Snezana Munk

High Glucose-suppressed Endothelin-1 Ca2+ Signaling via NADPH Oxidase and Diacylglycerol-sensitive Protein Kinase C Isozymes in Mesangial Cells

High glucose (HG) is the underlying factor contributing to long term complications of diabetes mellitus. The molecular mechanisms transforming the glomerular mesangial cell phenotype to cause nephropathy including diacylglycerol-sensitive protein kinase C (PKC) are still being defined. Reactive oxygen species (ROS) have been postulated as a unifying mechanism for HG-induced complications. We hypothesized that in HG an interaction between ROS generation, from NADPH oxidase, and PKC suppresses mesangial Ca2+ signaling in response to endothelin-1 (ET-1). In primary rat mesangial cells, growth-arrested (48 h) in 5.6 mm (NG) or 30 mm (HG) glucose, the total cell peak [Ca2+]i response to ET-1 (50 nm) was 630 ± 102 nm in NG and was reduced to 159 ± 15 nm in HG, measured by confocal imaging. Inhibition of PKC with phorbol ester down-regulation in HG normalized the ET-1-stimulated [Ca2+]i response to 541 ± 74 nm. Conversely, an inhibitory peptide specific for PKC-ζ did not alter Ca2+ signaling in HG. Furthermore, overexpression of conventional PKC-β or novel PKC-δ in NG diminished the [Ca2+]i response to ET-1, reflecting the condition observed in HG. Likewise, catalase or p47phox antisense oligonucleotide normalized the [Ca2+]i response to ET-1 in HG to 521 ± 58 nm and 514 ± 48 nm, respectively. Pretreatment with carbonyl cyanide m-chlorophenylhydrazone or rotenone did not restore Ca2+ signaling in HG. Detection of increased intracellular ROS in HG by dichlorofluorescein was inhibited by catalase, diphenyleneiodonium, or p47phox antisense oligonucleotide. HG increased p47phox mRNA by 1.7 ± 0.1-fold as measured by reverse transcriptase-PCR. In NG, H2O2 increased membrane-enriched PKC-β and -δ, suggesting activation of these isozymes. HG-enhanced immunoreactivity of PKC-δ visualized by confocal imaging was attenuated by diphenyleneiodium chloride. Thus, mesangial cell [Ca2+]i signaling in response to ET-1 in HG is attenuated through an interaction mechanism between NADPH oxidase ROS production and diacylglycerol-sensitive PKC.

High glucose activates PKC-ζ and NADPH oxidase through autocrine TGF-β1 signaling in mesangial cells

Conversion of normally quiescent mesangial cells into extracellular matrix-overproducing myofibroblasts in response to high ambient glucose and transforming growth factor (TGF)-beta(1) is central to the pathogenesis of diabetic nephropathy. Previously, we reported that mesangial cells respond to high glucose by generating reactive oxygen species (ROS) from NADPH oxidase dependent on protein kinase C (PKC) -zeta activation. We investigated the role of TGF-beta(1) in this action of high glucose on primary rat mesangial cells within 1-48 h. Both high glucose and exogenous TGF-beta(1) stimulated PKC-zeta kinase activity, as measured by an immune complex kinase assay and immunofluorescence confocal cellular imaging. In high glucose, Akt Ser473 phosphorylation appeared within 1 h and Smad2/3 nuclear translocation was prevented with neutralizing TGF-beta(1) antibodies. Neutralizing TGF-beta(1) antibodies, or a TGF-beta receptor kinase inhibitor (LY364947), or a phosphatidylinositol 3,4,5-trisphosphate (PI3) kinase inhibitor (wortmannin), prevented PKC-zeta activation by high glucose. TGF-beta(1) also stimulated cellular membrane translocation of PKC-alpha, -beta(1), -delta, and -epsilon, similar to high glucose. High glucose and TGF-beta(1) enhanced ROS generation by mesangial cell NADPH oxidase, as detected by 2,7-dichlorofluorescein immunofluorescence. This response was abrogated by neutralizing TGF-beta(1) antibodies, LY364947, or a specific PKC-zeta pseudosubstrate peptide inhibitor. Expression of constitutively active PKC-zeta in normal glucose caused upregulation of p22(phox), a likely mechanism of NADPH oxidase activation. We conclude that very early responses of mesangial cells to high glucose include autocrine TGF-beta(1) stimulation of PKC isozymes including PI3 kinase activation of PKC-zeta and consequent generation of ROS by NADPH oxidase.

Glomerular Mesangial Cell Altered Contractility in High Glucose Is Ca2+ Independent

In diabetes, loss of renal arteriolar smooth-muscle cell contractility leads to intraglomerular hypertension. In glomeruli isolated from streptozotocin (STZ)-induced diabetic rats, the mesangial cells (smooth muscle-like) display loss of contractile responsiveness to angiotensin II. This study examines the mechanistic relationship between altered mesangial cell contractility and vasopressor hormone-stimulated Ca2+ signaling in high glucose. Glomeruli were isolated from normal or STZ-induced diabetic rats to observe ex vivo mesangial cell contractile function. Also, rat mesangial cells were cultured (10–20 passages) in normal (5.6 mmol/1) or high (10–25.6 mmol/1) glucose for 1–5 days. Reduction of glomerular volume and decreased planar surface area of cultured mesangial cells in response to vasoconstrictor stimulation over 60 min were measured by videomicroscopy and personal computer—based morphometry. Contraction of glomeruli isolated from STZ-administered rats in response to endothelin (ET)-1 (0.1 μmol/1) or the Ca2+ ionophore A23187 (5 μmol/l) was impaired significantly compared with that in normal glucose. In the presence of arginine vasopressin (AVP) (1.0 μmol/1) or ET-1 (0.1 μmol/1), mesangial cells demonstrated a dose-dependent loss of contractile response to increasing glucose concentrations (5.6–25.6 mmol/1) within 24 h of high-glucose exposure, which was sustained for 5 days. Mesangial cells in high glucose were consistently smaller in size compared with those in normal glucose. Mesangial cells were preloaded with myo-[2-3H]inositol and intracellular [3H] inositol phosphate release in response to AVP (1.0 μmol/1) was analyzed by Dowex chromatography. Comparing cells in normal (5.6 mmol/1) versus high (25.6 mmol/1) glucose, we observed no significant difference in stimulated inositol phosphate levels from 10 to 60 s. The Ca2+-signaling response of cultured mesangial cells preloaded with Fura 2 or Indo 1 was measured by spectrofluorometry. After culture in 25.6 mmol/1 glucose, 0.1 μmol/l AVP or 0.1 μmol/1 ET-1 stimulated a cytosolic Ca2+ signal, with first and second phases, identical to that exhibited by mesangial cells in normal glucose. Fluorescence imaging of cultured mesangial cell cytoskeletal filamentous actin (F-actin) stained with rhodamine-phalloidin demonstrated reduced F-actin staining in the basal state and loss of the normal F-actin disassembly response to ET-1 in high glucose. Glomerular mesangial cells display glucose-induced altered contraction and F-actin disassembly to vasoactive stimuli, which occur in the presence of normal Ca2+ signaling.

Rosiglitazone Prevents High Glucose-Induced Vascular Endothelial Growth Factor and Collagen IV Expression in Cultured Mesangial Cells

Peroxisome proliferator-activated receptor (PPARγ), a ligand-dependent transcription factor, negatively modulates high glucose effects. We postulated that rosiglitazone (RSG), an activator of PPARγ prevents the upregulation of vascular endothelial growth factor (VEGF) and collagen IV by mesangial cells exposed to high glucose. Primary cultured rat mesangial cells were growth-arrested in 5.6 mM (NG) or 25 mM D-glucose (HG) for up to 48 hours. In HG, PPARγ mRNA and protein were reduced within 3 h, and enhanced ROS generation, expression of p22phox, VEGF and collagen IV, and PKC-ζ membrane association were prevented by RSG. In NG, inhibition of PPARγ caused ROS generation and VEGF expression that were unchanged by RSG. Reduced AMP-activated protein kinase (AMPK) phosphorylation in HG was unchanged with RSG, and VEGF expression was unaffected by AMPK inhibition. Hence, PPARγ is a negative modulator of HG-induced signaling that acts through PKC-ζ but not AMPK and regulates VEGF and collagen IV expression by mesangial cells.

Endothelin-1-induced mesangial cell contraction involves activation of protein kinase C-α, -δ, and -ε

In endothelin-1 (ET-1)-stimulated mesangial cells, to identify the independent roles of calcium and protein kinase C (PKC) causing contraction, the changes in planar surface area in response to ET-1, ionomycin, or phorbol 12-myristate 13-acetate (PMA) were compared. ET-1, PMA, and ionomycin reduced planar area to 49 ± 3%, 56 ± 3%, and 78 ± 2% of basal (means ± SE, n = 40-50 cells), respectively. ET-1 or ionomycin increased cytosolic calcium from 80 ± 7 to 220 ± 30 nM or 97 ± 10 to 192 ± 10 nM, respectively. The myosin light chain kinase inhibitor, ML-7, blunted ET-1- but not PMA-stimulated contraction (82 ± 3% and 48 ± 6% of time 0, respectively). Cells pretreated with 10 μM chelerythrine for 1 h or PMA for 24 h failed to contract to either ET-1 or PMA. To identify the specific PKC isoform response to ET-1, cytosolic, membrane, and particulate fractions of mesangial cell lysates were immunoblotted with PKC isoform-specific polyclonal antibodies. ET-1 increased membrane PKC-α, -δ, and -ε to 173 ± 30%, 162 ± 26%, and 166 ± 11% of basal ( P< 0.05 vs. basal), respectively, and decreased PKC-δ and PKC-ε in the cytosol to 56 ± 11% and 37 ± 6% of basal, respectively ( P < 0.05). ET-1 increased particulate PKC-δ and PKC-ε to 172 ± 15% and 187 ± 33% of basal ( P < 0.05), respectively. PKC-α in the cytosol and particulate fractions was not altered by ET-1, but translocation to the nucleus and cell periphery was observed by confocal immunofluorescence imaging. Ionomycin did not change PKC isoform distribution. PKC-ζ was expressed but unaltered by ET-1. Therefore, mesangial cell ET-1-stimulated contraction not only involves a calcium-dependent pathway but also includes the activation of one or more PKC-α, -δ, and -ε, but not PKC-ζ.

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

Background/Aims: We postulated that α-smooth muscle actin expressed in primary cultured mesangial cells is down-regulated in 3-dimensional (D) culture and up-regulated by high glucose and growth factors. Methods: Primary rat mesangial cells were growth-arrested in 5.6 mM (NG) or 30 mM (HG) glucose for 14 days in 3-D Matrigel. Alpha-SM actin expression was analyzed by immunoblotting, real-time PCR and by α-SM actin promoter activity in response to 24 h stimulation with endothelin-1 (ET-1), angiotensin II (Ang II) or HG. Results: Alpha-SM actin mRNA, protein and promoter activity were reduced to significantly lower levels in 3-D cells compared to cells in 2-D. Up-regulation of α-SM expression was stimulated by ET-1, Ang II and HG. Specific inhibitors of protein kinase C (PKC)-α, -β or -ζ prevented α-SM upregulation in HG. In NG, PKC and ERK1/2 activation were required for α-SM actin accumulation in 3-D in response to ET-1 or Ang II. In HG, enhanced expression of α-SM actin in response to ET-1 or Ang II was unchanged during PKC or ERK1/2 inhibition. Conclusion: Mesangial cells in 3-D express low levels of α-SM actin representing a more differentiated state. Regulation of α-SM actin expression is dependent on specific PKC isozyme and ERK1/2 signaling.