Expression and activity of lysosomal enzymes in cultured cells exposed to conditions that mimic diabetes

Abstract

Diabetic nephropathy is a microangiopathy that affects about 100 million of diabetic patients worldwide. The first clinical sign of diabetic nephropathy is microalbuminuria that may evolve to end stage renal disease. For sulfated polysaccharides (glycosaminoglycans - GAGs - and dextran sulfate), in contrast to proteins, an early and important decrease in urinary excretion occurs in diabetic rats, and these macromolecules accumulate in kidney and liver, suggesting their intracellular localization and decreased digestion.As a consequence of this observation, the activities of lysosomal enzymes (as well as metalloproteases) were analyzed in kidney and liver of diabetic rats. In kidney, both the expression and the activities of cathepsins B, L and other cysteine proteases were decreased. Also, on the 30th day of diabetes, high molecular weight peptides and proteins appeared in the urine, indicating the excretion of less digested proteins. These results show that the decreased lysosomal protease activities could explain, at least in part, the increased albuminuria detected by radial immunodifusion, due to the excretion of less degraded albumin. In contrast, decreased amounts of GAGs were found in diabetic urine, while metachromatic staining of tubular cells by toluidine blue occurred, suggesting build up of GAGs. The activities of lysosomal glycosidases - ²-D-glucuronidase, N-acetyl-²-D-glucosaminidase, and N-acetyl-²-D-galactosaminidase - were decreased on the 30th day of DM. Experimental evidences suggest that sulfated polysaccharides are filtered and internalized by kidney cells, although the receptor has not yet been characterized. In diabetes mellitus, due to a lower digestion rate, these compounds accumulate in the tissue, possibly in proximal tubule cells as well.In diabetic liver, an important decrease in the specific activities of cysteine proteases, especially cathepsin B, was also observed. The expression of these enzymes was also decreased on the 10th, but not on the 30th day. Sulfatase decreased on the 30th day, while glycosidases did not vary (except for b-glucuronidase that presented a transitory decrease). There were no apparent changes in the liver morphology, and immunohistochemistry revealed cathepsin B in hepatocyte cytoplasmatic granules. The sulfatase decrease could be responsible for the dextran sulphate build up in diabetic liver, since the action of sulfatases precedes glycosidases in the digestive pathway of sulfated polysaccharides. However, it seems that the decreased activities of cathepsins and sulfatase did not result from general lysosomal failure, since other lysosomal enzymes did not vary in diabetic liver.Concerning metalloproteases (MMPs), although the expressions of MMP-2 and MMP-9 have decreased, the activities of MMPs did not vary in diabetic kidney and liver. The aim of the present study is to investigate the possible mechanisms that lead to decrease of lysosomal enzymes in diabetes. This study will be performed in proximal and distal tubular cells, as well as in mesangial cells exposed to conditions that mimic diabetes: high glucose, different concentrations of insulin, and presence of plasmatic and extracellular matrix proteins submitted to non-enzymatic glycation (AGEs - advanced glycation endproducts). The expression and activity of lysosomal enzymes will be analyzed. We believe that this study will shed some light on the mechanisms leading to diabetic nephropathy, and may suggest useful alternatives for prevention and therapeutic strategies. (AU)