Excess collagen IV expression by mesangial cells contributes to diabetic glomerulosclerosis. We hypothesized that in high glucose reactive oxygen species (ROS) generation by NADPH oxidase is PKC dependent and required for collagen IV expression by mesangial cells. In rat mesangial cells cultured in 5 mM (NG) or 25 mM d-glucose (HG), RT-PCR and Western immunoblotting detected p22(phox) and p47(phox) mRNA and protein, respectively. Quantitative real-time RT-PCR analyzed collagen IV mRNA. With the use of confocal microscopy, ROS were detected with dichlorofluorescein and intracellular collagen IV by immunofluorescence. In HG, ROS were generated within 1 h, sustained up to 48 h, and prevented by a NADPH oxidase inhibitor, diphenylenechloride iodonium (DPI), or a conventional PKC isozyme inhibitor, Gö6976. In NG, phorbol myristate acetate stimulated ROS generation that was inhibited with DPI. In HG, expression of p22(phox) and p47(phox) was increased within 3 to 6 h and inhibited by Gö6976. In HG, Gö6976 or transfection with antisense against p22(phox) reversed the 1.8-fold increase in collagen IV mRNA. In HG, the antioxidants Tempol or Tiron, or transfection with antisense against p22(phox) or p47(phox), prevented ROS generation and the 2.3-fold increase in collagen IV protein. Increased mitochondrial redox potential in HG was unaffected by transfection with antisense against p22(phox). We conclude that in HG, mesangial cell ROS generation by upregulated NADPH oxidase is dependent on conventional PKC isozymes and also required for collagen IV expression.

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 Ca 2؉ 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 High glucose (HG) 1 is the key factor contributing to long term complications of diabetes mellitus (1). One of the phenotypic changes observed in mesangial cells exposed to HG is altered Ca 2ϩ signaling. Several groups have shown that the Ca 2ϩ signal induced by vasoactive compounds, including endothelin-1 (ET-1), is markedly reduced in the presence of HG. The mechanism(s) by which HG may depress Ca 2ϩ signaling is unknown. One possible candidate is the activation of protein kinase C (PKC) in HG. Mené et al. (2) have shown that HG inhibits Ca 2ϩ influx through store-operated channels via a PKC-dependent mechanism. An alternative postulate is the involvement of reactive oxygen species (ROS), which have been demonstrated to modify intracellular Ca 2ϩ signaling responsiveness depending on the cell type, the species of ROS, and the magnitude and duration of ROS generation. HG induces dysfunction in mesangial cells and other target cells through enhanced synthesis of autocrine growth factors such as transforming growth factor-␤ 1 , ET-1, and altered signaling via pathways such as PKC (3, 4). In the last few years, enhanced production of ROS in response to HG, identified in many target cells including mesangial cells (5, 6), has been postulated as a unifying mechanism causing diabetes complications (7-9).Although ROS have been implicated in causing cell damage and apoptosis, they also play a physiological role in intracellular signaling pathways (10, 11). In particular, several growth factors including ET-1, angiotensin II, plateletderived growth factor, and epidermal growth factor stimulate production of ROS as second messengers (12,13). In several cell types, signaled ROS production is due to activation of NADPH oxidase, a multicomponent enzyme (14). In phagocytic cells, the multiple subunits of NADPH oxidase are localized in subcellular compartments. gp91 phox , the catalytic moiety of the phagocyte oxidase, and p22 phox associate to form a flavocytochrome in the plasma membrane. The cytosol components p47 phox , p67 phox , p40 phox , and the small GTPase, Rac1 (or Rac2), are recruited to the membrane for assembly of a fully active oxidase (15)(16)(17). In nonphagocytic cells, most of the subunits of NADPH oxidase have been identified, although the precise mechanisms of regulation are not completely understood. A functional glomerular mesangial NADPH oxid...

In high glucose, mesangial cell PKC-zeta is required for ROS generation from NADPH oxidase similar to PDGF stimulation of PKC-zeta-dependent ROS generation through a pathway independent of PI3 kinase. F-actin disassembly in high glucose also requires ROS. A positive feedback loop occurs between ROS and the activation of PKC-zeta in high glucose.

IG, Whiteside CI. Reactive oxygen species, PKC-␤ 1, and PKCmediate high-glucose-induced vascular endothelial growth factor expression in mesangial cells.

CI. High glucose activates PKC-and NADPH oxidase through autocrine TGF-␤ 1 signaling in mesangial cells.

Hua, Hong, Snezana Munk, and Catharine I. Whiteside. Endothelin-1 activates mesangial cell ERK1/2 via EGFreceptor transactivation and caveolin-1 interaction. Am J Physiol Renal Physiol 284: F303-F312, 2003. First published September 17, 2002 10.1152/ajprenal.00127.2002.-Endothelin-1 (ET-1) stimulates glomerular mesangial cell proliferation and extracellular matrix protein transcription through an ERK1/2-dependent pathway. In this study, we determined whether ET-1 activation of glomerular mesangial cell ERK1/2 is mediated through EGF receptor (EGF-R) transactivation and whether intact caveolae are required. We showed that ET-1 stimulated tyrosine phosphorylation of the EGF-R in primary cultured, growth-arrested rat mesangial cells. In response to ET-1, ERK1/2 phosphorylation was increased by 27 Ϯ 1-fold and attenuated by AG-1478, a specific EGF-R inhibitor, to 9 Ϯ 1-fold. Moreover, filipin III and ␤-cyclodextrin, two cholesterol-depleting drugs known to disrupt caveolae, significantly reduced ET-1-induced phosphorylation of ERK1/2. In addition, preincubation of mesangial cells with a myristoylated peptide that binds to the caveolin-1 scaffolding domain diminished ET-1 activation of ERK1/2. ET-1 caused interaction of caveolin-1 with phosphorylated ERK1/2 identified by coimmunoprecipitation. Activation of ERK1/2 and its interaction with caveolin-1 were reduced by AG-1478, ␤-cyclodextrin, or inhibition of PKC. Phosphorylated ERK1/2 localized at focal adhesion complexes along with phospho-caveolin-1, suggesting specific sites of compartmentalization of these signaling molecules. Hence, ET-1 activates mesangial cell ERK1/2 predominantly through a pathway involving EGF-R transactivation, leading to a mechanism involving attachment to caveolin-1, presumably in caveolae.caveolae; epidermal growth factor receptor transactivation

In high glucose (HG), mesangial cells (MCs) lose their contractile response to endothelin-1 (ET-1) coincidently with filamentous (F)-actin disassembly. We postulated that these MC phenotypic changes are mediated by altered protein kinase C (PKC) isozyme activity, myosin light chain (MLC(20)) phosphorylation, or Ca(2+) signaling. MCs were growth arrested for 24 h in 0.5% fetal bovine serum (FBS)-DMEM in 5.6 (normal glucose; NG) or 30 mM glucose (high glucose; HG). In HG, the planar area was reduced [2,608 +/- 135 vs. 3,952 +/- 225 (SE) microm(2) in NG, P < 0.01, n = 31] with no contractile response to 0.1 microM ET-1. Mannitol did not affect cell size or ET-1 response. Confocal imaging of fluo 3- loaded cells revealed that the peak intensity of ET-1-induced Ca(2+) signaling was not altered in HG vs. NG. Immunoblotting of phosphorylated MLC(20) showed that HG increased mono- and decreased unphosphorylated MLC(20) (42 +/- 16 and 49 +/- 15 vs. 13 +/- 3 and 80 +/- 4% of total in NG, P < 0.05, n = 3), but the peak phosphorylation responses to ET-1 were identical in NG and HG. ET-1 stimulated translocation of PKC-delta and -epsilon from cytosolic to membrane and particulate fractions identically in NG and HG but did not cause PKC-zeta translocation. In HG, membrane accumulation of PKC-zeta was observed. Membrane PKC-zeta activity measured by immunoprecipitation and (32)P phosphorylation of PKC-epsilon pseudosubstrate peptide was 190 +/- 18% of NG (P < 0.01, n = 4), which was completely inhibited by pretreatment with a myristoylated peptide inhibitor (ZI). In HG, pretreatment with ZI for 24 h restored normal MC size and contractile and F-actin disassembly responses to ET-1. In conclusion, in HG, decreased MC size is due to decreased F-actin assembly, and loss of contractile response to ET-1 occurs in the presence of normal Ca(2+) signaling and normal MLC(20) phosphorylation. In HG, altered F-actin and contractile functions in MCs are mediated by PKC-zeta.

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