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Am J Physiol Renal Physiol 292: F1182-F1189, 2007. First published January 2, 2007; doi:10.1152/ajprenal.00268.2006
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Profiling of human mesangial cell subproteomes reveals a role for calmodulin in glucose uptake

Satish P. Ramachandra Rao,1,3 Richard Wassell,2 M. Alexander Shaw,2 and Kumar Sharma1,3

1Dorrance H. Hamilton Research Laboratories, Department of Medicine, 3Center for Novel Therapies for Kidney Disease, and 2Proteomics Core Facility, Department of Cancer Biology, Thomas Jefferson University, Philadelphia, Pennsylvania

Submitted 15 July 2006 ; accepted in final form 29 December 2006

Proteomics combined with cell fractionation was used to identify proteins regulated by high glucose (HG) in human mesangial cells (HMC). Total membrane and cytosolic fraction proteins derived from HMC after 7 days of HG exposure were resolved by a two-dimensional gel electrophoresis approach. DeCyder software was used to analyze the HG-induced protein spot dysregulation. In the membrane subproteome, of the 92 spots that were matched across all gels, HG induced significant downregulation of only 4 protein spots. The dysregulated spots from the membrane subproteome included binding protein (BiP), calreticulin precursor protein, a 63-kDa transmembrane protein from a ER/Golgi intermediate, and beta-subunit of collagen proline 4-hydroxylase. In the cytosolic subproteome, of the 122 spots that were matched across all gels, HG induced downregulation of 3 protein spots and upregulation of 2 protein spots significantly. Enolase 1, annexin VI, and {gamma}2-actin were decreased, whereas heat shock protein-70 kDa and calmodulin (CaM) were increased. Further confocal microscopy and Western immunoblotting of mesangial cells validated the increase in CaM. Immunoblotting of diabetic mouse and rat kidneys exhibited a marked increase in CaM at both early and late stages of diabetes, reflecting the potential physiological relevance of CaM upregulation. CaM-specific inhibitors blocked glucose transport stimulated by transforming growth factor-beta and insulin in mesangial cells. In conclusion, using a combination of cell fractionation and protein expression profiling, we identified a cohort of HG-dysregulated proteins in the HMC and identified a critical and as yet unrecognized role for CaM in glucose transport in mesangial cells.

proteomics; subcellular fraction; diabetic nephropathy


Address for reprint requests and other correspondence: S. P. Ramachandra Rao, Dept. of Medicine, Center for Novel Therapies for Kidney Disease, Suite 365 Jefferson Alumni Hall, 1020 Locust St., Philadelphia, PA 19107 (e-mail: satish.rao{at}jefferson.edu and Kumar.Sharma{at}jefferson.edu)






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