Biosynthetic regulation of renal glomerular heparan sulfate-proteoglycans by various aldohexoses (mannose, glucose, and galactose) and laminin. Cellular ATP levels were dramatically reduced in all groups, and the maximal depletion was caused by mannose. Addition of ATP (0.1-1.0 mM) to the perfusion medium resulted in the normalization of the de novo synthesis and of the biochemical characteristics of heparan sulfate-proteoglycans. The relevance of decreased de novo synthesis of proteoglycans due to the depletion of ATP in hyperglycemic states is discussed in terms of increased glomerular permeability to plasma proteins, as seen in diabetes mellitus.During ultrafiltration, the glomerular basement membrane (GBM) restricts the passage of circulating plasma proteins into the urinary space (1). Thus, a compromise in the integrity of the GBM would be expected to lead to the leakage of proteins into the urine, as observed in a variety of renal diseases, including diabetic nephropathy (2, 3). In diabetes, there is a remarkable thickening of the GBM, and an increase in the mesangial matrices associated with proteinuria (4, 5). The GBM alterations may be due to the metabolic derangements in its various components-i.e., type IV collagen, laminin, entactin, and proteoglycans (PGs) (3, 6, 7). In this context, several studies have indicated a loss or a decrease in the synthesis of PGs in diabetes (3, 6-10). The loss of PGs has been regarded as one of the key events responsible for the proteinuric response seen in diabetes (4, 5). Although the deficiency of PGs/glycosaminoglycans (GAGs) is well documented, the mechanism(s) responsible for their decreased synthesis remains to be elucidated. This investigation relates to the study of one of the possible mechanisms-i.e., ATP depletion caused by high aldohexose levels-that may be responsible for the decreased de novo synthesis of heparan sulfate (HS)-PGs in diabetes.
METHODSRadiolabeling of Glomerular PGs. An ex vivo organ perfusion system was utilized for radiolabeling of PGs with [35S]sulfate (100 ,uCi/ml; 1 ,uCi = 37 kBq) (11). The hexoses used in various experiments were mannose, glucose, and galactose. They were individually added (10-50 mM) to the perfusion medium. Mannitol (25 mM) was included in the medium as a control. After 5 hr of radiolabeling of the rat kidney, a small cortical piece was processed for tissue autoradiography and the remaining kidney was perfused with Hanks' balanced salts solution containing a mixture of protease inhibitors (11). The kidney was then bisected, the cortex was dissected out, and glomeruli were isolated. Both the isolated glomeruli and the perfusion medium were processed for isolation and characterization of PGs/GAGs.Isolation and Characterization of PGs/GAGs. The radiolabeled PGs from isolated glomeruli were extracted with 4 M guanidinium chloride containing a mixture of protease inhibitors (11). An aliquot of the extract was passed through a Sephadex G-25 column equilibrated with 4 M guanidinium chloride/0.05 M sodium acetate/0...
