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

Abstract

Glomerular epithelial cells (GECs) are known to play a key role in maintaining the structure and function of the glomerulus. GEC injury induced by hyperglycemia is present in early-stage diabetic nephropathy (DN), which is the most common cause of renal failure. In an attempt to identify target proteins involved in the pathogenesis of GEC injury at early DN, we performed the proteomic analysis using primary cultures of GECs, prepared from the dissected rat glomeruli. The protein expression profiles in the two-dimensional electrophoresis gels were compared between GECs treated for three days with normal glucose (5 mM) and those with high glucose (30 mM) concentrations. These concentrations correspond to blood glucose concentrations under normoglycemia and hyperglycemia, respectively. Proteins with differential expression levels were identified using ESI-Q-TOF tandem mass spectrometry. The primary GECs cultured in hyperglycemic conditions showed cellular hypertrophy and increased production of reactive oxygen species, both of which reflect the GEC injury. Our proteomic analysis identified eight proteins with differential expression profiles, depending on glucose concentrations. Among them, we selected ATP synthase β subunit and enolase 2 that are related to energy metabolism and are down-regulated under hyperglycemia, and confirmed that hyperglycemia decreased the expression levels of ATP synthase β subunit and enolase 2 proteins by western blotting analysis. Hyperglycemia may impair mitochondrial function and alter glycolysis in GECs by down-regulating the expression of ATP synthase β subunit and enolase 2. The present study may provide a better understanding of the pathogenic mechanisms of GEC injury in early DN.

GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes

“…Total RNA was extracted from all specimens (podocytes and Sprague Dawley rats) using the TRIzol reagent (Invitrogen) and reversed transcribed using the SuperScript III reverse transcriptase (Invitrogen). 24 The sequences of the primers used are as follows: GLCCI1 sense 5′-GCCTTCACCAGTGAAATGACAA-3′ and antisense 5′-TTCCTGACCACTTACTGTCCCA-3′ Nephrin sense 5′-GTTCAGCTGGGAGAGACTGG-3′ and antisense 5′-AATCGGACGACAAGACGAAC-3′ Podocin sense 5′-TCTCCCAAACCTGTTGAACC-3′ and antisense 5′-GCGTGTGTCCATCTTGTGAC-3′ Synaptopodin sense 5′-GCAGAGGAAGTGAGGTCCAG-3′ and antisense 5′-GATGCCACTAGGGTGCTAGG-3′ Podocalxyin sense 5′-GGTGGCAGTGAAGAGATTC-3′ and antisense 5′-CTGTGAGTCGTTGTTGGTC-3′ and GAPDH sense 5′-CTCTACCCACGGCAAGTTCAA-3′ and antisense 5′-GGATGACCTTGCCCACAGC-3′. The PCR products were visualized on 1.5% agarose gels, and the band density was measured using the Gel-Doc image analysis system 2000 (Bio-Rad, Hercules, CA, USA).…”