The dystrophin-glycoprotein complex, which comprises ␣-and ␤-dystroglycan, sarcoglycans, and utrophin/dystrophin, links the cytoskeleton to agrin and laminin in the basal lamina in muscle and epithelial cells. Recently, agrin was identified as a major heparan sulfate proteoglycan in the glomerular basement membrane. In the present study, we found mRNA expression for agrin, dystroglycan, and utrophin in kidney cortex, isolated glomeruli, and cultured podocytes and mesangial cells. In immunofluorescence, agrin was found in the glomerular basement membrane. The antibodies against ␣-and ␤-dystroglycan and utrophin revealed a granular podocyte-like staining pattern along the glomerular capillary wall. With immunoelectron microscopy, agrin was found in the glomerular basement membrane, dystroglycan was diffusely found over the entire cell surface of the podocytes, and utrophin was localized in the cytoplasm of the podocyte foot processes. In adriamycin nephropathy, a decrease in the glomerular capillary wall staining for dystroglycan was observed probably secondary to the extensive fusion of foot processes. Immunoelectron microscopy showed a different distribution pattern as compared to the normal kidney, with segmentally enhanced expression of dystroglycan at the basal side of the extensively fused podocyte foot processes. In passive Heymann nephritis we observed no changes in the staining intensity and distribution of the dystrophin-glycoprotein complex by immunofluorescence and immunoelectron microscopy. From these data, we conclude that agrin, dystroglycan, and utrophin are present in the glomerular capillary wall and their ultrastructural localization supports the concept that these molecules are involved in linking the podocyte cytoskeleton to the glomerular basement membrane. (Am J Pathol 2000, 156:1749 -1765) Dystroglycan (DG) is an important member of the dystrophin-glycoprotein complex (DGC) which links the subsarcolemmal cytoskeleton to the basal lamina in skeletal muscle.1 The importance of this link becomes clear from the severe muscular dystrophies resulting from mutations in genes that encode different members of the DGC. 2-5

Heparan sulfate (HS) proteoglycans are major anionic glycoconjugates of the glomerular basement membrane and are thought to contribute to the permeability properties of the glomerular capillary wall. In this study we evaluated whether the development of (micro)albuminuria in early human and experimental diabetic nephropathy is related to changes in glomerular HS expression or structure. Using a panel of recently characterized antibodies, glomerular HS expression was studied in kidney biopsies of type I diabetic patients with microalbuminuria or early albuminuria and in rat renal tissue after 5 months diabetes duration. Glomerular staining, however, revealed no differences between control and diabetic specimens. A significant ( p < 0.05) ϳ60% increase was found in HS N-deacetylase activity, a key enzyme in HS sulfation reactions, in diabetic glomeruli. Structural analysis of glomerular HS after in vivo and in vitro radiolabeling techniques revealed no changes in HS N-sulfation or charge density. Also HS chain length, protein binding properties, as well as disaccharide composition did not differ between control and diabetic glomerular HS samples. These results indicate that in experimental and early human diabetic nephropathy, increased urinary albumin excretion is not caused by loss of glomerular HS expression or sulfation and suggest other mechanisms to be responsible for increased glomerular albumin permeability.One of the first signs of diabetic nephropathy (DNP) 2 is a discrete increase in urinary excretion of albumin called microalbuminuria (1, 2). In this early phase of DNP mainly the charge-dependent permeability of the glomerular capillary wall seems to be affected, while during progression to overt proteinuria also the size-dependent permeability becomes disturbed (3). These findings suggest an early loss of glomerular basement membrane (GBM) charge with a subsequent increase in pore size. This charge-dependent permeability of the GBM is probably related to the presence of anionic constituents of the GBM, mainly heparan sulfates (HS), the glycosaminoglycan side chains of heparan sulfate proteoglycans (HSPGs) (4). Some years ago, we identified agrin as a major HSPG present in the GBM (5, 6), using antibodies toward either the core protein or the HS polysaccharide side chains (7-9). The relevance of HS for the charge-dependent permeability is illustrated by several observations: (i) enzymatic removal of HS from the GBM leads to albuminuria (10); (ii) an acute selective albuminuria was induced in rats by a monoclonal antibody to HS (4); (iii) a reduction of HS expression in the GBM was documented in several human and experimental proteinuric glomerular diseases, which inversely correlated with proteinuria (11,12).In overt DNP, the expression of HS in the GBM was decreased and correlated with proteinuria (13,14). However, these studies were performed in advanced stages of DNP. In a rat diabetes model resembling early human DNP, we documented a significant loss in HS-related anionic sites in the GBM; howeve...