Proteomic analysis of human proximal tubular cells exposed to high glucose concentrations

Abstract

Hyperglycemia is a major key factor in the pathogenesis of microvascular complications of diabetes, including diabetic nephropathy (DN). Most studies to date have focused on the glomerular abnormalities found in DN. However, nephromegaly in the early stages of diabetes and the correlation of tubulointerstitial pathology rather than glomerular pathology with declining renal function in DN suggests the involvement of the tubulointerstitium. The etiology of the tubulointerstitial pathology in DN, however, is not fully understood. In this study, to understand the DN pathways, we constructed an initial 2-DE reference map for primitively cultured human proximal tubule (HK-2) cell in the presence of 5 mM and 25 mM glucose, which correspond to blood glucose concentrations during the normal and hyperglycemia conditions, respectively. Differentially expressed HK-2 cell cellular proteins at the high glucose concentration were identified via ESI-Q-TOF MS/MS and confirmed by Western blotting; enolase 1 (up-regulated) and lactate dehydrogenase (down-regulated). The regulation of these proteins will help in understanding DN mechanism through the glycolysis metabolic pathways in high glucose stimulated HK-2 cells.

Hyperglycemia Decreases the Expression of ATP Synthase <b><i>β</i></b> Subunit and Enolase 2 in Glomerular Epithelial Cells

“…Several analyses of the renal proteome with regard to DN have been conducted, including studies of the renal cortex (Tilton et al 2007), renal proximal tubular epithelial cells (So et al 2008), and urinary proteins (Rao et al 2007). Furthermore, one current study showed differential proteomic changes in an immortalized mouse podocyte cell line after long term exposure (two weeks) to HG (Schordan et al 2009).…”

“…2-DE analysis and protein identification were conducted following a previously described procedure (So et al 2008). Isoelectric focusing (IEF) was performed using Immobiline DryStrips (24 cm, pH 3-10 NL, GE healthcare, USA) and fifty micrograms of the sample were loaded onto each strip.…”

“…SDS-PAGE was performed at 55 V for 1 hr, 160 V for 1 hr and 330 V for 6 hr using the Ettan DALT II system (GE healthcare). Proteins were detected using the silver staining method (So et al 2008). Spot detection and matching between gels were automatically analyzed using Image Master 2D Platinum 6.0 Software (GE healthcare) for identification of proteins with statistically differential expression (using student's t-test analysis ( p < 0.05)) between NG-and HG-treated GECs.…”

“…After obtaining MS/MS spectra data, protein sequences were searched from NCBI databases using the MASCOT search program (www.matrixscience.com) (So et al 2008). The search parameters were as follows; MS/MS Ion Search for search type, trypsin in enzyme, carbamidomethyl (c) in fixed modifications, oxidation (M) in variable modifications, monoisotoic mass values, ± 1 Da for peptide mass tolerance, ± 0.8 Da for fragment mass tolerance, and 1 for maximum missed cleavage.…”

“…Analysis of proteomes allows description of translational regulation and protein modification, which cannot be explained by genomic analysis of changes in gene expression at the mRNA level (Ransom et al 2005). Proteomic investigations of DN have been applied recently and these studies have revealed new mechanisms of the pathology of DN (Kreidberg et al 1996;Wolf and Ziyadeh 2007;So et al 2008). In addition, potential blood and urinary markers of DN have also been investigated by proteomic analysis; in fact, our group proposed DN markers by serum proteome analysis Kim et al 2007).…”