Effects of transforming growth factor-beta1 on renal extracellular matrix components and their regulating proteins.
Transforming growth factor-beta1 (TGF-beta1) is widely regarded as a potent fibrogenic renal growth factor. In cell culture, TGF-beta1 has been shown to increase various extracellular matrix (ECM) proteins and tissue inhibitors of metalloproteinases (TIMP), while decreasing matrix metalloproteinases (MMP), providing the optimum environment for progressive ECM accumulation. This study, which uses the isolated perfused rat kidney (IPRK), describes for the first time in a whole kidney preparation the action of TGF-beta1 on factors associated with ECM processing. This model allows the study of the intact rat kidney with physiologic cell-cell interactions in the absence of confounding systemic influences. Left kidneys were removed from male Wistar rats by a nonischemic technique and perfused with a sterile, apyrogenic, endotoxin-free perfusate, based on the plasma volume expander Hemaccel (polygeline), at constant pressure in a recirculating IPRK system. Kidneys were perfused for 1 h either with (n = 3) or without (n = 3) recombinant human TGF-beta1 (20 ng/ml). The effects of perfusion were controlled by comparison with the nonperfused contralateral kidney (n = 6). TGF-beta1 was measured in the perfusate and urine, at the start and end of the experiment using an enzyme-linked immunosorbent assay to its biologically active form. After perfusion, sections of the kidneys were analyzed for changes in mRNA by Northern blotting. Significant increases in mRNA for fibronectin (7.5-fold, P < 0.01), heparan sulfate proteoglycan core protein (53-fold, P < 0.001), laminin beta1 (12-fold, P < 0.001), collagen alpha1(IV) (17-fold, P < 0.001), collagen alpha1(III) (fourfold, P < 0.001), and MMP9 (twofold, P < 0.05) were observed after perfusion with TGF-beta1. Measurement of TIMP1, TIMP2, TIMP3, MMP1, and MMP2 mRNA demonstrated no detectable change, whereas determination of mRNA for tissue transglutaminase, an enzyme capable of cross-linking many ECM components, showed an eightfold increase (P < 0.01). This study suggests that in the IPRK and in the absence of other exogenous growth factors, TGF-beta1 selectively increases the synthesis of ECM and tissue transglutaminase without changes that would result in the reduction of ECM degradation.
Douthwaite
J A
JA
Sheffield Kidney Institute, Northern General Hospital Trust, United Kingdom.
Johnson
T S
TS
Haylor
J L
JL
Watson
P
P
El Nahas
A M
AM
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D007677:Kidney Function Tests; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000187:drug effects; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D012016:Reference Values; D012680:Sensitivity and Specificity; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology
D016326:Extracellular Matrix Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D008666:Metalloendopeptidases; D019696:Peptidylprolyl Isomerase