title: Visfatin reduces gap junction mediated cell-to-cell communication in proximal tubule-derived epithelial cells.
abstract: OBJECTIVE
  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.
authors: School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK.
authors: Claire E
authors: CE
authors: Hills
authors: Michael I
authors: MI
authors: Kerr
authors: Mark J
authors: MJ
authors: Wall
authors: Paul E
authors: PE
authors: Squires
mesh_terms: D002450:Cell Communication / Q000187:drug effects*; D002460:Cell Line / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D000072259:Connexin 26; D018031:Connexin 43 / Q000378:metabolism; D017630:Connexins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D017629:Gap Junctions / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D054409:Nicotinamide Phosphoribosyltransferase / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
chemical_list: D018031:Connexin 43; D017630:Connexins; C000607042:GJB2 protein, human; D053773:Transforming Growth Factor beta1; D000072259:Connexin 26; D054409:Nicotinamide Phosphoribosyltransferase