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.
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.
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.
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.
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 (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 heterotypic interactions. Results Tonabersat partially negated glucose/TGFβ1‐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 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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.