Connexin 43: a New Therapeutic Target Against Chronic Kidney Disease

Chronic kidney disease is an incurable to date pathology with a continuously growing incidence that contributes to the increase of the number of deaths worldwide. With currently no efficient prognostic or therapeutic options being available, the only possibility for treatment of end-stage renal disease is renal replacement therapy through dialysis or transplantation. Understanding the molecular mechanisms participating in the progression of renal diseases and uncovering the pathways implicated will permit the identification of novel and more efficient targets of therapy. Connexin43 was recently identified as a novel player in the development of chronic kidney disease. It was found de novo expressed and/or differentially localized in various renal cell populations during progression of renal disease, indicating an abnormal connexin signaling, both in patients and animal models. Subsequent in vivo studies demonstrated that connexin43 is involved in mediating inflammatory and fibrotic processes contributing to renal damage. Genetic, pharmaco-genetic or peptide-based inhibition of connexin43 in animal models and cell culture systems was successful in preventing the progression of the pathology and preserving the cell phenotypes. This review will summarize the recent advances on connexin43 in the field of kidney diseases and discuss the potential of future connexin43-based therapies against chronic kidney disease

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

Crosstalk mechanisms between glomerular endothelial cells and podocytes in renal diseases and kidney transplantation

The glomerular filtration barrier (GFB), composed of endothelial cells, glomerular basement membrane, and podocytes, is a unique structure for filtering blood while detaining plasma proteins according to size and charge selectivity. Structurally, the fenestrated endothelial cells, which align the capillary loops, are in close proximity to mesangial cells. Podocytes are connected by specialized intercellular junctions known as slit diaphragms and are separated from the endothelial compartment by the glomerular basement membrane. Podocyte-endothelial cell communication or crosstalk is required for the development and maintenance of an efficient filtration process in physiological conditions. In pathological situations, communication also has an essential role in promoting or delaying disease progression. Podocytes and endothelial cells can secrete signaling molecules, which act as crosstalk effectors and, through binding to their target receptors, can trigger bidirectional paracrine or autocrine signal transduction. Moreover, the emerging evidence of extracellular vesicles derived from various cell types engaging in cell communication has also been reported. In this review, we summarize the principal pathways involved in the development and maintenance of the GFB and the progression of kidney disease, particularly in kidney transplantation

Deletion of Notch3 Impairs Contractility of Renal Resistance Vessels Due to Deficient Ca2+ Entry

Notch3 plays an important role in the differentiation and development of vascular smooth muscle cells. Mice lacking Notch3 show deficient renal autoregulation. The aim of the study was to investigate the mechanisms involved in the Notch3-mediated control of renal vascular response. To this end, renal resistance vessels (afferent arterioles) were isolated from Notch3(-/-) and wild-type littermates (WT) and stimulated with angiotensin II (ANG II). Contractions and intracellular Ca2+ concentrations were blunted in Notch3(-/-) vessels. ANG II responses in precapillary muscle arterioles were similar between the WT and Notch3(-/-) mice, suggesting a focal action of Notch3 in renal vasculature. Abolishing stored Ca2+ with thapsigargin reduced Ca2+ responses in the renal vessels of the two strains, signifying intact intracellular Ca2+ mobilization in Notch3(-/-). EGTA (Ca2+ chelating agent), nifedipine (L-type channel-blocker), or mibefradil (T-type channel-blocker) strongly reduced contraction and Ca2+ responses in WT mice but had no effect in Notch3(-/-) mice, indicating defective Ca2+ entry. Notch3(-/-) vessels responded normally to KCl-induced depolarization, which activates L-type channels directly. Differential transcriptomic analysis showed a major down-regulation of Cacna1h gene expression, coding for the alpha(1H) subunit of the T-type Ca2+ channel, in Notch3(-/-) vessels. In conclusion, renal resistance vessels from Notch3(-/-) mice display altered vascular reactivity to ANG II due to deficient Ca2+-entry. Consequently, Notch3 is essential for proper excitation-contraction coupling and vascular-tone regulation in the kidney

Deletion of Notch3 Impairs Contractility of Renal Resistance Vessels Due to Deficient Ca2+ Entry

Notch3 plays an important role in the differentiation and development of vascular smooth muscle cells. Mice lacking Notch3 show deficient renal autoregulation. The aim of the study was to investigate the mechanisms involved in the Notch3-mediated control of renal vascular response. To this end, renal resistance vessels (afferent arterioles) were isolated from Notch3−/− and wild-type littermates (WT) and stimulated with angiotensin II (ANG II). Contractions and intracellular Ca2+ concentrations were blunted in Notch3−/− vessels. ANG II responses in precapillary muscle arterioles were similar between the WT and Notch3−/− mice, suggesting a focal action of Notch3 in renal vasculature. Abolishing stored Ca2+ with thapsigargin reduced Ca2+ responses in the renal vessels of the two strains, signifying intact intracellular Ca2+ mobilization in Notch3−/−. EGTA (Ca2+ chelating agent), nifedipine (L-type channel-blocker), or mibefradil (T-type channel-blocker) strongly reduced contraction and Ca2+ responses in WT mice but had no effect in Notch3−/− mice, indicating defective Ca2+ entry. Notch3−/− vessels responded normally to KCl-induced depolarization, which activates L-type channels directly. Differential transcriptomic analysis showed a major down-regulation of Cacna1h gene expression, coding for the α1H subunit of the T-type Ca2+ channel, in Notch3−/− vessels. In conclusion, renal resistance vessels from Notch3−/− mice display altered vascular reactivity to ANG II due to deficient Ca2+-entry. Consequently, Notch3 is essential for proper excitation–contraction coupling and vascular-tone regulation in the kidney.publishedVersio

Evaluating a role for Connexin-43 hemichannel mediated ATP release in priming and activation of the NLRP3 inflammasome in a human model of diabetic kidney disease

Aims: Sterile inflammation in diabetic nephropathy is mediated by the Nod-like receptor pyrin domain contain- ing-3 (NLRP3) inflammasome, an integral driver of the innate immune response that mediates tissue damage in multiple age-associated diseases. We recently demonstrated that increased connexin-43 (Cx43) hemichannel activity drives inflammation in nephropathy. Here we evaluate a role for Cx43 hemichannel-mediated ATP re- lease in NLRP3 priming and activation. Methodology: Primary proximal tubule epithelial cells (hPTECs) isolated from renal biopsy, were cultured in 5mM or 25mM glucose ± interleukin-1β (IL1β; 10ng) and tumour necrosis factor alpha (TNFα; 10ng) ± Cx43 hemichannel blocker Tonabersat for 48hrs. Carboxyfluorescein dye uptake and ATPlite assays assessed hemichannel mediated ATP release. Quantitative real time PCR (qRT-PCR), caspase Glo-1 and inflammation arrays assessed NLRP3 priming and activation. Results: Carboxyfluorescein dye uptake and ATP re- lease were increased in 25mM glucose+cytokine treated hPTECs by 61±3.2% (P < 0.001, N = 4) and 54±6.4% (P < 0.001, N = 6) respectively compared to control, effects attenuated by Tonabersat (P < 0.001, N = 6). Quantitative RT-PCR evaluated priming of the NLRP3 inflammasome with IL1β (97.7±0.69%, P < 0.001, N = 5) and NLRP3 (81±9.4%, P < 0.001, N = 5) expression increased in high glucose+cytokine treated cells compared to control. Tonabersat reduced expression by 51±8.4%, (P < 0.001, N = 5) and 57±5.3% (P < 0.001, N = 5) respectively. In 25mM glucose+cytokine treated hPTECs, Tonabersat decreased caspase1 activity by 24±6%, (P < 0.05, N = 3) and IL1β secretion by 18±5.3% (P < 0.01, N = 4) respectively compared to control. Conclusion: Cx43 hemichannel-mediated ATP release primes and activates the NLRP3 inflammasome, effects negated by Tonabersat. Our data highlights Cx43 hemichannels as a target for dampening NLRP3-induced inflammation in the diabetic kidney

The RenTg mice: a powerful tool to study renin-dependent chronic kidney disease.

BACKGROUND: Several studies have shown that activation of the renin-angiotensin system may lead to hypertension, a major risk factor for the development of chronic kidney disease (CKD). The existing hypertension-induced CDK mouse models are quite fast and consequently away from the human pathology. Thus, there is an urgent need for a mouse model that can be used to delineate the pathogenic process leading to progressive renal disease. The objective of this study was dual: to investigate whether mice overexpressing renin could mimic the kinetics and the physiopathological characteristics of hypertension-induced renal disease and to identify cellular and/or molecular events characterizing the different steps of the progression of CKD. METHODOLOGY/PRINCIPAL FINDINGS: We used a novel transgenic strain, the RenTg mice harboring a genetically clamped renin transgene. At 3 months, heterozygous mice are hypertensive and slightly albuminuric. The expression of adhesion markers such as vascular cell adhesion molecule-1 and platelet endothelial cell adhesion molecule-1 are increased in the renal vasculature indicating initiation of endothelial dysfunction. At 5 months, perivascular and periglomerular infiltrations of macrophages are observed. These early renal vascular events are followed at 8 months by leukocyte invasion, decreased expression of nephrin, increased expression of KIM-1, a typical protein of tubular cell stress, and of several pro-fibrotic agents of the TGFβ family. At 12 months, mice display characteristic structural alterations of hypertensive renal disease such as glomerular ischemia, glomerulo- and nephroangio-sclerosis, mesangial expansion and tubular dilation. CONCLUSIONS/SIGNIFICANCE: The RenTg strain develops CKD progressively. In this model, endothelial dysfunction is an early event preceding the structural and fibrotic alterations which ultimately lead to the development of CKD. This model can provide new insights into the mechanisms of chronic renal failure and help to identify new targets for arresting and/or reversing the development of the disease

MiR‐21 is up‐regulated in urinary exosomes of chronic kidney disease patients and after glomerular injury

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