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- Title
Protein Degradation -- A Common Mechanism in Podocyte Damage?
- Authors
Blattner, S. M.; Cohen, C. D.; Rastaldi, M. P.; Schlöndorff, D.; Kretzler, M.
- Abstract
Objective: Mutations of alpha-actinin-4 have been shown to be responsible for a familial form of focal segmental glomerulonephritis (FSGN). The mechanism by which this mutated alpha-actinin-4 acts may involve disturbances of the cytoskeleton, however detailed information about its function and regulation in podocyte is lacking. In this study we analyzed the regulation of alpha-actinin-4 and a novel associated cytoskeletal adaptor molecule, CLP-36, in acquired proteinuric disease. Methods: The mRNA and protein expression of alpha-actinin-4 and CLP-36 were analyzed in several glomerular diseases (Minimal Change Disease n = 13, benign nephrosclerosis n = 16, Membranous GN n = 31, FSGS n = 9, renal tumor nephrectomies n = 6, pretransplant biopsies n = 4) in vivo by immunohistochemistry and realtime RT-PCR. Furthermore, we used in vitro models of podocyte damage (puromycin and xantin/xantin-oxidase) to functionally characterize the expression regulation. Results: A significant reduction of glomerular alpha-actinin-4 and CLP-36, but not synaptopodin, staining was observed in all proteinuric glomerulopathies with a maximal repression in sporadic FSGS. To further define alpha-actinin-4 and CLP-36 regulation, mRNA levels were determined on glomeruli microdissected from renal biopsies. Surprisingly, alpha-actinin-4 and CLP-36 transcription showed a reciprocal regulation to the protein levels, with maximal mRNA induction in FSGN glomeruli for alpha-actinin-4 and stable mRNA levels for CLP36. Further, alpha-actinin-4 expression showed a significant correlation with proteinuria in FSGN. In addition to co-regulation, a co-localization and molecular interaction of alpha-actinin-4 and CLP-36 could be shown with immunofluorescence and co-immunoprecipitation studies. In oxidative stress to cultured podocytes, mRNA levels remained stable, but protein levels declined in a dose dependant manner, as observed in vivo. The oxidative stress induced reduction in protein levels could be blocked by lactacystin, an inhibitor of proteasome function. Conclusions: For several podocyte specific molecules a discrepant regulation of protein and mRNA has been reported by us and others. In this study, we could show a proteasome dependant degradation to be responsible for the loss of alpha-actinin-4 and CLP-36 protein in podocyte damage. The decreased protein levels might be responsible for a compensatory up-regulation of the corresponding mRNAs. As proteasome inhibition is already evaluated in clinical oncology, intervention of proteasome function could be a novel therapy target in podocytes damage.
- Subjects
GLOMERULONEPHRITIS; PROTEINURIA; CYTOSKELETAL proteins; MESSENGER RNA; OXIDATIVE stress; RENAL biopsy; IMMUNOFLUORESCENCE
- Publication
Kidney & Blood Pressure Research, 2004, Vol 27, Issue 5/6, p287
- ISSN
1420-4096
- Publication type
Article