Abstract
ObjectiveConnexin 43 (Cx43) is a gap junction‐forming protein which mediates the intercellular passage of molecules < 1000 Da. In the kidney, Cx43 was reported in glomerular vessels and mesangium. Own results prompted us to reevaluate the distribution of Cx43 and its phosphorylated form, phospho‐S368‐Cx43 (pCx43), within the different zones of the rat kidney and in cultured fibroblasts in more detail.MethodsCx43 and pCx43 were localized by immunofluorescence in rat kidney sections and cultured human fibroblasts. High‐resolution immunostaining of Cx43 was achieved by ultrastructural immunogold labelling on electron microscopy sections. Double‐labelings with anti‐aquaporin 1 (AQP1), aquaporin 2 (AQP2), zonula occludens‐1 (ZO‐1), and cyclooxygenase‐2 (COX‐2) were conducted to clarify Cx43 distribution in the rat renal medulla. Cultured human fibroblasts served to study intercellular transfer of fluorescent lucifer yellow.ResultsCx43 was distributed in renal arterial and arteriolar endothelia, lymphatic vessel walls, peritubular capillaries, and glomerular compartments. In the interstitium, Cx43 was present as linear or punctate signals of the cell membrane of fibroblasts, in colocalization with pCx43. Cx43‐positive medullary fibroblasts co‐expressed COX‐2. No colocalization was detected between Cx43 and AQP1 or AQP2. Ultrastructural analysis showed Cx43 immunoreactivity at membrane contact zones between fibroblasts and in isolated cell processes of inner medullary interstitium. Cultured fibroblasts showed extensive, punctate Cx43 and pCx43 signals between adjacent cells. Signal was further distributed in non‐junctional areas of plasma membrane, suggesting Cx43 hemichannel functions. Lucifer yellow spread rapidly (10 min) between fibroblasts suggesting intercellular exchange via gap junctions, which was blocked by the gap junctional inhibitor, 18β‐glycyrrhetinic acid.ConclusionsCx43 in rat kidney shows prominent glomerular, vascular, and interstitial distribution, while epithelia, with exception of the podocytes, were negative. Focusing on interstitial fibroblasts, Cx43‐mediated information transfer was demonstrated. A constitutive nature of Cx43 phosphorylation was established in these cells. Our data suggest a cell‐specific heterogeneity of Cx43‐positive gap junctions mediating information exchange among renal cell types.
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