Effects of Rebamipide in a Model of Experimental Diabetes and on the Synthesis of Transforming Growth Factor-b and Fibronectin, and Lipid Peroxidation Induced by High Glucose in Cultured Mesangial Cells

Oxidative stress has been proposed as the basis for the pathogenesis of diabetic nephropathy. Rebamipide is a novel antiulcer drug that has, in addition, oxygen radical scavenging activity. Our study examines whether rebamipide could ameliorate the pathophysiology associated with experimental diabetes in vivo, such as microalbuminuria, and to reverse the increased production of transforming growth factor-b1 and fibronectin in SV-40 transformed murine mesangial cells in culture that were stimulated with high glucose. Chronic administration of rebamipide (100 mg/kg/day, p.o., for 3 wk) to rats, in which diabetes was previously induced by the i.v. injection of streptozotocin 50 mg/kg, reversed hyperglycemia, which would contribute to prevent the increases in urinary excretion rates of albumin and lipid peroxides observed in this experimental model. Rebamipide, at this dose level, did not cause any discernible effect on age-matched control rats. Rebamipide 2 mM was as effective as 20 mM of dimethylthiourea, a known hydroxyl radical scavenger, in inhibiting the increase in lipid peroxides, transforming growth factor-b1, fibronectin mRNAs and proteins induced by incubation of cultured mesangial cells with high glucose. Our data suggest that rebamipide attenuates high glucose-induced nephropathy, which is attributable, in part, to its antioxidative property and, in part, to its effect on reversing hyperglycemia. Hyperglycemia, a well-recognized pathogenetic factor of long-term complications of diabetes mellitus (the diabetes control and complications trial research group, 1993), would not only generate more reactive oxygen metabolites but also attenuate antioxidative mechanisms through glycating scavenging enzymes. Therefore, oxidative stress has been considered to be the basis for the pathogenesis of diabetic complications (Baynes, 1991; Wolff et al., 1991; Wolff, 1993), including nephropathy (Larkins and Dunlop, 1992). This hypothesis has been supported by studies from this laboratory (Ha et al., 1991, 1994a, 1994b, 1996) and those of others (Trachtman et al., 1993, 1995; Trachtman, 1994). Two indices of oxidative stress, LPO and 8-hydroxydeoxyguanosine, were increased in kidneys of experimental diabetic rats (Ha et al., 1991, 1994a; Trachtman et al., 1995). In addition, high glucose directly increased lipid peroxidation of glomeruli (Ha et al., 1994) and glomerular mesangial cells (Ha et al., 1996; Trachtman et al., 1993; Trachtman, 1994). Moreover, antioxidants reversed the increase in collagen synthesis in mesangial cells cultured under high glucose (Trachtman et al., 1993; Trachtman, 1994). Rebamipide(2-(4-chlorobenzoylamino)-3-[2-(1H)-quinolinon-4-yl] propionic acid) is a novel antiulcer drug that has, in addition, oxygen radical scavenging activity (Yoshikawa et al., 1993; Kim and Hong, 1995; Lee and Kim 1995). Our study was conducted to examine whether rebamipide could ameliorate the pathophysiology of experimental diabetes, such as microalbuminuria, and high glucose-induced increases in TGF-b1 and fibronectin production in cultured mesangial cells. Microalbuminuria is an accepted predictor of overt diabetic nephropathy (Mogensen, 1989; Viberti et al., 1982). Expansion of the renal glomerular mesangium appears to be most closely associated with the loss of renal function in diabetes mellitus (Mauer et al., 1984). Several studies (Ayo et al., 1990, 1991) have shown that high glucose stimulated increased production of extracellular matrix proteins, viz., Received for publication September 25, 1996. 1 This study was supported in part by a grant from the Korea Otsuka Pharmaceutical Co., Ltd., Seoul, Korea and by a grant from Korea Science and Engineering Foundation (KOSEF 94-0403-07-01-3). ABBREVIATIONS: CR, control rats; DMEM, Dulbecco’s modified Eagle’s media; DMTU, dimethylthiourea; EDTA, ethylenediamine tetraacetic acid, FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethane sulfonic acid; LPO, lipid peroxides; PBS, phosphate buffered saline; SDS, sodium dodecyl sulfate; SSC, sodium chloride and sodium citrate; STZR, streptozotocin induced diabetic rats; TGF-b1, transforming growth factor-b1. 0022-3565/97/2813-1457$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 281, No. 3 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 281:1457–1462, 1997 1457 at A PE T Jornals on A uust 1, 2017 jpet.asjournals. org D ow nladed from fibronectin, laminin and type IV collagen synthesis, and mRNA levels, in mesangial cell cultures. At the same time, the close similarity of the effects of TGF-b1 and high ambient glucose on the growth and extracellular matrix metabolism of renal cells strongly suggests that the increased activity of TGF-b1 system in the kidney is implicated in the development of diabetic nephropathy (Wolf et al., 1992; Ziyadeh and Sharma, 1995).