GLUTATHIONE REVERSES THE GROWTH ABNORMALITIES OF SKIN FIBROBLASTS FROM INSULIN-DEPENDENT DIABETIC-PATIENTS WITH NEPHROPATHY

Oxidative stress has been proposed as a possible pathogenic factor for diabetic complications. It is relevant in determining cell replicativ e capacity and life span, and in vitro antioxidant treatment is able t o reverse the impaired proliferative activity of different cell types. It was recently demonstrated that cultured skin fibroblasts from insu lin-dependent diabetic patients with nephropathy age prematurely and h ave a shorter life cell cycle. To test whether the growth phenotype of cells from patients with diabetic nephropathy was related to a lack o f protection from oxidative stress, the effect of reduced glutathione (GSH) on cultured skin fibroblasts from 13 insulin-dependent diabetes mellitus (IDDM) patients with nephropathy (DN), 10 IDDM patients witho ut kidney disease (D), and 10 nondiabetic control subjects (C), in nor mal (5 mM) glucose (NG) and high (22 mM) glucose (HG) medium was studi ed. After 6 to 8 passages, fibroblasts from DN showed impaired growth both in NG (mean +/- SD fold increase over baseline counts in DN 1.17 +/- 0.6 versus D 1.7 +/- 0.5 versus C 1.95 +/- 0.8; P = 0.04 by ANOVA) and in HG (mean +/- SD fold increase over baseline counts DN 1.16 +/- 0.41 versus D 1.89 +/- 0.66 versus C 2.24 +/- 0.9; P = 0.003 by ANOVA ). GSH prevented the growth abnormalities of cells from DN restoring i t to values similar to that of the other two groups (mean +/- SD fold increase over baseline counts NG + GSH: DN 1.68 +/- 0.9 versus D 1.78 +/- 0.49 versus C 1.99 +/- 0.7, P = 0.6; and in HG + GSH: DN 1.66 +/- 0.69 versus D 1.87 +/- 0.75 versus C 2.2 +/- 0.9, P = 0.3). Growth rat es were not affected by the addition of GSH in fibroblasts from D and C. The treatment of fibroblasts from D and C with the inhibitor of the gamma-glutamylcysteine synthetase activity, L-buthionine-S,R-sulfoxim ine, resulted in growth impairment, and the addition to the culture me dium of another antioxidant, superoxide dismutase, corrected the growt h abnormalities in fibroblasts from DN. The impaired growth of culture d fibroblasts from IDDM patients with nephropathy is prevented by GSH and superoxide dismutase and is independent of prevailing glucose conc entrations. This suggests that oxidative stress is an important mechan ism of intrinsic cell dysfunction in these patients.