Amadori-configurated albumin induces nitric oxide-dependent apoptosis of endothelial cells: a possible mechanism of diabetic vasculopathy.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Amore
Alessandro
A
Nephrology, Dialysis and Transplantation Department, Regina Margherita Children's Hospital, Piazza Polinia 94, 10126 Turin, Italy. amorenefro@oirmsantanna.piemonte.it
Cirina
Paola
P
Conti
Giovanni
G
Cerutti
Franco
F
Bagheri
Nayer
N
Emancipator
Steven Noel
SN
Coppo
Rosanna
R
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D003925:Diabetic Angiopathies / Q000503:physiopathology*; D003928:Diabetic Nephropathies / Q000503:physiopathology*; D042783:Endothelial Cells / Q000502:physiology*; D017127:Glycation End Products, Advanced / Q000096:biosynthesis; D006801:Humans; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D012709:Serum Albumin / Q000378:metabolism*; D000093423:Glycated Serum Albumin
D017127:Glycation End Products, Advanced; D012709:Serum Albumin; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D000093423:Glycated Serum Albumin