Visfatin Reduces Gap Junction Mediated Cell-to-Cell Communication in Proximal Tubule-Derived Epithelial Cells

Abstract

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-β1 secretion was confirmed by ELISA, and the effects of both TGF-β1 (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-β1 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-β1 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-β secretion and the pattern of change for connexins expression was mimicked by exogenous application of TGF-β1. The effect of visfatin on Cx-expression and dye transfer were negated in the presence of a TGF-β1 neutralising antibody. These data suggest that visfatin reduces connexin-mediated intercellular communication in proximal tubule-derived epithelial cells via a TGF-β dependent pathway.

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

“…Having confirmed that in both acute and chronic time points, glucose/TGF-β1 evoked changes in CX expression are paralleled by reduced GJIC, we wanted to determine if hemi-channel activity increased as a compensatory mechanism to maintain cell-to-cell communication. Carboxyfluorescein is a membrane impermeant dye that has been previously used to assess hemi-channel opening in a variety of cell types [31]. An increase in carbenoxleone-sensitive dye uptake suggested that the number and/or activity of hemi-channels was elevated in cells cultured in TGF-β1 for 48h and 7days.…”

“…Described previously [31], cells cultured on glass cover-slips were transferred to the recording bath and perfused (2ml/min) with physiological saline solution composed of (mM): 127 NaCl, 1.9 KCl, 1.2 KH 2 PO 4 , 26 NaHCO 3 , 1 MgCl 2 , 2CaCl 2 and 10 D-Glucose bubbled with 95% O 2 / 5 % CO 2 and maintained at 32°C. Cells were visualized (x600 magnification) using IR-DIC optics (Olympus BX51W1 microscope and Hitachi CCD camera).…”

Epithelial-to-Mesenchymal Transition in Diabetic Nephropathy: Fact or Fiction?

“…ZO-1 and N-cadherin) and have been shown to be downregulated in podocytes, endothelial cells, and tubular cells in vitro , in diabetic mouse models, and human DN [ 64 ]. Interestingly, it has been found that visfatin, an adipokine that is elevated in patients with type 2 diabetes, inhibits the expression of CX43 in proximal tubular cells, illustrating the intricacy involved in understanding the in vivo situation of diabetic patients [ 65 ].…”

Cell-Cell Communication in the Tumor Microenvironment, Carcinogenesis, and Anticancer Treatment

“…Different types of junctions, which connect cells to their microenvironment, are part of a communication network essential for signaling. The loss of cell-cell adhesion can be associated with a subsequent reduction of gap junctions or with local changes in the environment and these changes can then activate ion-related receptors in neighboring cells [5]. Such events demonstrate that different stimuli can have vastly different effects.…”