- 33.6% (TGFb1) and 415.4 +/- 58.7% (ATPyS) (n=3, p < 0.001). CY09 and ACYVAD-CHO restored expression of NLRP3, caspase1, IL1b and IL18 to 139.6 +/- .6% and 108 +/- 2.2%, 139.6 +/- 6.8% and 202.5 +/- 36.2%, 138.4 +/- 13.0% and 152.6 +/- 12.9%, and 160.8 +/- 6.% and 146.6 +/- 4.3% respectively (n=3, p < 0.001). A438079 negated TGF-b1-evoked upregulation, returning the expression of NLRP3 to 120.9 +/- 4.9%, caspase1 to 132.3 +/- 13.7%, IL1b to 160.0 +/- 39.7% and IL18 to 154.5 +/- 14.3% (n=3, p < 0.001). Caspase1 activity decreased to 243.2 +/- 34.2% and 210.2 +/- 22.0% respectively (n=3, p < 0.001). Finally, peptide5 negated TGF-b1-evoked changes in primary hPTECs, returning the expression of NLRP3 to 117.3 +/- 8.6%, caspase1 to 120.1 +/- 39.2%, IL1b to 105.9 +/- 16.8%, and IL18 to 122.8 +/- 18.7% (n=3, p < 0.001). Conclusions: TGF-b1-induced hemichannel ATP release activates the NLRP3 inflammasome, leading to inflammation. Inhibition of Cx43 highlights potential as a future therapeutic target

Inhibition of intern A2B1 negates collagen-1 induced changes in connexion-mediated hemichannel activity in an in vitro model of diabetic nephropathy

Aims: Tubulointerstitial fibrosis, the underlying pathology of diabetic nephropathy, is characterised by extracellular matrix accumulation. Aberrant connexin (Cx) expression and increased adenosine triphosphate (ATP) have been linked to inflammation and fibrosis, and we have previously reported that glucose-evoked transforming growth factor-beta1 (TGF-b1) increases connexin mediated hemichannel ATP release. While extracellular matrix modification has been linked to altered cell phenotype, this study explores a role for collagen-I in regulating connexin-mediated hemichannel activity. Methods: Human kidney (HK-2) proximal tubule cells were cultured on collagen-I (50μg/ml) +/- TGF-b1 (10ng/ml) +/- anti-a2b1 integrin neutralising antibody (2.5μg/ml) for 48h. Uncoated plastic served as the control. The expression of candidate proteins was determined by immunoblotting. Carboxyfluorescein (200μM) dye uptake confirmed hemichannel activity. Results: TGF-b1 decreased Cx26 to 57.0 +/- 3.4% and increased collagen-I expression to 187.0 +/- 3.1% as compared to control (n-4, p < 0.01). Co-incubation of HK-2 cells with TGF-b1 + a2b1 (neutralising antibody against the principle collagen integrin), restored Cx26 expression to 76.2 +/- 6.1% (n-4, p < 0.01). In support of this data, when cultured on collagen-I, Cx26 expression decreased to 58.7 +/- 10.1%; and a further 31.0 +/- 8.9% (n-4, p < 0.01) in the presence of TGF-b1. When cultured on collagen-I +/- TGF-b1, HK-2 cells demonstrated increased carboxyfluorescein uptake to 260.0 +/- 10.9% and 182.5 +/- 8.1%, respectively, while incubation with neutralising a2b1 antibody significantly reduced dye uptake by 27.0 +/- 3.7% and by 24.0 +/- 2.6%, respectively, as compared to uncoated control (n-4, p < 0.01). Conclusion: Collagen-I +/- TGF-b1 decreases Cx26 expression, an effect paralleled by increased hemichannel activity. Inhibition of integrin a2b1 negates collagen/TGF-b1 induced dye uptake, suggesting a role for collagen-I in regulation of connexin-mediated communication

Additional file 1 of Connexin-43 hemichannels orchestrate NOD-like receptor protein-3 (NLRP3) inflammasome activation and sterile inflammation in tubular injury

Additional file 1: Figure 1. Generation of a transgenic (Pax8-rtTA-cre:cx43 flox Cx43-/-) mouse model. (Panel a) Scheme of the transgenic construct used to delete Cx43 in renal tubular cells. The reverse tetracycline-dependent transactivator (rtTA) drives the expression of Cre recombinase specifically in the renal tubular compartment (controlled by Pax8 promoter) after doxycycline administration. (Panel b) An illustration of the experimental design using WT controls and mice with and without Cx43 tubule-specific deletion, subjected to UUO. Kidneys were collected after 10 days of UUO for RNA extraction, renal morphometry and immunostainings. (Panel c) Connexin 43 immunostainings (in green) were performed on cryosections from injured kidneys of mice with and without Cx43 tubule-specific deletion after 10 days of UUO. Connexin 43 is undetectable in UUO-Cx43-/- mice. Sections were counterstained with Evans Blue (red). (Panel d) Connexin 43 mRNA increased expression was blunted in injured kidneys of mice with Cx43 tubule-specific deletion after 10 days of UUO