title: Profiling of human mesangial cell subproteomes reveals a role for calmodulin in glucose uptake.
abstract: 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.
authors.affiliation: Dept. of Medicine, Center for Novel Therapies for Kidney Disease, Suite 365 Jefferson Alumni Hall, 1020 Locust St., Philadelphia, PA 19107, USA. satish.rao@jefferson.edu
authors.forename: Satish P
authors.initials: SP
authors.lastname: Ramachandra Rao
authors.forename: Richard
authors.initials: R
authors.lastname: Wassell
authors.forename: M Alexander
authors.initials: MA
authors.lastname: Shaw
authors.forename: Kumar
authors.initials: K
authors.lastname: Sharma
mesh_terms: D000818:Animals; D002147:Calmodulin / Q000502:physiology*; D003921:Diabetes Mellitus, Experimental / Q000503:physiopathology; D003922:Diabetes Mellitus, Type 1 / Q000503:physiopathology; D015536:Down-Regulation; D020869:Gene Expression Profiling; D005947:Glucose / Q000008:administration & dosage / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D050527:Mesangial Cells / Q000378:metabolism*; D051379:Mice; D020543:Proteome / Q000032:analysis*; D051381:Rats; D016212:Transforming Growth Factor beta / Q000494:pharmacology
chemical_list: D002147:Calmodulin; D007328:Insulin; D020543:Proteome; D016212:Transforming Growth Factor beta; D005947:Glucose