Rat Glomerular Epithelial Cells Research Articles

Regulation of basement membrane heparan sulfate proteoglycan, perlecan, gene expression in glomerular epithelial cells by high glucose medium

Proteinuria in diabetic nephropathy has been correlated with reduction in heparan sulfate proteoglycan (HSPG) content of the glomerular basement membrane. We have previously shown that the underlying mechanism probably involves reduction in the synthesis by glomerular epithelial cells. In this study we explored whether high glucose medium regulates basement membrane HSPG gene expression. Northern analysis demonstrated that rat glomerular epithelial cells in vitro constitutively express mRNA for basement membrane HSPG, similar to that observed in rat kidney glomerulus. RNase protection assay showed that incubation of glomerular epithelial cells with 30 mM glucose for 24 h and 7 days resulted in reduction in HSPG mRNA abundance. The decrease in mRNA abundance correlated with reduction in the synthesis of 35 SO4-labeled basement membrane HSPG as measured by immunoprecipitation. Reduction in synthesis of HSPG could not be entirely accounted for by decrease in mRNA abundance, suggesting both transcriptional and posttranscriptional mechanisms may be involved in reduction of glomerular basement membrane HSPG synthesis by glomerular epithelial cells in diabetic nephropathy. © 1996 Wiley-Liss, Inc.

Characterization of a glomerular epithelial cell metalloproteinase as matrix metalloproteinase-9 with enhanced expression in a model of membranous nephropathy.

The role of the glomerular visceral epithelial cell in the physiologic turnover and pathologic breakdown of the glomerular extracellular matrix has remained largely unexplored. In this study a 98-kD neutral proteinase secreted by cultured rat visceral glomerular epithelial cells was shown to be a calcium, zinc-dependent enzyme secreted in latent form. In addition, the protein was heavily glycosylated and demonstrated proteolytic activity against Type I gelatin, Type IV collagen gelatin, and fibronectin. The similarity in molecular mass and substrate specificities to the 92-kD human matrix metalloproteinase-9 (MMP-9, or gelatinase B) suggested the identity of this activity, which was confirmed by immunoprecipitation and Northern blot analysis. The differences in molecular mass (98 vs. 92 kD) were not due to species-specific differences in glycosylation patterns, since cultured rat peritoneal macrophages secreted MMP-9 as a 92-kD enzyme. Furthermore, transfection of the human MMP-9 cDNA into rat glomerular epithelial cells yielded the 98-kD product. Using a specific monoclonal anti-MMP-9 antibody and in situ reverse transcription (ISRT) analysis of MMP-9 mRNA, the expression of this enzyme was evaluated in vivo. Normal rat glomeruli expressed little immunohistochemical or ISRT staining for MMP-9, while in rats with passive Heymann nephritis there was a major increase in MMP-9 protein and mRNA staining within the visceral epithelial cells. The temporal patterns of MMP-9 expression correlated with the period of proteinuria associated with this model, suggesting that a causal relationship may exist between GEC MMP-9 expression and changes in glomerular capillary permeability.

Cyclic AMP regulates basement membrane heparan sulfate proteoglycan, perlecan, metabolism in rat glomerular epithelial cells.

Perlecan, the basement membrane heparan sulfate proteoglycan (HSPG), has been fully cloned from mouse and human tissues. When a cRNA probe of murine perlecan cDNA was employed in RNase protection assay to test whether rat glomerular epithelial cells (GEC) constitutively express perlecan, several bands of hybridization were seen, suggesting that sequences between rat and murine perlecan may not be identical. Using primers based on published cDNA sequences of murine and human perlecan and poly A+ RNA of rat GEC, we synthesized a 497 bp product (RPD-I) by RT-PCR. The deduced aminoacid sequence showed an 85% and 88% homology with domain I of murine and human perlecan, respectively. The three putative sites containing the consensus sequence SGD for attachment of heparan sulfate chains were fully conserved in the rat perlecan as was a site (NFT) for attachment of N-linked oligosaccharide. RPD-I detected a > 9.5 kb transcript of perlecan in RNA of GEC, similar in size to that present in rat glomeruli. Employing a riboprobe synthesized from RPD-I in RNase protection assay we examined whether dbcAMP regulated perlecan expression in the GEC. At 1, 6, 24 and 48 h of incubation, 1 mM dbcAMP caused 43%, 32%, 47% and 40% reduction in mRNA abundance of perlecan, respectively. Immunoprecipitation showed a corresponding reduction of 61%, 70% and 65% in the synthesis of 35SO4 labeled basement membrane HSPG by the GEC following 12, 24 and 48 h of incubation with dbcAMP. Following incubation for 1 and 24 h prostaglandins, PGE1 and PGE2 (1 uM), known activators of glomerular adenylate cyclase, reduced perlecan mRNA abundance to a similar extent as dbcAMP on northern analysis. Our results show that glomerular basement membrane HSPG synthesized by the GEC belongs to the perlecan family. Decrease of GEC perlecan gene expression and synthesis by cAMP and prostaglandins may be of relevance to proteinuric states characterized by activation of these mediators.

Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.

In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which, in some models, is partially mediated by eicosanoids. By analogy, sublytic C5b-9 injures plasma membranes and releases arachidonic acid (AA) and eicosanoids in cultured rat GEC. In this study, we demonstrate that, in GEC, sublytic C5b-9 stably increased the activity of a high-molecular-mass cytosolic phospholipase A2 (PLA2), which we identified as "cPLA2." This increase was abolished with inhibitors of protein kinase C. C5b-9 did not affect low-molecular-mass membrane-associated or secretory PLA2 activities. In GEC that stably overexpress cPLA2 activity and protein (produced by transfection of cPLA2 cDNA), immunoblot analysis showed that sublytic C5b-9 induced a decreased mobility of cPLA2, consistent with cPLA2 phosphorylation. Incubation of cPLA2-transfected GEC with sublytic C5b-9 significantly increased production of free AA and prostaglandin E2, whereas, in control GEC, the C5b-9-induced changes in free AA and prostaglandin E2 were small. Furthermore, both C5b-9-dependent sublytic cytotoxicity and cytolysis were enhanced in GEC overexpressing cPLA2, compared with control cells. Thus C5b-9 increased cPLA2 activity, probably via phosphorylation involving a protein kinase C-dependent pathway. Phospholipid hydrolysis by cPLA2 resulted in release of substrate for eicosanoid synthesis and in enhancement of C5b-9-dependent GEC injury. Both processes may facilitate glomerular damage in membranous nephropathy.

TGF-<italic>β</italic> upregulates interleukin 6 production by rat glomerular epithelial cells <italic>in vitro</italic>

Glomerular epithelial cells (GECs) play an important role in maintaining normal glomerular permselectivity in vivo. Recent in-vitro studies have suggested that GECs are able to secrete substances which may modulate glomerular injury. Interleukin 6 (IL-6) has been shown to be a potent mediator of glomerular injury. It is also known that IL-6 could be produced by various cells. IL-6 production by rat GECs in culture was examined in this study. IL-6 bioactivity in conditioned medium collected from cultured GECs (GEC-CM) was measured using IL-6 dependent murine hybridoma cell line, namely B9 cells. IL-6 gene expression by GECs was analysed by reverse transcriptase polymerase chain reaction (RT-PCR). Effects of recombinant IL-6 on the proliferation of GECs and type IV collagen secretion by GECs were evaluated to examine the possible role of GECs derived IL-6. GEC-CM stimulated B9 cells growth in a dose dependent fashion. The mitogenic activity was inhibitable by anti-murine IL-6 antibody. De-novo synthesis of IL-6 was suggested by the demonstration of IL-6 mRNA by GECs using the RT-PCR. Secretion of IL-6 by GECs was increased by transforming growth factor beta but not by IL-1 beta. Recombinant murine IL-6 stimulated GECs growth and their type IV collagen secretion. These results indicate that rat GECs could produce IL-6 which may modulate glomerular inflammation and that IL-6 may function as an autocrine factor for GECs.

Molecular characterization of rat Crry: widespread distribution of two alternative forms of Crry mRNA.

The rat complement regulator 5I2 antigen/Crry is present in a widespread distribution as at least two discrete proteins of Mr 65000-70000 and 75000-85000. The molecular basis for the different proteins has not been determined. We screened a cDNA library derived from cultured rat glomerular epithelial cells with a polymerase chain reaction (PCR)-generated nucleotide probe for CR1 and Crry. Two identical clones with 1.8 kilobase inserts were obtained. Clone 6.1 consisted of 1811 nucleotides. The sequence was identical to nucleotides 42-1666 of the cDNA for rat 5I2 antigen/Crry except for 11 nucleotides at the extreme 5' and 3' ends and an exact duplication of 186 bases that would encode an additional complete short consensus repeat (SCR). By reverse transcription PCR, we show that the two forms of Crry mRNA exist in all rat cells/tissues examined. It is likely that these two Crry mRNA species differ by the absence or presence of a 186 base repeat that encodes a complete SCR. These are translated into Crry proteins, containing six and seven SCRs, respectively, which explains the different sizes of Crry proteins. The role of the SCR duplication remains to be defined.

Regulation of manganese superoxide dismutase in glomerular epithelial cells: Mechanisms for interleukin 1 induction

Reactive oxygen species have been implicated as mediators of tissue injury in glomerular inflammation. The expression of the antioxidant enzyme, manganese superoxide dismutase (MnSOD), was examined in primary cultures of rat glomerular epithelial cells (GEC) in response to inflammatory mediators. The results demonstrate that GEC respond to interleukin-1 (IL-1) and bacterial lipopolysaccharride (LPS) with an increase in MnSOD steady-state mRNA levels. The IL-1 alpha-mediated induction of MnSOD mRNA levels was both time- and dose-dependent. Maximal levels approximately 40-fold above controls, were observed at 12 hours with 2 ng/ml of IL-1 alpha. MnSOD protein levels were also markedly elevated by IL-1 alpha. The induction of MnSOD mRNA by IL-1 alpha required de novo transcription as well as some degree of protein synthesis. To elucidate the potential intracellular signal that mediates IL-1 alpha-dependent MnSOD expression, three classical signaling pathways were examined. We found no evidence that MnSOD induction by IL-1 alpha is mediated by either the cyclooxygenase or lipoxygenase pathway or via activation of protein kinase C. Based on the presence of IL-1 alpha in several forms of glomerular inflammation, the observed increase in MnSOD expression by this immunoregulatory cytokine must have an important role in the antioxidant defense of glomerular epithelial cells.

Analysis of the Plasminogen System on Rat Glomerular Epithelial Cells

Rat glomerular epithelial cells were grown to confluency on semipermeable tissue culture inserts and the plasminogen system of these cells was analyzed using enzyme assays, Western analysis, zymography, and reverse transcriptase-PCR. The glomerular epithelial cells are capable of activating exogenous plasminogen to plasmin by endogenous plasminogen activators. The cells produce both tissue-plasminogen activator and urokinase-plasminogen activator with urokinase being the prominent activator. Both activators are present primarily on the basolateral side of the cells with urokinase found primarily at the cell surface presumably bound to its receptor and tissue-plasminogen activator found primarily in the matrix secreted by the cells on the semipermeable insert. The cells also produce plasminogen activator inhibitor-1 and urokinase-plasminogen activator receptor. Inhibition of plasminogen activation occurred with plasminogen activator inhibitor-1, anti-catalytic anti-tissue-plasminogen activator antibody, ϵ-aminocaproic acid, which inhibits the binding of plasminogen through its lysine binding sites, and amiloride, which specifically inhibits urokinase.

Crry and CD59 regulate complement in rat glomerular epithelial cells and are inhibited by the nephritogenic antibody of passive Heymann nephritis.

Human glomerular epithelial cells (GEC) contain CD59, decay-accelerating factor, and membrane cofactor protein. Crry is the rodent analogue to the latter two proteins. We have previously shown that the nephritogenic Ab of passive Heymann nephritis, anti-Fx1A, impairs C regulation in rat GEC. Here we examined rat GEC C regulation. 125I-labeled GEC membrane proteins were immunoprecipitated with anti-Crry, anti-CD59, or anti-Fx1A. Crry and CD59 were present in GEC. Anti-Fx1A reacted with both Crry and CD59 from GEC, as well as with purified rCrry and CD59. The alternative C pathway was studied by incubating GEC in rat serum in Mg(++)-EGTA buffer. To inhibit the function of the C regulators, anti-Crry or anti-CD59 Ab were added to GEC. Inhibition of CD59 function alone had no effect on C regulation, whereas inhibition of Crry led to significant cytotoxicity from alternative pathway activation. Under conditions in which Crry was inactive, inhibition of CD59 further enhanced cytotoxicity. When the classical pathway of C was activated by GEC-bound IgG Ab, inhibition of either Crry or CD59 enhanced cytotoxicity, whereas inhibition of both Crry and CD59 together was additive. Therefore, Crry and CD59 are present and functionally active in GEC. Crry restricts C activation via both alternative and classical pathways. When the classical pathway of C is activated, or when Crry function is inhibited, CD59 limits C5b-9-mediated cytotoxicity. Anti-Fx1A binds to both Crry and CD59, which may account for its ability to activate the alternative pathway in vitro, and for its superior nephritogenicity in vivo.

Effect of Insulin on High-Glucose Medium-Induced Changes in Rat Glomerular Epithelial Cell Metabolism of Glycoconjugates

We have previously reported that incubation of rat glomerular epithelial cells in vitro for 8 days with 30 mM glucose without insulin results in reduction in the synthesis of a cell layer heparan sulfate proteoglycan (HSPG) species that has hydrodynamic size and antigenic characteristics of glomerular basement membrane HSPG (1994, Arch. Biochem. Biophys 309, 149-159). In these studies, reduction in HSPG synthesis could be attributed either to high-glucose medium or to insulin deficiency. In this study we investigated the effects of insulin replacement on changes in glomerular epithelial cell metabolism of glycoconjugates induced by high-glucose medium. Addition of pharmacologic concentrations of insulin prevented the following changes induced by 30 mM glucose: (a) increment in 35SO 4 incorporation into macromolecules in cell layer and medium, (b) increment in the synthesis of low anionic macromolecules, probably glycoproteins, in both cell layer and medium, (c) increment in synthesis of small-sized glycosaminoglycans ( K av 0.75 on Sephrose CL-4B) associated with the cell layer. Insulin was unable to correct the 30 mM glucose-induced reduction in the synthesis of cell layer HSPG that resembles glomerular basement membrane HSPG. Physiologic concentrations of insulin did not affect any of the changes in glycopeptide metabolism induced by 30 mM glucose, These findings suggest that (a) inhibition of glomerular epithelial cell synthesis of 35SO 4-labeled low anionic macromolecules, probably glycoproteins, may be involved in insulin-induced reversal of glomerular hypertrophy seen in early diabetes, and (b) mechanisms other than insulin lack are involved in reduction in the synthesis of glomerular basement membrane HSPG in diabetic nephropathy.

Phenotypic modulation of rat glomerular visceral epithelial cells by culture substratum.

The interaction of cells with their supporting extracellular matrix influences cellular phenotype, cellular proliferation, protein synthetic profile, and specific gene activation. To examine the ability of culture substratum to modulate the phenotype expressed by glomerular epithelial cells (GEC) in culture, GEC were grown on plastic culture plates coated with collagen gels (Type I collagen, Vitrogen) or a complex matrix from the Englebreth-Holm-Swarm tumor (Matrigel). Cultures were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). On untreated plastic, GEC grew in a random pattern. Cells were flat and thin with many filamentous processes. When grown on collagen I gels, GEC grew to confluence as a tight monolayer with typical cobblestone appearance. These cells demonstrated surface microvilli and a central cilium. TEM showed an epithelial appearance with tight junctions. When plated on the surface of Matrigel, GEC formed nests of cells that gradually burrowed into the gel. Proliferation on this matrix was extremely slow. TEM demonstrated that there are surface projections that abut the matrix and that the nests of cells are hollow with a central lumen. SEM demonstrated nests of cells that formed a sphere. Surface microvilli were not as abundant as cells grown on Vitrogen, and cilia were not seen. Cells could be removed from one surface, plated onto another, and would shift phenotype to that observed for subcultures primarily plated onto that surface. Cells on each complex substrate, as well as GEC plated on tissue culture plates coated with individual matrix proteins were biosynthetically labeled with (35S)methionine. The profile and rate of protein synthesis were modified by the plating substrate. These observations demonstrate that rat GEC can be induced to display variable phenotypes in culture that are determined by the plating substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

IL-1 beta increases laminin B2 chain mRNA levels and activates NF-kappa B in rat glomerular epithelial cells.

The amount and distribution of laminin in the glomerular basement membrane (GBM) change in the course of many types of glomerular disease. Because interleukin-1 (IL-1) is thought to play a role in the pathogenesis of glomerulonephritis, it raises the possibility that this and other cytokines might regulate laminin gene expression. To determine whether laminin B2 chain mRNA levels change in response to cytokines, total mRNA from rat glomerular epithelial cells (GEC) grown in culture was analyzed by Northern blots. These studies showed an increase in laminin B2 chain mRNA levels in GEC treated with IL-1 beta. Nuclear factor kappa B (NF-kappa B) is a cytokine-responsive factor that regulates transcription of many genes from the cognate kappa B enhancer element. The mouse laminin B2 chain promoter contains several kappa B-like motifs, suggesting that NF-kappa B might be involved in IL-1-induced laminin B2 chain gene expression. Nuclear extracts from IL-1 beta-treated GEC showed increased binding to the immunoglobulin kappa B enhancer element and to a kappa B consensus sequence from the murine laminin B2 chain promoter in an electrophoretic mobility-shift assay (EMSA). The immunoglobulin kappa B and the laminin B2 chain kappa B-like motifs competed for the same DNA binding activity in nuclear extracts from IL-1 beta-treated GEC. Pretreatment of these nuclear extracts with antibodies to either the p50 or p65 subunits of NF-kappa B abrogated the DNA binding activity recognized by either of the two DNA motifs.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidants induce transcriptional activation of manganese superoxide dismutase in glomerular cells

Cultured rat glomerular mesangial and epithelial cells and bovine glomerular endothelial cells were exposed to various concentrations of hydrogen peroxide (H2O2). Mesangial cells treated with 10 to 100 microM H2O2 for 24 hours showed a two- to ninefold increase in Mn-SOD mRNA expression associated with significantly (P < 0.005) increased Mn-SOD activity (22.2 +/- 1.2 and 12.2 +/- 0.7 mu/mg protein for H2O2 100 microM treated and untreated cells, respectively). In contrast, expression of Cu-Zn SOD and beta-actin mRNA was not affected by H2O2. Induction of Mn-SOD mRNA by H2O2 was inhibited by actinomycin-D (4 microM) treatment. Glomerular endothelial cells also showed an increase in Mn-SOD mRNA expression following 100 microM H2O2 treatment, as did glomerular epithelial cells following treatment with 500 and 1000 microM H2O2 but not with 100 microM. Transcriptional activity of the Mn-SOD gene was assessed with a fusion reporter gene consisting of a luciferase gene (pGL2P) and a 1.2 kb fragment from the rat Mn-SOD genomic DNA (-806 to +408 bp of the transcription initiation site, -806:+408). The construct was transfected into rat glomerular mesangial and epithelial cells. Mesangial and epithelial cells transfected with pGL2P (-806:+408) and treated with H2O2 (100 microM and 1 mM for mesangial and epithelial cells, respectively) demonstrated some threefold increase in luciferase activity, whereas cells transfected with pGL2P lacking the Mn-SOD fragment did not show changes in luciferase activity following H2O2 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

An ultrastructural study of rat glomerular epithelial cells in culture

Cultured, rat glomerular podocytes were examined by electron microscopy and immunohistochemistry in order to determine their origin. The outgrowth of polygonal cells was noted first. Large arboroid cells were occasionally observed around the polygonal cells. Immunostaining of these large arboroid cells for the intermediate filament proteins showed an intensely positive reaction for vimentin, whereas cytokeratin was not detected. In the polygonal cells, however, both cytokeratin and vimentin were sometimes detected. Although scanning electron microscopy did not detect any specific irregularities in podocytes on the surface of the glomerulus, developing polygonal cells were observed. Both transmission and scanning electron microscopies revealed the polygonal cells to be similar to Bowman's capsule epithelial cellsin situ. The vascular poles of the cultured glomeruli were always in contact with the culture dish. A residual Bowman's capsule was also observed. These results suggest that polygonal cells originate from the epithelial cells of the residual Bowman's capsule, whereas large arboroid cells arise from podocytes.

Effects of tumour necrosis factor alpha and interleukin 1 beta on the proliferation of cultured glomerular epithelial cells.

Rat glomerular epithelial cells were cultured with human monocyte supernatant or with recombinant cytokines. A primary glomerular culture and a glomerular epithelial cell culture were made; supernatant from monocyte cultures derived from healthy humans, and recombinant tumour necrosis factor alpha (TNF alpha) or recombinant interleukin 1 beta (IL-1 beta) were added. Cell proliferation rates were assayed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. In serum-free media, consistent proliferation of glomerular epithelial cells (GEC) was observed throughout the 3 week culture period. Significant growth-stimulatory effects were induced by lipopolysaccharide-treated monocyte conditioned medium and by 1-50 ng/ml of TNF alpha, growth being up to 400% more than in the control culture. The effect of TNF alpha depended mainly on its interaction with epidermal growth factor (EGF). In contrast to TNF alpha, IL-1 beta inhibited GEC proliferation; this was due to the early appearance and proliferation of mesangial cells, despite the culture being serum-free. This study showed that activated monocytes secrete growth factors for GEC in vitro, and that interaction between both TNF alpha and IL-1 beta and between TNF alpha and EGF can modulate GEC proliferation. These findings suggest that, under pathological conditions, monocytes or macrophages affect GEC proliferation, probably being involved in crescent formation.

Extracellular matrix modulates epidermal growth factor receptor activation in rat glomerular epithelial cells.

To understand how glomerular epithelial cell (GEC) proliferation may be regulated in health and disease, we studied the effects of type I collagen extracellular matrices (ECM) on EGF receptor (EGF-R) activation in cultured rat GEC. EGF stimulated proliferation of GEC adherent to ECM, but not of GEC on a plastic substratum. Significant and prolonged EGF-R tyrosine autophosphorylation (which reflects receptor kinase activation) was induced by EGF in GEC adherent to collagen, but EGF did not stimulate EGF-R autophosphorylation in GEC on plastic (at 37 degrees C). However, EGF-R autophosphorylation increased significantly in plastic-adherent GEC that were stimulated with EGF at 4 degrees C or in the presence of vanadate, an inhibitor of phosphotyrosine phosphatases. Furthermore, dephosphorylation of EGF-R was enhanced in GEC on plastic as compared with collagen. At 4 degrees C, [125I]EGF binding was not different between substrata, and there was negligible accumulation of intracellular [125I]EGF (which reflects EGF-R internalization). At 37 degrees C, EGF-R internalization was reduced significantly in collagen-adherent GEC as compared with GEC on plastic. Thus, contact with ECM facilitates proliferation and EGF-R activation in GEC. The enhanced activity of EGF-R tyrosine kinase may be due to ECM-induced reduction in EGF-R internalization and dephosphorylation by phosphotyrosine phosphatase(s). Signals from ECM to growth factor receptors may regulate cell turnover in the glomerulus under normal conditions and during immune glomerular injury.

Molecular characterization of rat glomerular epithelial cell complement receptors.

Complement receptor type 1 (CR1) has previously been isolated from cultured rat glomerular epithelial cells (GEC) by C3b affinity chromatography. In addition, the presence of Crry in GEC and in rat glomeruli has been demonstrated. Crry appears to be the rodent analogue of human decay accelerating factor, which was previously described in human GEC and in human glomeruli. In this study, the molecular biology of these rat complement receptors is examined. A specific cDNA probe for rat CR1 was generated by reverse transcription of GEC mRNA, followed by polymerase chain reaction (PCR). The oligonucleotide primers were chosen from conserved regions spanning 271 bases in human and mouse CR1. A 271-base-pair PCR product was generated from rat GEC cDNA, the nucleotide sequence of which was 70.1% and 77.2% identical to those of the respective mouse and human sequences. This PCR product, designated rCR1-p, was then used to probe for CR1 mRNA. By northern blot analysis, rCR1-p hybridized to 4.5-kilobase (kb) mRNA from both cultured GEC and rat glomeruli and also weakly hybridized to 4.5-kb CR1 mRNA from mouse spleen. In additional northern blots, a nucleotide probe for mouse Crry hybridized to mRNA of 2.1 to 2.4 kb from rat GEC, slightly larger than the 1.9- to 2.1-kb mouse Crry mRNA. Therefore, mRNA for CR1 and Crry are present in cultured rat GEC and in rat glomeruli in vivo. To further investigate the composition of rat CR1 mRNA, northern hybridizations were performed with nucleotide probes for mouse and human CR1.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Rat Glomerular Epithelial Cell Proteoglycans by High-Glucose Medium

In diabetic nephropathy the heparan sulfate proteoglycan (HSPG) content of the glomerular basement membrane (GBM) is reduced but the cellular mechanisms involved have not been studied. Glomerular epithelial cells (GEC) are thought to be the source of HSPG present in the GBM. In this study we examined if proteoglycan metabolism of the rat GEC in culture is dysregulated in a metabolic environment simulating diabetes. Following incubation for 8 days with a serum-supplemented medium containing 30 mM glucose and no added insulin, a significant increase in the overall synthesis of 35SO 4-labeled molecules by the GEC was seen compared to control monolayers incubated with medium containing 5 mM glucose and insulin. Ion exchange chromatography revealed that 30 mM glucose did not alter the anionic charge density of proteoglycans, but significantly increased the amount of 35S-labeled low-anionic macromolecules in the medium; mannitol induced similar changes. Sepharose CL-4B chromatography, glycosaminoglycan analysis and immunoprecipitation of control cell layer proteoglycans demonstrated the presence of HSPG of hydrodynamic size, K av 0.4, resembling rat GBM HSPG in size and antigenic nature. Incubation of GEC with 30 mM glucose resulted in a significant reduction (58%) in this HSPG species, an effect not seen with equimolar mannitol. Additionally, 30 mM glucose induced a significant increment in synthesis of a small HS species ( K av 0.71 on Sepharose CL-4B column) present in the cell layer. Our findings suggest that both osmotic and nonosmotic mechanisms are operative in dysregulation of glycopeptide metabolism by high-glucose medium and that reduced synthesis by the GEC may contribute to decreased content of GBM HSPG in diabetic nephropathy.

Ultrastructural changes in cultured glomerular epithelial cells induced by puromycin aminonucleoside: a quick-freezing and deep-etching study.

The effects of puromycin aminonucleoside (PAN) on rat glomerular epithelial cells (GECs) in primary culture were studied using a quick-freezing and deep-etching method. On the 5th day after seeding, GECs were treated with PAN for 24 hours. The three-dimensional structure of the cell organelles, microtubules, and intermediate filaments were observed, primarily in the perinuclear areas of untreated GECs. Microfilaments were observed in marginal and basal areas. The GECs treated with PAN became rounded and partially detached from the substratum. Intermediate filaments formed bundled structures. A slight decrease in the number of microfilaments and a significant widening of their networks were observed in the basal areas of the GECs treated with PAN. These results suggest that PAN may affect the cytoskeletal components of GECs, such as the intermediate filaments and microfilaments, and may induce detachment of the cells from the substratum.

Cytokine-stimulated secretion of group II phospholipase A2 by rat mesangial cells. Its contribution to arachidonic acid release and prostaglandin synthesis by cultured rat glomerular cells.

Potent pro-inflammatory cytokines, such as interleukin 1 (IL-1) or tumor necrosis factor (TNF) alpha have been found to increase group II phospholipase A2 (PLA2) synthesis and secretion by mesangial cells. In all cases 85-90% of the enzyme is secreted from the cells and a parallel increase in prostaglandin (PG)E2 synthesis is observed. We report here that co-incubation with a monoclonal antibody that specifically binds and neutralizes rat group II PLA2 attenuates IL-1 beta and TNF alpha-stimulated PGE2 production by 45% and 52%, respectively. CGP43182, a specific inhibitor of group II PLA2, potently blocks mesangial cell group II PLA2 in vitro with a half-maximal inhibitory concentration (IC50) of 1.5 microM, while only slightly affecting mesangial cell high molecular weight PLA2. CGP 43182 markedly attenuates IL-1 beta- and TNF alpha-stimulated PGE2 synthesis in intact mesangial cells with IC50's of 1.3 and 1.0 microM, respectively. PLA2 secreted from cytokine-stimulated mesangial cells was purified to homogeneity. Addition of the purified enzyme to unstimulated mesangial cells causes a marked release of arachidonic acid and a subsequent increased synthesis of PGE2. Moreover, addition of purified PLA2 to a cloned rat glomerular epithelial cell line and cultured bovine glomerular endothelial cells augmented both arachidonic acid release and PGE2 synthesis, with the endothelial cells being especially sensitive. Thus, cytokine-triggered synthesis and secretion of group II PLA2 by mesangial cells contributes, at least in part, to the observed synthesis of PGE2 that occurs in parallel to the enzyme secretion. Furthermore, extracellular PLA2 secreted by mesangial cells is able to stimulate arachidonic acid release and PGE2 synthesis by the adjacent endothelial and epithelial cells. These data suggest that expression and secretion of group II PLA2 triggered by pro-inflammatory cytokines may crucially participate in the pathogenesis of inflammatory processes within the glomerulus.