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      Altered Cultured Mesangial Cell Phenotypes from RF/J Mice: A Spontaneous Immune Complex Mediated Glomerulonephritis with Progressive Glomerulosclerosis

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          Background/Aim: RF/J mice are a model of spontaneous immune complex mediated glomerulonephritis showing massive extracellular matrix accumulation and progressive glomerulosclerosis. The aim of this study was to investigate whether there is an altered cultured mesangial cell (MC) phenotype in RF/J mice associated with these glomerular changes. Methods: The nature of cultured MCs from RF/J mice in the proliferative response to platelet-derived growth factor (PDGF) BB was compared with that of normal mice (BALB/c) by <sup>3</sup>H-thymidine incorporation. The binding of PDGF-BB was examined with Scatchard analysis, and the messenger RNAs (mRNAs) of PDGF β-receptor, collagen I, collagen IV, and fibronectin were detected using Northern blot analysis in the MCs of each mouse. Results: The <sup>3</sup>H-thymidine incorporation of MCs from RF/J mice showed significantly lower responses to PDGF-BB stimulations with concentrations ranging from 0.5 to 10.0 ng/ml in comparison with those of BALB/c mice which exhibited a proportional dose- dependent increase of the incorporation (p < 0.05 for 0.5 ng/ml PDGF-BB, p < 0.01 for 1.0–10.0 ng/ml). According to the Scatchard analysis, MCs from BALB/c mice showed aKD of 105 p M of PDGF-BB binding to its receptors, and the density of receptors was 5.82 fmol/10<sup>5</sup> cells. However, no binding PDGF-BB site on the surface of MCs from RF/J mice was noted. Northern blot analysis of MCs from RF/J mice indicated negative expression of detectable PDGF-β receptor mRNA. As for matrix protein messages, MCs from RF/J mice did not express mRNA of type I collagen, but did express a higher amount of type IV collagen and fibronectin in comparison with MCs from normal BALB/c mice. Conclusions: An altered phenotype in MCs of RF/J mice was demonstrated, possibly contributing to the characteristic pathological glomerular changes. However, the precise association remains to be clarified.

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          Most cited references 4

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          Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

          A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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            Glycated albumin stimulates fibronectin gene expression in glomerular mesangial cells: involvement of the transforming growth factor-beta system.

             J. Guo,  F Ziyadeh,  D Han (1998)
            Albumin modified by Amadori glucose adducts, formed in increased amounts in diabetes, stimulates collagen IV production and gene expression in renal glomerular mesangial cells, and induces mesangial matrix accumulation accompanied by increased mRNA encoding alpha 1 (IV) collagen and fibronectin in diabetic animals. These effects contribute to the pathogenesis of diabetic nephropathy, and resemble biologic activities of the cytokine TGF-beta 1, which also has been causally implicated in diabetic renal disease. We postulated that Amadori-modified glycated albumin modulates TGF-beta 1 expression in mesangial cells, and that TGF-beta 1 participates in mediating the glycated albumin-induced increases in mesangial cell matrix production. To test this hypothesis, we measured mRNA encoding TGF-beta 1, the TGF-beta Type II receptor and fibronectin, a key matrix component of the TGF-beta 1 tissue response, after incubation of mesangial cells with glycated albumin. Steady state levels of the mRNAs encoding for these proteins were stimulated when mesangial cells were cultured in the presence of albumin containing Amadori glucose adducts compared with levels in cells cultured with the nonglycated, glucose-free counterpart. The glycated protein-induced changes in mRNA expression were observed with concentrations of glycated albumin encompassing those found in clinical specimens and in media containing physiologic (5.5 mM) glucose concentrations, indicating that they were due to the glucose-modified protein and not to a hyperglycemic milieu. Further, they were accompanied by increased translated fibronectin protein, which was prevented with TGF-beta neutralizing antibody, as was the glycated albumin-induced increase in fibronectin mRNA. The findings indicate that Amadori-modified glycated albumin stimulates mesangial cell TGF-beta 1 gene expression by mechanisms that are operative under normoglycemic conditions. These data provide the first link between elevated glycated serum albumin concentrations and increased TGF-beta 1 bioactivity in the pathogenesis of mesangial matrix accumulation in diabetes.
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              Estradiol suppresses type I collagen synthesis in mesangial cells via activation of activator protein-1.

              Estradiol suppresses the synthesis of type I collagen by murine mesangial cells. However, neither the alpha 1(I) nor the alpha 2(I) collagen gene contains an estrogen-response element. Because estradiol modulates the transcription of several genes that lack an estrogen-response element but contain a regulatory activator protein-1 (AP-1) binding motif, we hypothesized that AP-1 may mediate estradiol-induced suppression of type I collagen synthesis. We measured type I collagen synthesis in murine mesangial cells exposed to estradiol, phorbol 12-myristate 13-acetate (an activator of AP-1), or curcumin (an inhibitor of AP-1). We also assessed the effects of estradiol on the steady-state level of c-fos and c-jun mRNA and on the binding of mesangial cell nuclear extracts to an AP-1 consensus binding site oligonucleotide. Estradiol (10(-10) M to 10(-7) M) suppressed type I collagen synthesis by murine mesangial cells in a dose-dependent manner (10(-7) M, 43.7 +/- 8.2% of control values, P < 0.001). Phorbol 12-myristate 13-acetate (10 microM, four-hr exposure) also decreased type I collagen in the media. In contrast, curcumin (1 microM) increased type I collagen. Estradiol increased the steady-state level of c-fos mRNA twofold at 30 minutes, with a return to basal levels at one hour. This was associated with a greater than threefold increase in the binding of nuclear extracts from estradiol-treated mesangial cells to an AP-1 consensus binding site oligonucleotide. Estradiol-enhanced binding of nuclear extracts to the AP-1 oligonucleotide was reversed by cycloheximide. These data suggest that estradiol suppresses collagen I synthesis by murine mesangial cells via enhanced AP-1 activity.

                Author and article information

                Nephron Exp Nephrol
                Cardiorenal Medicine
                S. Karger AG
                07 November 2001
                : 9
                : 6
                : 420-427
                aDivision of Nephrology, Kyoto City Hospital, and bThird Division, Department of Internal Medicine, Kyoto University Hospital, Kyoto, Japan, and cClinical Laboratory Medicine, Fukui Medical University, Fukui, Japan
                52641 Exp Nephrol 2001;9:420–427
                © 2001 S. Karger AG, Basel

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                Page count
                Figures: 7, Tables: 1, References: 25, Pages: 8
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/52641
                Original Paper


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