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Glomerular epithelial protein 1 (GLEPP1) is a receptor tyrosine phosphatase present on the apical cell surface of the glomerular podocyte. The GLEPP1 gene (PTPRO:) was disrupted at an exon coding for the NH(2)-terminal region by gene targeting in embryonic stem cells. Heterozygote mating produced the expected genotypic ratio of 1:2:1, indicating that the Ptpro(-/-) genotype does not lead to embryonic or neonatal lethality. Kidney and glomerular structure was normal at the gross and light microscopic levels. Scanning and transmission electron microscopy showed that Ptpro(-/-) mice had an amoeboid rather than the typical octopoid structure seen in the wild-type mouse podocyte and that there were blunting and widening of the minor (foot) processes in association with altered distribution of the podocyte intermediate cytoskeletal protein vimentin. Reduced filtration surface area in association with these structural changes was confirmed by finding reduced glomerular nephrin content and reduced glomerular filtration rate in Ptpro(-/-) mice. There was no detectable increase in the urine albumin excretion of Ptpro(-/-) mice. After removal of one or more kidneys, Ptpro(-/-) mice had higher blood pressure than did their wild-type littermates. These data support the conclusion that the GLEPP1 (Ptpro) receptor plays a role in regulating the glomerular pressure/filtration rate relationship through an effect on podocyte structure and function.

Many studies have suggested that enhanced glucose uptake protects cells from hypoxic injury. More recently, it has become clear that hypoxia induces apoptosis as well as necrotic cell death. We have previously shown that hypoxia-induced apoptosis can be prevented by glucose uptake and glycolytic metabolism in cardiac myocytes. To test whether increasing the number of glucose transporters on the plasma membrane of cells could elicit a similar protective response, independent of the levels of extracellular glucose, we overexpressed the facilitative glucose transporter GLUT-1 in a vascular smooth muscle cell line. After 4 h of hypoxia, the percentage of cells that showed morphological changes of apoptosis was 30.5 +/- 2.6% in control cells and only 6.0 +/- 1.1 and 3.9 +/- 0.3% in GLUT-1-overexpressing cells. Similar protection against cell death and apoptosis was seen in GLUT-1-overexpressing cells treated for 6 h with the electron transport inhibitor rotenone. In addition, hypoxia and rotenone stimulated c-Jun-NH(2)-terminal kinase (JNK) activity >10-fold in control cell lines, and this activation was markedly reduced in GLUT-1-overexpressing cell lines. A catalytically inactive mutant of MEKK1, an upstream kinase in the JNK pathway, reduced hypoxia-induced apoptosis by 39%. These findings show that GLUT-1 overexpression prevents hypoxia-induced apoptosis possibly via inhibition of stress-activated protein kinase pathway activation.

Glycosphingolipids have been proposed to be critical components of clustered lipids within cell membranes that serve as rafts for the attachment and sorting of proteins to the cell membrane. Density gradient centrifugation was used to isolate and to ascertain the lipid composition of caveolin-enriched membranes. These membranes demonstrated a significant enrichment of sphingolipids and cholesterol containing up to 20 and 30%, respectively, of the cellular glucosylceramide and lactosylceramide. A specific inhibitor of glucosylceramide synthase, d-threo-1-phenyl-2-palmitoyl-3-pyrrolidino-propanol, was used to test the hypothesis that glycosphingolipids are required for the sorting of proteins to caveolae. When NIH 3T3 cells were depleted of their glucosylceramide based glycosphingolipid mass, the caveolar structure remained intact as determined by electron microscopy and confocal microscopy. The caveolar proteins caveolin and annexin II sorted normally to caveolae, as determined by immunoblotting and confocal microscopy. When the GPI-linked protein B61 was inducibly expressed in these cells, sorting to caveolar membranes occurred normally, even in the presence of glucosylceramide depletion. These observations suggest that protein sorting to caveolae in fibroblasts occurs independently of glycosphingolipid synthesis., (Copyright 2000 Academic Press.)

mAb 5-1-6 identifies an antigen on rat podocyte slit-diaphragms and induces severe proteinuria when injected into rats. Nephrin, an Ig-like transmembrane protein that is mutated in congenital nephrotic syndrome of the Finnish type, has been localized to the slit-diaphragm on human podocytes. Here we document that the mAb 5-1-6 antigen is rat nephrin. After incubation of rat glomeruli with this mAb, the antibody/antigen complex was chemically cross-linked, extracted, and immunoprecipitated, prior to Western analysis. By mass spectrometry and 2D gel electrophoresis, we identified several peptides with complete identity to human nephrin. In addition, the 185-kDa protein immunoprecipitated by mAb 5-1-6 from rat glomerular extracts reacts with a rabbit anti-mouse nephrin antibody. Finally, nephrin and the mAb 5-1-6 antigen have identical glomerular localization patterns on immunofluorescence of rat kidney. These results demonstrate that the nephritogenic mAb 5-1-6 identifies the extracellular domain of nephrin, thereby documenting the importance of the slit-diaphragm and its component, nephrin, in the regulation of glomerular permselectivity.

Signal transducers and activators of transcription (STATs) are transcription factors that mediate normal biologic responses to cytokines and growth factors. However, abnormal activation of certain STAT family members, including Stat3, is increasingly associated with oncogenesis. In fibroblasts expressing the Src oncoprotein, activation of Stat3 induces specific gene expression and is required for cell transformation. Although the Src tyrosine kinase induces constitutive Stat3 phosphorylation on tyrosine, activation of Stat3-mediated gene regulation requires both tyrosine and serine phosphorylation of Stat3. We investigated the signaling pathways underlying the constitutive Stat3 activation in Src oncogenesis. Expression of Ras or Rac1 dominant negative protein blocks Stat3-mediated gene regulation induced by Src in a manner consistent with dependence on p38 and c-Jun N-terminal kinase (JNK). Both of these serine/threonine kinases and Stat3 serine phosphorylation are constitutively induced in Src-transformed fibroblasts. Furthermore, inhibition of p38 and JNK activities suppresses constitutive Stat3 serine phosphorylation and Stat3-mediated gene regulation. In vitro kinase assays with purified full-length Stat3 as the substrate show that both JNK and p38 can phosphorylate Stat3 on serine. Moreover, inhibition of p38 activity and thus of Stat3 serine phosphorylation results in suppression of transformation by v-Src but not v-Ras, consistent with a requirement for Stat3 serine phosphorylation in Src transformation. Our results demonstrate that Ras- and Rac1-mediated p38 and JNK signals are required for Stat3 transcriptional activity induced by the Src oncoprotein. These findings delineate a network of tyrosine and serine/threonine kinase signaling pathways that converge on Stat3 in the context of oncogenesis.

Background: The transcription factor Pax-2 is known to play a key regulatory role during embryonic development of the nervous and excretory systems in mammals and flies. During mouse kidney development, Pax-2 is expressed in the undifferentiated mesenchyme in response to ureter induction and continues to be expressed in the developing comma- and s-shaped bodies. These structures harbor the immediate precursors of the proximal tubular epithelial cells. Pax-2 expression is down-regulated as the differentiation of the functional units of the nephron proceeds. In the adult mammalian kidney, the Pax-2 protein is detectable exclusively in the epithelium of the collecting ducts. We sought to test the hypothesis that tissue regeneration is characterized by re-expression of developmentally important regulatory genes such as Pax-2., Methods: The expression pattern of Pax-2 in kidneys after experimentally-induced acute tubular necrosis caused by intraperitoneally injected folic acid in mice was tested by indirect immunofluorescence, Western blotting, reverse transcriptase-polymerase chain reaction, and in situ hybridization analysis., Results: A transient, temporally and locally restricted re-expression of Pax-2 in regenerating proximal tubular epithelial cells was observed following kidney damage., Conclusions: These data indicate that during the regeneration processes, developmental paradigms may be recapitulated in order to restore mature kidney function.

Background: Recognition that mutation of the protein nephrin, encoded by the NPHS1 gene, singly results in the cellular alterations that result in foot process effacement, and nephrotic range proteinuria emphasizes the pivotal role that this protein plays in regulating glomerular filter integrity. This article reports the development of reagents necessary to study the biology of nephrin in mouse, and describes the initial characterization of the nephrin protein., Methods: A cDNA including the full-length mouse nephrin open reading frame was cloned and sequenced. Immuno-affinity purified polyclonal antiserum directed against the cytoplasmic domain of mouse nephrin was developed., Results: Nephrin identified in mouse glomerular extract was found to be a glycoprotein with an apparent molecular mass of 185 kDa. As detected by indirect immunofluorescence microscopy and immunogold electron microscopy, nephrin was located only in visceral glomerular epithelial cells, where it was targeted to intercellular junctions of mature podocyte foot processes. In developing glomeruli of newborn mouse, antinephrin immunolocalized to the earliest slit pore regions between differentiating podocytes, sites where slit diaphragms first become visible., Conclusion: As a putative cell adhesion molecule of the immunoglobulin superfamily, nephrin likely participates in cell-cell interactions between podocyte foot processes and may represent a component of the slit diaphragm.

Despite of the increased availability of genetically modified mouse strains, the experimental models in the rat have provided the most widely employed and versatile models for the study of renal pathophysiology and functional genetics. The identification of the human gene mutated in the congenital nephrotic syndrome of the Finnish type (NPHS1) has recently been reported, and its protein product has been termed nephrin. Here we report the molecular cloning and characterization of rat nephrin cDNA. Rat nephrin cDNA has an open reading frame of 3705 bp, shows 82% sequence identity with human nephrin cDNA, and shows characteristic rat-specific splicing variants. The translated nucleotide sequence has 89% sequence identity at the amino acid level. The signal sequence, glycosylation, and cysteine localization patterns are nearly identical to those of human nephrin. As in the human, the rat nephrin transcript is expressed in a tissue-restricted pattern. Antipeptide antibodies raised to the intracellular nephrin-specific domain identified immunoreactivity exclusively within the rat kidney glomerulus by indirect immunofluorescence. Initial results with semiquantitative reverse transcriptase-polymerase chain reaction analysis showed a remarkable down-regulation of nephrin-specific mRNA in the puromycin nephrosis of the rat.

Previous studies have shown that the SA gene is expressed at higher levels in the kidney of genetically hypertensive rats than in control strains and that in hybrid crosses of genetically hypertensive rats and normotensive controls, markers in or close to the SA gene cosegregate with blood pressure. The present studies examine the localization of the SA gene product in the kidney by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). cDNA was prepared from microdissected nephron segments from Sprague-Dawley (SD) rats, spontaneously hypertensive rats (SHRs), and Wistar-Kyoto (WKY) rats, and RT-PCR was performed using specific primers. In all three strains, SA gene mRNA was found to be abundantly expressed in proximal tubules. SA PCR product was occasionally detected at approximately 100-fold lower abundance in glomeruli, while no signal was obtained from the collecting duct, thick ascending limb of the loop of Henle, or arcuate artery. Within the proximal tubule of normotensive rats, distribution of SA mRNA was found to be strain dependent: in SD rats it was expressed at high levels in the proximal convoluted tubule, whereas in WKY rats it was restricted to the proximal straight tubule. In SHRs, SA PCR product was detected along the entire proximal tubule. Induction of hypertension by renal artery clamping (two-kidney, one-clamp Goldblatt model) did not alter the pattern of expression observed in the SD rat. These results indicate that an extension of SA gene expression to the full length of the proximal tubule accompanies spontaneous hypertension and that in nonhypertensive animals the pattern of gene product expression is more restricted but shows substantial strain variability.

Podocytes are specialized epithelial cells with delicate interdigitating foot processes which cover the exterior basement membrane surface of the glomerular capillary. They are in part responsible for the extraordinary charge and size filtration characteristics of the glomerulus. To better understand disease processes affecting the glomerular filter, we searched for proteins with relative specificity to the podocyte using a monoclonal antibody strategy. The first such protein characterized (designated glomerular epithelial protein 1 (GLEPP1)) is a membrane protein-tyrosine phosphatase (PTPase) with a large extracellular domain containing eight fibronectin type III-like repeats, a hydrophobic transmembrane segment, and a single PTPase domain. The GLEPP1 PTPase domain shows homology with two other single domain transmembrane PTPases (PTP beta and Drosophila central nervous system PTP10D). This homology includes 2 cysteines in the PTPase domain not present in intracellular or tandem domain membrane PTPases. GLEPP1 PTPase protein is distributed to the podocyte foot processes themselves. RNase protection assay shows that GLEPP1 mRNA is also present in brain. By analogy with the CD45 PTPase of T cells, we expect that this receptor might play a role in maintaining foot process structure and/or function by regulating tyrosine phosphorylation of podocyte proteins.