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Background: Renal inflammation is regulated by a network of local and systemic mediators. Of them, transforming growth factor-beta1 (TGF-beta 1) and glucocorticoids play an important role in deactivating monocytes/macrophages. We examined the hypothesis that TGF-beta 1 effects may be partially achieved through modulation of the sensitivity of these cells to glucocorticoids., Methods: Human promonocytic U 937 cells differentiated to a mature macrophage-like phenotype were exposed to recombinant TGF-beta 1 before specific binding of [3H] dexamethasone was measured. The expression of glucocorticoid receptor (GR) was examined by RNase protection assay and Western blot analysis. The role of Smad 2/3 and activator protein 1 (AP-1) in the response to TGF-beta 1 was determined by introducing transdominant negative mutants and decoy oligodeoxynucleotides, respectively., Results: U 937 cell exposure to TGF-beta 1 caused a dose- and time-dependent increase in [3H] dexamethasone binding to these cells, with a < or =twofold increase in the number of binding sites per cell, without modification of the affinity. The changes in glucocorticoid binding were associated with identical changes in GR protein and mRNA levels, that were explained by an increase in GR gene transcription rather than by posttranscriptional mechanisms. Functional inactivation of Smad 2/3 and AP-1 limited the response to TGF-beta 1, indicating a role for these transcription factors. Finally, increases in glucocorticoid binding to GR were responsible for increases in the ability of GR to transactivate minimal promoters containing glucocorticoid-responsive elements (GRE) [MMTV-Luc and (GRE)2 TK-Luc]., Conclusion: TGF-beta 1 increases glucocorticoid binding and signaling in inflammatory cells through a Smad 2/3- and AP-1-mediated process. This may represent a new target for intervention to increase glucocorticoid responsiveness.

Background: The growth hormone (GH)/insulin-like growth factor (IGF) system is thought to participate in the glomerulosclerosis process. Because IGF-binding proteins (IGFBPs) modulate IGF actions and hence GH secretion, this study assessed whether mice transgenic for human IGFBP-1 have altered susceptibility to glomerulosclerosis., Methods: A line of transgenic mice that express human IGFBP-1 mRNA in the liver under the control of the alpha1-antitrypsin promoter has been obtained, and morphological changes in the kidney tissue were assessed. Glomerulosclerosis was identified using light microscopy, light microscopic morphometry, and electron microscopy. Extracellular matrix components were analyzed by immunohistochemistry., Results: There was a marked increase in mesangial extracellular matrix area in homozygous transgenic mice at three months of age as compared with heterozygous transgenic mice and nontransgenic littermates. These changes were not associated with alterations in glomerular volume or cellularity. The expansion of extracellular matrix area was related to a marked increase in laminin and type IV collagen and to the appearance of type I collagen., Conclusions: These observations indicate that the enhanced expression of IGFBP-1 may result in the development of glomerulosclerosis without glomerular hypertrophy. The changes are potentially related to a decrease in IGF-I availability and/or to an IGF-I-independent role of IGFBP-1.

IGF-I regulates renal growth and development. Insulin-like growth factor binding proteins (IGFBPs) are synthesized by the kidney and may modulate the local autocrine and/or paracrine actions of IGF-I. We have previously demonstrated that mesangial cells (MC) release IGF-I and IGF-binding activity; however, the specific IGFBPs produced by these cells and the factors involved in their regulation are unknown. We examined MC for expression of IGFBP-1 to -6 mRNAs and proteins. RNase protection assays using total RNA demonstrated that MC express all of the IGFBPs. [125I]IGF-I Western ligand blot of conditioned medium demonstrated that MC release IGFBPs of 24, 29, 32 kDa, and a doublet at 46 kDa, consistent with IGFBP-4, -5, -2 and -3, respectively. IGFBP species of 28 and 34 kDa were also detected. Since IGF-I and TGF-beta are implicated in glomerular hypertrophy and matrix expansion, we tested their effect on IGFBPs released by MC. IGF-I (100 ng/ml), TGF-beta (2 ng/ml) and forskolin (10(-5) M) differentially regulated the abundance of IGFBPs released in the conditioned medium in a time-dependent manner. IGF-I and TGF-beta were potent inducers of the release of IGFBP3 protein; however, TGF-beta, but not IGF-I, increased IGFBP3 mRNA levels. Recombinant IGFBP3 was tested for its effect on IGF-I-induced mitogenesis. IGFBP3 inhibited IGF-I-stimulated DNA synthesis in a dose-dependent manner with a peak effect observed at 50 nM IGFBP3. Although TGF-beta is a potent inhibitor of IGF-I-stimulated DNA synthesis, this effect is not mediated via IGFBPs. Expression of IGFBP-1 to -6 by MC suggests that these proteins may modulate IGF-I bioavailability in the glomerulus. IGF-I itself, TGF-beta and cAMP agonists may indirectly modulate the effects of IGF-I via the release of IGFBPs by MC.

Both fish oil-derived omega-3 polyunsaturated fatty acid (omega 3 PUFA) supplementation and essential fatty acid (EFA) deficiency have been shown to exert anti-inflammatory effects and, hence, to ameliorate immune-mediated glomerulonephritis. The mechanisms underlying these effects include alterations in the production of eicosanoids, cytokines (that is, tumor necrosis factor, TNF-alpha) and reactive oxygen species by blood borne cells. Because, in addition to these mediators nitric oxide (NO) is also implicated in glomerular injury, we have examined if both diets affected macrophage NO production as well. Rats were fed a standard chow, an omega 3 PUFA-supplemented diet, or an EFA-deficient diet for six weeks before resident peritoneal macrophages were isolated. These cells were exposed to lipopolysaccharide (LPS) and the NO metabolite, nitrite (NO2-), was measured in the medium using the Griess reagent. Release of NO2- was enhanced by LPS in a dose-dependent manner. With 10 ng/ml LPS challenge, NO2- release was reduced by 37% and 57% by omega 3 PUFA supplementation and EFA deficiency, respectively. NO2- returned to control levels two weeks after the end of diet. Macrophage production of TNF-alpha responded in a similar manner. Diet-induced reduction of NO2- release was neither attributable to a reduction of inducible NO synthase mRNA levels as shown by Northern blot analysis, nor to an increased competition of NO synthase and arginase for the substrate (L-arginine). Indeed, arginase activity of macrophages was even slightly reduced by both omega 3 PUFA-supplemented diet and EFA-deficient diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet-derived growth factor (PDGF) stimulates mitogenesis and exerts other biologic activities in glomerular mesangial cells. The precise mechanism of PDGF-induced mitogenesis in these cells is not clear. The activation of a signal transducing enzyme, phosphatidylinositol 3 kinase (PI 3 kinase) is associated with mitogenesis. Activation of PI 3 kinase results from stimulation of tyrosine kinase and G-protein-coupled classes of receptors. The synthesis of D3 phosphorylated inositides, the products of this enzymatic reaction, in non-nucleated cells such as blood platelets is dependent upon protein kinase C activation and G-proteins. We studied the activation of PI 3 kinase in response to PDGF in human glomerular mesangial cells. Using a PI 3 kinase 85 kD subunit specific antibody, we detected mesangial cell PI 3 kinase protein as 110 and 85 kD heterodimer. PDGF stimulated PI 3 kinase activity in antiphosphotyrosine immunoprecipitates in a dose-dependent manner showing maximum activation at 12 ng/ml. The antiphosphotyrosine associated PI 3 kinase activity showed biphasic kinetics with a fast peak within two minutes followed by a second peak at 10 minutes. Antiphosphotyrosine and PI 3 kinase immunoprecipitation studies indicated the association of the 85 kD PI 3 kinase subunit with PDGFR. Direct immunoprecipitation with PDGFR beta antibody showed the association of PI 3 kinase activity with the PDGF-receptor. The isoquinoline sulfonyl piperazine compound H7 at concentrations that inhibit PDGF-stimulated PKC activity had no effect on PDGF-stimulated PI 3 kinase activity in antiphospotyrosine immunoprecipitates. These data indicate that PI3 kinase activation is insensitive to PKC. Treatment of mesangial cells with pertussis toxin at concentrations that partially inhibited PDGF-induced DNA synthesis in human mesangial cells did not inhibit PDGF-induced PI 3 kinase activation. These data indicate that PDGF activates PI 3 kinase in mesangial cells and that pertussis toxin-sensitive G-proteins are not involved in PI 3 kinase activation. The data further dissociate activation of PI 3 kinase from mitogenesis in human mesangial cells.

Cultured rat mesangial cells have been demonstrated to express tumor necrosis factor alpha (TNF alpha) mRNA and to release TNF activity into the medium upon stimulation by bacterial lipopolysaccharide (LPS). The present study was undertaken to determine whether TNF was only secreted by mesangial cells or was also present as a cell-associated molecule. LPS-activated mesangial cells which had been fixed in paraformaldehyde lysed the TNF-sensitive L-929 fibroblasts, as assessed by 51Cr release. This cytotoxic activity was inhibited by anti-TNF alpha antiserum. Cell-associated TNF expression was demonstrable after less than one hour of exposure to LPS, peaked at two hours and decreased progressively thereafter, while TNF activity increased in the medium. Mesangial cell-associated TNF was localized at the cell surface, as shown by immunohistochemical demonstration and by the ability of plasma membranes purified from LPS-activated mesangial cells to lyse L-929 fibroblasts. Flow cytometry experiments revealed that two-thirds of LPS-activated mesangial cells were stained by anti-TNF alpha antiserum. The major part of these cell-associated TNF molecules persisted after low pH treatment, indicating that they were integral membrane proteins. As assessed by immunoprecipitation analysis, these proteins were 26 kDa molecules, whereas the released forms of TNF were 17 kDa molecules. Pretreatment of mesangial cells with desferrioxamine (DFX), an iron chelator preventing the synthesis of hydroxyl radicals (OH.), delayed the release of TNF from the membranes into the medium, and enhanced its cell surface expression. It also subsequently accelerated its decay in the medium.(ABSTRACT TRUNCATED AT 250 WORDS)