Glomerular basement membrane degradation by endogenous cysteine proteinases in isolated rat glomeruli
| Autor: | Youwen Zhou, Quoc C. Le, Sudhir V. Shah, Sally O'Connor, Rudene M. Dicarlo, Shirley L. Cortez, William H. Baricos |
|---|---|
| Rok vydání: | 1990 |
| Předmět: |
Male
Renal glomerulus Kidney Glomerulus In Vitro Techniques urologic and male genital diseases Basement Membrane Cathepsin B Dithiothreitol chemistry.chemical_compound Cathepsin H medicine Animals Cathepsin Basement membrane urogenital system Chemistry Glomerular basement membrane Rats Inbred Strains Cathepsins Rats Cysteine Endopeptidases Kidney Tubules medicine.anatomical_structure Microscopy Fluorescence Biochemistry Nephrology Chromatography Gel Cysteine |
| Zdroj: | Kidney International. 38:395-401 |
| ISSN: | 0085-2538 |
| DOI: | 10.1038/ki.1990.218 |
| Popis: | Glomerular basement membrane degradation by endogenous cysteine proteinases in isolated rat glomeruli. Recent in vitro and in vivo studies suggest that cysteine proteinases may play an important role in degradation of the glomerular basement membrane (GBM) 1 by renal glomeruli. However, little information is available concerning the cysteine proteinases present in glomeruli, the distribution of cysteine proteinases in other areas of the kidney, or the potential role of endogenous glomerular cysteine proteinases in GBM degradation. Using well characterized fluorogenic substrates, we have documented the presence of the cysteine proteinases, cathepsins B, H, and L, in glomeruli (0.45 ± 0.06, 0.39 ± 0.05, and 0.66 ± 0.14 mU/mg protein, mean ± SEM, N=8) and other fractions prepared from normal rat kidney. The presence of cysteine proteinases in glomeruli was verified by fluorescence microscopy. For each proteinase, the activity was: proportional to the amount of tissue protein and time of incubation; dependent on the presence of exogenously added dithiothreitol; and completely inhibited by the specific cysteine proteinase inhibitor, E-64. The pH optimum for cathepsin B (substrate: Z-Arg-Arg-HNMec) and L (substrate: Z-Phe-Arg-HNMec in the presence of Z-Phe-Phe-CHN 2 ) was approximately pH 6.0 for both glomeruli and renal cortex; while that for cathepsin H (substrate: Arg-HNMec) was approximately 6.5. Incubation of sonicated glomeruli with 3 H-GBM under conditions optimal for cysteine proteinase activity (pH 4.5, 1mM EDTA, and 1mM dithiothreitol, 37°C) resulted in significant GBM degradation as measured by the release of non-sedimentable (10,000 × g, 10 min) radioactivity or hydroxyproline, and by Sephadex gel chromatography of the degradation products. GBM degradation by glomeruli: increased with increasing amounts of glomerular protein (25 to 200 µ g) and incubation time (0 to 24 hr); was maximal at pH 4.5 to 5.0; was dependent on the presence of exogenously added dithiothreitol; and was markedly inhibited by E-64. These data document the presence of the cysteine proteinases, cathepsins B, H, and L, in glomeruli and other fractions prepared from normal rat kidney and demonstrate the ability of endogenous glomerular cysteine proteinases to degrade intact glomerular basement membrane in vitro. |
| Databáze: | OpenAIRE |
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