Abstract
We have demonstrated previously that Amadori-configurated glycated albumin (GA) enhances nitric oxide synthase (NOS) activity, and this action may modulate glomerular hyperfiltration in early phases of diabetic nephropathy. Since the late stage of diabetic vasculopathy is characterized by reductions in viable cells within an expanded and disorganized matrix, we tested the hypothesis that GA enhances endothelial cell (EC) apoptosis. Murine (t End.1) or human umbilical vein ECs (HUVECs) were incubated with graded GA concentrations (furosine 0.48-96 nmol/ml) at levels that approximated those reported in sera of diabetic patients (76 +/- 0.02 nmol/ml). Apoptosis was evaluated using terminal uridine nick end labelling (TUNEL) to detect DNA fragmentation in gel electrophoresis and p53 expression in immunoperoxidase. Transcription of the inducible (i) and constitutive (c) isoforms of NOS was detected by northern analysis, and total NOS activity was measured as [(3)H]citrulline production from [(3)H]arginine. Cells were also incubated with the NOS inhibitors L-nitromethylarginine (L-NAME) at 0.01 M and aminoguanidine (AMG) at 0.01 M, the protein synthesis inhibitor cycloheximide (CHX) at 1 micro g/ml, and the NO donor sodium nitroprusside (SNP) at 0.01 M. ECs cultured in the presence of GA at furosine concentrations corresponding to levels in diabetic patients showed a significant enhancement of apoptosis. GA also caused parallel dose-dependent increases in iNOS mRNA expression and total NOS activity. The pro-apoptotic effect of GA was inhibited by L-NAME, AMG and CHX, but enhanced by SNP. We found that Amadori-configurated GA at furosine concentrations similar to those in diabetic patients favoured EC apoptosis through enhancement of iNOS activity. We propose that this process may be involved in the progressive cellular loss occurring in vascular and glomerular diabetic sclerosis.
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