Interaction between mesangial cell signaling and cytoskeletal function and high glucose in response to endothelin-1 and mechanical stretch

grantor: University of TorontoIn diabetes, glomerular mesangial cells (MCs) are transformed into a sclerotic phenotype. This is due to the direct effect of high glucose, mechanical stretch due to raised intraglomerular pressure, and autocrine growth factors such as transforming growth factor ß1 (TGF-ß1), angiotensin II (ANG II) and endothelin-1 (ET-1). Activation of protein kinase C (PKC) and mitogen-activated protein kinases (MAPKs) has also been implicated. The loss of afferent arteriolar smooth muscle cell contractility in high glucose, leading to intraglomerular hypertension, is mimicked by the vascular smooth muscle cell-like MCs. The purpose of these studies was to: (1) determine the role of PKC isozymes in ET-1-stimulated MC contraction; (2) identify the mechanism of high glucose-induced loss of MC contractile responsiveness to ET-1; and, (3) examine the interaction of high glucose and mechanical stretch on MC PKC and MAPK signaling. In 5.6 mM normal glucose (NG), ET-1 stimulated MC contraction, ' in situ' PKC activity, and the membrane and particulate translocation of PKC-Ã, -[delta], -[epsilon], but not PKC-ßI, -ßII or -[zeta]. In 30 mM high glucose (HG), MCs did not respond to ET-1, despite normal translocation of PKC-isozymes. This was not due to altered Ca2+ signaling or ETA receptor expression. HG increased basal myosin light chain (MLC 20) phosphorylation, but this was not PKC-dependent. HG stimulated F-actin disassembly and the membrane accumulation and activity of PKC-[zeta]. Pretreatment for 24 h with a PKC-[zeta] inhibitor (ZI) restored PKC-[zeta] activity, MC size, F-actin assembly and the contractile responsiveness to ET-1, suggesting that HG-induced MC dysfunction is mediated by PKC-[zeta]-dependent cytoskeletal disorganization and not altered signaling. HG for 48 h enhanced 5 min stretch-induced ERK1/ERK2 activation but not p38, in MCs subjected to 15% stretch on fibronectin. Stretch-induced activation of MAPKs required an intact cytoskeleton and was ß1 integrin-dependent, but was independent of PKC, tyrosine kinase, Ca2+, PI3-kinase or 'src' kinase. Dephosphorylation of ERK1/ERK2 but not p38, was diminished in HG following inhibition of cellular kinases by ATP depletion. This implies reduced phosphatase activity in HG. Enhanced MC ERK1/ERK2 signaling in response to the combined effects of mechanical stretch and HG may contribute to the pathogenesis of diabetic nephropathy.Ph.D