Proteomic identification of early changes in the renal cytoskeleton in obstructive uropathy

Lene Stødkilde, Johan Palmfeldt, Line Nilsson, Inge Carlsen, Yan Wang, Rikke Nørregaard, Jørgen Frøkiær
2014 AJP - Renal Physiology  
Bilateral ureteral obstruction (BUO) is associated with renal damage and impaired ability to concentrate urine and is known to induce alterations in an array of kidney proteins. The aim of this study was to identify acute proteomic alterations induced by BUO. Rats were subjected to BUO for 2, 6, or 24 h. Mass spectrometry-based proteomics was performed on the renal inner medulla, and protein changes in the obstructed group were identified. Significant changes were successfully identified for
more » ... proteins belonging to different biological classes. Interestingly, proteins belonging to the cytoskeleton and proteins related to cytoskeletal regulation were found to be biologically enriched in BUO using online-accessible tools. Western blots confirmed the selected results, demonstrating acute downregulation of proteins belonging to all three cytoskeletal components. The microfilament protein ␤-actin and the intermediate filament proteins pankeratin and vimentin were all downregulated. ␤-Tubulin, an important microtubular protein, was found to be significantly downregulated after 24 h. Also, there was significant upregulation of cofilin, an actin-binding protein known to be upregulated in other nephropathy models. Furthermore, both upregulation and downregulation of cytoskeletal motor and regulatory proteins were observed. These findings were confirmed by immunohistochemistry, which clearly showed alterations in labeling in the inner medulla. Interestingly, we were able to confirm selected results in mpkCCD cells exposed to mechanical stretch. Our findings add to the knowledge of BUO-induced acute changes in the renal cytoskeleton and suggest that these molecular changes are partly mediated by increased stretch of the cells during obstruction. ureteral obstruction; mass spectrometry; acute proteome changes URINARY TRACT OBSTRUCTION is a common condition associated with reduced renal blood flow, glomerular filtration rate (GFR), and an impaired ability to concentrate urine (21). Impairment of the urinary concentrating capacity involves multiple elements including altered expression of key renal membrane water and ion proteins located in different segments of the nephron (14, 24). Furthermore, mechanical stretch due to urine accumulation, hypoxia caused by reduced renal perfusion, and oxidative stress all contribute to the renal damage associated with obstruction (41). Moreover, several proteins involved in cellular stress and inflammation have been shown to be regulated during obstruction (7, 26, 35, 49) . These findings show Address for reprint requests and other correspondence: J. Frøkiaer,
doi:10.1152/ajprenal.00244.2013 pmid:24761003 fatcat:dtlypn7h6rff3npioyvfqkxov4