Mesangial cell-reduced Ca²⁺signaling in high glucose is due to inactivation of phospholipase C-β₃by protein kinase C

Abstract

In high glucose, glomerular mesangial cells (MCs) demonstrate impaired Ca(2+) signaling in response to seven-transmembrane receptor stimulation. To identify the mechanism, we first postulated decreased release from intracellular stores. Intracellular Ca(2+) was measured in fluo-3-loaded primary cultured rat MCs using confocal fluorescence microscopy. In high glucose (HG) 30 mM for 48 h, the 25 nM ionomycin-stimulated intracellular Ca(2+) response was reduced to 82% of that observed in normal glucose (NG). In NG 5.6 mM, Ca(2+) responses to endothelin (ET)-1 and platelet-derived growth factor (PDGF) were unchanged in cells cultured in 50 nM Ca(2+) vs. 1.8 mM Ca(2+). Depletion of intracellular Ca(2+) stores with thapsigargin eliminated ET-1-stimulated Ca(2+) responses. Incubation in 30 mM glucose (HG) for 48 h or stimulation with phorbol myristate acetate (PMA) for 10 min eliminated the Ca(2+) response to ET-1 but had no effect on the PDGF response. Downregulation of protein kinase C (PKC) with 24-h PMA or inhibition with Gö6976 in HG normalized the Ca(2+) response to ET-1. Because ET-1 and PDGF stimulate Ca(2+) signaling through different phospholipase C pathways, we hypothesized that, in HG, PKC selectively phosphorylates and inhibits PLC-beta(3). Using confocal immunofluorescence imaging, in NG, a 1.6- to 1.7-fold increase in PLC-beta(3) Ser(1105) phosphorylation was observed following PMA or ET-1 stimulation for 10 min. In HG, immunofluorescent imaging and immunoblotting showed increased PLC-beta(3) phosphorylation, without change in total PLC-beta(3), which was reversed with 24-h PMA or Gö6976. We conclude that reduced Ca(2+) signaling in HG cannot be explained by reduced Ca(2+) stores but is due to conventional PKC-dependent phosphorylation and inactivation of PLC-beta(3).

Downregulation of TRPC6 protein expression by high glucose, a possible mechanism for the impaired Ca2+signaling in glomerular mesangial cells in diabetes

“…For instance, Mené et al (25) demonstrated a reduced Ca 2ϩ peak in response to multiple vasoconstrictor peptides following 3 to 5 days of high glucose in cultured rat MCs. This observation was supported by several other groups (10,29). Our results from the present study are consistent with those studies in that agonist-stimulated Ca 2ϩ entry is significantly inhibited by high glucose in cultured MCs (Fig.…”

Regulation of Mesangial Cell Alpha-Smooth Muscle Actin Expression in 3-Dimensional Matrix by High Glucose and Growth Factors

“…Mesangial cells were characterized as previously described [9, 10]. Mesangial cells contracted in response to Ang II, arginine vasopressin and ET-1.…”

“…Equal amounts of protein from total cells lysates (5 µg) with appropriate molecular weight markers (Bio/Can Scientific, Mississauga, Ont., Canada) were separated by 10% SDS-PAGE at 120 V for 90 min as previously reported [9, 10]. Alpha-SM actin antibody (1:50,000) was used to detect α-SM actin.…”

Mesangial cell NADPH oxidase upregulation in high glucose is protein kinase C dependent and required for collagen IV expression

“…Nox4, also referred to as Renox, is expressed predominantly in kidney epithelium and recently reported to be expressed also by mesangial cells (16,17). Mesangial cells express p22 phox and p47 phox , and the activity of these subunits in high glucose has been implicated in contributing to the oxidative stress associated with the pathogenesis of diabetic nephropathy (13,30,33,35,62).We demonstrated previously that transfection of primary cultured rat mesangial cells with antisense oligodeoxynucleotides (ODNs) against the NADPH oxidase p47 phox subunit prevents production of ROS and reverses PKC-dependent depressed Ca 2ϩ signaling in response to high glucose (24). However, the molecular mechanisms underlying high glucoseenhanced NADPH oxidase activity, in particular the regulation of NADPH oxidase subunit expression, have not been fully explored.…”

“…Nox4, also referred to as Renox, is expressed predominantly in kidney epithelium and recently reported to be expressed also by mesangial cells (16,17). Mesangial cells express p22 phox and p47 phox , and the activity of these subunits in high glucose has been implicated in contributing to the oxidative stress associated with the pathogenesis of diabetic nephropathy (13,30,33,35,62).…”