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      The Renal TGF-β System in the db/db Mouse Model of Diabetic Nephropathy

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          Abstract

          The prosclerotic cytokine transforming growth factor beta 1 (TGF-β<sub>1</sub>) has been causally implicated in renal pathobiology in diabetes. We sought evidence that the TGF-β system participates in the nephropathic process in the db/db mouse, a hyperinsulinemic model of genetic diabetes that develops abnormalities in renal morphology and function that parallel those in human diabetic nephropathy. In support of this hypothesis, we found that steady state levels of mRNA encoding the TGF-β type II receptor were significantly increased in renal cortex from db/db diabetic mice. Additionally, the translated TGF-β type II receptor protein, assessed by immunoblot, also was increased in diabetic kidneys. However, in contrast to rodents with insulin-deficient diabetes, steady state levels of mRNA encoding TGF-β<sub>1</sub> in the renal cortex of diabetic db/db mice did not differ from those in cortex from nondiabetic (db/m) littermate controls. Further, concentrations of TGF-β protein, measured by immunoassay and bioassay, were significantly lower in extracts prepared from renal cortex of diabetic animals compared with those from nondiabetic controls. Urine and serum concentrations of immunoreactive TGF-β<sub>1</sub> also were reduced in diabetic mice. The findings are consistent with upregulation of TGF-β type II receptor activity as a consequence of hyperglycemia in the hyperinsulinemic db/db mouse and suggest that hyperinsulinemia inhibits TGF-β<sub>1</sub> production. The results further suggest that type II receptor upregulation is a contributing factor to the increased gene expression of renal cortical mRNAs encoding the extracellular matrix proteins fibronectin and alpha 1 (IV) collagen and to the renal abnormalities observed in this animal model.

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          Most cited references 3

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          Transforming growth factor beta in tissue fibrosis.

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            An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct.

            Transforming growth factor-beta (TGF-beta) is a potent regulator of cellular differentiation, proliferation, migration, and protein expression. These properties have been exploited to create a variety of bioassays for detecting the mature growth factor. In this paper, we describe a highly sensitive and specific, nonradioactive quantitative bioassay for TGF-beta based on its ability to induce plasminogen activator inhibitor-1 (PAI-1) expression. Mink lung epithelial cells (MLEC) were stably transfected with an expression construct containing a truncated PAI-1 promoter fused to the firefly luciferase reporter gene. Addition of TGF-beta (0.2 to > 30 pM) to the transfectants resulted in a dose-dependent increase in luciferase activity in the cell lysates. Although responsive to TGF-beta, this promoter fragment was only minimally influenced by other known inducers of PAI-1 expression. When compared to the widely used MLEC assay, this assay demonstrated greater sensitivity and specificity, allowing quantification of TGF-beta in complex biological solutions.
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              Production and characterization of monoclonal antibodies against human glycoalbumin.

              Hybridomas secreting monoclonal antibodies specific for nonenzymatically glycated albumin were produced by fusion of SP2/0 myeloma cells with spleen cells from BALB/c mice immunized with unreduced nonenzymatically glycated albumin prepared from human plasma. Wells containing hybridomas secreting antibodies against glycoalbumin were identified by binding, in an enzyme-linked immunosorbent assay, to glycoalbumin isolated from human plasma or to albumin that had been glycated in vitro. The colony designated A717, which secreted antibodies discriminating between glycated versus unglycated albumin, was cloned four times by limiting dilution and used for further study, performed with monoclonal antibody purified from mouse ascites fluid. Specificity of A717 was demonstrated by immunoblotting and by ELISA, wherein the monoclonal antibody reacted preferentially with glycated albumin but insignificantly with unglycated albumin. Immunoblotting of human plasma with A717 on nitrocellulose yielded a single band, the electrophoretic mobility of which corresponded with that of authentic glycated albumin, indicating site specificity for glycated epitopes residing in albumin but not in other nonenzymatically glycated serum proteins. A717 differs from other antibodies raised against glycated albumin and other proteins, which recognize glycated residues only after reductive conversion to glucitol-lysine and which do not discriminate between different glycated proteins. Thus, this report describes the establishment of the first hybridoma secreting monoclonal antibody raised against unreduced glycated albumin, which is the physiologic form occurring in vivo, and for the epitope when it resides in albumin but not other proteins.
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                Author and article information

                Journal
                EXN
                Nephron Exp Nephrol
                10.1159/issn.1660-2129
                Cardiorenal Medicine
                S. Karger AG
                1660-2129
                1998
                June 1998
                22 May 1998
                : 6
                : 3
                : 226-233
                Affiliations
                Departments of Medicine and Biochemistry, University of Pennsylvania and Thomas Jefferson University, Philadelphia, Pa., USA
                Article
                20527 Exp Nephrol 1998;6:226–233
                10.1159/000020527
                © 1998 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 3, Tables: 2, References: 51, Pages: 8
                Product
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/20527
                Categories
                Original Paper

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