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-induced activation of mesangial cell protein kinase C (PKC) contributes significantly to the pathogenesis of diabetic nephropathy. Excess glucose metabolism through the polyol pathway leads to de novo synthesis of both diacylglyerol (DAG) and phosphatidic acid, which may account for increased mesangial cell PKC-α, -β, -δ, -ε, and -ζ activation/translocation observed within 48-h exposure to high glucose. Raised intracellular glucose causes generation of reactive oxygen species that may directly activate PKC isozymes and enhance their reactivity to vasoactive peptide signaling. In both diabetic rodent models of diabetes and cultured mesangial cells, PKC-β appears to be the key isozyme required for the enhanced expression of transforming growth factor-β1, initiation of early accumulation of mesangial matrix protein, and increased microalbuminuria. Enhanced collagen IV expression by mesangial cells in response to vasoactive peptide hormone stimulation, e.g., endothelin-1, requires PKC-β, -δ, -ε and -ζ. Loss of mesangial cell contractility to potent vasoactive peptides and coincident F-actin disassembly are due to high-glucose-activation of PKC-ζ. Inhibition of mesangial cell PKC isozyme activation in high glucose may prove to be the next important treatment for diabetic nephropathy.

Podocytes are responsible in part for maintaining the size and charge filtration characteristics of the glomerular filter. The major sialoprotein of the podocyte foot process glycocalyx is a 140-kDa sialoprotein named podocalyxin. Monoclonal antibodies raised against isolated rabbit glomeruli that recognized a podocalyxinlike protein based upon size, Alcian blue staining, wheat germ agglutinin binding, and distribution in renal cortex were used to expression clone cDNAs from a rabbit glomerular library. On Northern blot the cDNAs hybridized to a 5.5-kilobase pair transcript predominantly present in glomerulus. The overlapping cDNAs spanned 5,313 base pairs that contained an open reading frame of 1,653 base pairs and were not homologous with a previously described sequence. The deduced 551-amino acid protein contained a putative 21-residue N-terminal signal peptide and a 26-amino acid transmembrane region. The mature protein has a calculated molecular mass of 55 kDa, an extracellular domain that contains putative sites for N-and O-linked glycosylation, and a potential glycosaminoglycan attachment site. The intracellular domain contains potential sites for phosphorylation. Processing of the full-length coding region in COS-7 cells resulted in a 140-kDa band, suggesting that the 55-kDa core protein undergoes extensive post-translational modification. The relationship between the cloned molecule and the monoclonal antibodies used for cloning was confirmed by making a fusion protein that inhibited binding of the monoclonal antibodies to renal cortical tissue sections and then raising polyclonal antibodies against the PCLP1 fusion protein that also recognized glomerular podocytes and endothelial cells in tissue sections in a similar distribution to the monoclonal antibodies. We conclude that we have cloned and sequenced a novel transmembrane core glycoprotein from rabbit glomerulus, which has many of the characteristics of podocalyxin. We have named this protein podocalyxin-like protein 1.

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...