Differential proteomic analysis of proteins induced by glucocorticoids in cultured murine podocytes.
BACKGROUND
The glomerular podocyte is the kidney cell most affected during the development of nephrotic syndrome, and mutations in podocyte proteins are responsible for a variety of inherited forms of nephrotic syndrome. Although glucocorticoids are a primary treatment for nephrotic syndrome, neither their target cell nor mechanism of action are known. In order to describe the proteome of the podocyte, and to identify podocyte proteins whose expression is altered by glucocorticoids, we performed a differential proteomic analysis of control and dexamethasone-treated cultured murine podocytes.


METHODS
Podocyte proteins were separated by two-dimensional-polyacrylamide gel electrophoresis (PAGE) and identified by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry and peptide fingerprinting. Comparisons of stained two-dimensional-PAGE separations were used to identify proteins whose expression was altered by treatment with the glucocorticoid dexamethasone, and these results were confirmed by quantitative Western blotting.


RESULTS
A total of 106 protein spots yielded MALDI-TOF results, and 92 were identified by protein fingerprinting. Of the 88 unique proteins and four protein isoforms identified, six proteins were found whose expression was altered by dexamethasone. The proteome of cultured murine podocytes is particularly rich in actin cytoskeletal proteins and proteins involved in responses to cellular stress. The change in expression of three proteins [ciliary neurotrophic factor (CNTF), alphaB-crystallin, and heat shock protein 27 (hsp27)] was confirmed by quantitative Western blotting.


CONCLUSIONS
Three proteins with known roles in protecting cells from injury were up-regulated by dexamethasone, demonstrating that glucocorticoids exert a direct effect on cultured podocytes resulting in changes in the expression of proteins with potential relevance to the therapeutic action of glucocorticoids in diseases such as nephrotic syndrome.
Ransom
Richard F
RF
Pediatric Nephrology Division, University of Michigan, Ann Arbor, Michigan 48109, USA. rransom@umich.edu
Vega-Warner
Virginia
V
Smoyer
William E
WE
Klein
Jon
J
D000818:Animals; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology*; D005938:Glucocorticoids / Q000494:pharmacology*; D007678:Kidney Glomerulus / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005938:Glucocorticoids; D011506:Proteins; D003907:Dexamethasone