Targeting senescence to prevent diabetic kidney disease: Exploring molecular mechanisms and potential therapeutic targets for disease management.

Background/aims: As a microvascular complication, diabetic kidney disease is the leading cause of chronic kidney disease and end-stage renal disease worldwide. While the underlying pathophysiology driving transition of diabetic kidney disease to renal failure is yet to be fully understood, recent studies suggest that cellular senescence is central in disease development and progression. Consequently, understanding the molecular mechanisms which initiate and drive senescence in response to the diabetic milieu is crucial in developing targeted therapies that halt progression of renal disease.

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.

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.

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.

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.

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.

Danegaptide Prevents TGFβ1-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.

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.

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.

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-β1) ± apyrase, or ATPγS for 48 h.

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.

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.

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.

Transforming Growth Factor Beta 1 Drives a Switch in Connexin Mediated Cell-to-Cell Communication in Tubular Cells of the Diabetic Kidney.

Background/aims: Changes in cell-to-cell communication have been linked to several secondary complications of diabetes, but the mechanism by which connexins affect disease progression in the kidney is poorly understood. This study examines a role for glucose-evoked changes in the beta1 isoform of transforming growth factor (TGFβ1), on connexin expression, gap-junction mediated intercellular communication (GJIC) and hemi-channel ATP release from tubular epithelial cells of the proximal renal nephron.

Methods: Biopsy material from patients with and without diabetic nephropathy was stained for connexin-26 (CX26) and connexin-43 (CX43).

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

Unlabelled: 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-β1 treated kidney cells of the human proximal tubule (HK2).

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.

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 β-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).

The calcium-sensing receptor and β-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 β-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.

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.

'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.

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 Ca²⁺-dependent mechanical stimulation within a novel system of the human collecting duct (HCD).

Calcium-sensing receptor activation increases cell-cell adhesion and β-cell function.

Background/aims: The extracellular calcium-sensing receptor (CaR) is expressed in pancreatic β-cells where it is thought to facilitate cell-to-cell communication and augment insulin secretion. However, it is unknown how CaR activation improves β-cell function.

Methods: Immunocytochemistry and western blotting confirmed the expression of CaR in MIN6 β-cell line.