Diabetic patients, who consume Vacor (N-3-pyridylmethyl-N’-p-nitrophenylurea, MW272), experience microangiopathy causing retinopathy and nephropathy¹⁾. The width of the capillary basement membrane in these patients thickens significantly compared to normal controls after 6 years of Vacor ingestion, on average.

A number of animal models of diabetes have suggested that hyperglycemia can produce microangiopathic changes²⁾. Rodents made hyperglycemic with streptozotocin (STZ) or alloxan developed glomerular changes that were similar to, but not strictly comparable to, those of humans³⁾. Vacor-induced diabetic rats developed acute glomerulopathy within a few months and the glomerulus revealed prominent thickening of the basement membrane⁴⁾.

The glomerular filtration barrier consists of complex matrix constituents interposed between the glomerular endothelial and epithelial cells, which constitute the lining of the glomerular capillaries. They include collagen (mostly type IV), laminin, polyanionic proteoglycans (mostly heparan sulfate), fibronectin, entactin and several other glycoproteins⁵⁾. These individual components of glomerular basement membrane (GBM) were immunohistochemically tagged using specific monoclonal antibodies and observed by light and electron microscopy ^6–8). Changes of four major components of GBM, (type IV collagen, laminin, fibronectin and proteoglycan), were studied in Vacor-induced diabetic rats in this study.

Adult male Wistar rats (n=42), weight 200–250g, were used for this study. A single dose of Vacor, 80 mg/kg of body weight, was administered to the experimental animals by an orogastric canule, while distilled water was given to controls. Blood glucose was measured by glucose oxidase before and after the treatment.

The experimental animals were sacrificed 0.5, 1, 3, 7, 14, 28 and 56 days after the administration of Vacor. Animals were perfused through the jugular vein with 300 ml of physiologic saline followed by an equal amount of 10% buffered formalin. Both kidneys were taken, and then routine formalin-fixed, paraffin-embedded tissue blocks were made for the immunohistochemical studies of glomerular basement membrane (GBM) components. Tissue sections, 4 μm in thickness, were obtained, and stained with hematoxylin-eosin (H&E) and periodic acid Schiff (PAS) stain.

For immunohistochemical studies of the GBM components, monoclonal antibodies for collagen type IV (Monosan, Netherlands), laminin (Monosan, Netherlands), fibronectin (Sigma Bioscience, U.S.A.) and proteogylcan chondroitin sulfate (CSPG, Biogenesis, U.K.) were used. Immunohistochemical studies were performed by using the peroxidase-antiperoxidase technique⁹⁾. Paraffin sections were cut to 4μm in thickness. Deparaffined sections were soaked in absolute methanol containing 0.3% hydrogen peroxide for 30 minutes at room temperature in order to block endogenous peroxidase activity. After washing, the sections were incubated with 0.4% pepsin (Sigma) in 0.01 N HCI for 1 hour at 37°C, then treated with 0.05% protease type VII (Sigma) in phosphate-buffered saline (PBS), pH 7.2, for 15 minutes at 37°C. With these enzyme pretreatments, consistent visualization of collagen type IV in the formalin-fixed tissues was possible. Enzyme digestion was terminated with cold running tap water. After further washing in PBS, the sections were exposed to 1:20 diluted normal swine serum (DAKO-Immunoglobulins Ltd., Denmark) for 30 minutes at room temperature. Then sections were incubated with each primary antibody at a dilution of 1: 500 overnight at 4°C. After washing with PBS, treatment followed with anti-rabbit IgG swine serum (at a dilution of 1:20, DAKO), and PAP solution (at a dilution of 1:80, DAKO), for 30 minutes, respectively, at room temperature. Finally, the sections were soaked in 0.05 M Tris-HCI buffer, pH 7.6, containing 3,3′-diaminobenzidine hydrochloride (40 mg/100 ml) and hydrogen peroxide (0.015%) for 10 minutes, and counterstained with hematoxylin. Vascular walls and renal tubules in the specimens served as positive controls for the primary antibodies. As the negative control, the primary antibodies were replaced by nonimmune rabbit serum by the same procedure.

In humans, characteristic morphological changes of the glomeruli in diabetes mellitus include GBM thickening, diffuse glomerulosclerosis and nodular glomerulosclerosis ¹⁰⁾. Diffuse thickening of the GBM occurs in virtually all diabetics, irrespective of the presence of proteinuria, and constitutes a part of diabetic microangiopathy. GBM thickening can be detected only by electron microscopy. Careful morphometric studies demonstrate that this thickening begins as early as 2 years after the onset of type I diabetes and, by 5 years, develops to about a 30% increase¹¹⁾. Diffuse and nodular glomerulosclerosis are late events for diabetics, and become pronounced after 10 to 20 years of diabetes¹⁰⁾.

In Vacor-treated rats, the glomeruli reveal prominent thickening of the GBM within a short period. The width of the GBM is more than twice that of normal controls, 28 and 56 days after Vacor administration. Increased expressions of collagen IV, laminin, fibronectin and neutral mucopolysaccharide engendered the thickening of the GBM in this study. Proteoglycan immunoreactivity was not significantly different between Vacor-treated and normal control groups. In a study of human diabetic diffuse glomeruloscleosis, the enlarged mesangial matrix also revealed increased staining reactions for collagen IV, V, laminin and fibronectin, whereas the staining pattern for heparan sulfate proteoglycan (HSPG) was markedly reduced¹²⁾. These data suggest that Vacor-induced glomerulopathy in rats can provide a useful experimental model to study human diabetic glomerulopathy.

Expression of type IV collagen and its mRNA in glomeruli and the interstitium of diabetic rats increased at 3, 7 and 14 days after streptozotocin (STZ) administration¹³⁾. The expression was associated with up-regulation of transforming growth factor (TGF)-β. Administration of TGF-β increased collagen synthesis in normal rat glomeruli in a dose-dependent manner up to 5 ng/ml¹⁴⁾. These data indicate a regulatory role for TGF-β in renal glomerular collagen synthesis in normal and diabetic rats. Inhibition of TGF-β and type IV collagen expression by insulin treatment suggests that they may be useful structural markers for determining the efficacy of therapeutic intervention during early diabetic nephropathy¹³⁾. Concomitant increased staining patterns for laminin and type IV collagen have been reported in human diabetic glomerular lesions¹²⁾ and experimental diabetic nephropathy¹⁵⁾. Over-expression of TGF-β was also partly associated with the up-regulation of glomerular laminin gene expression in diabetic mice. The occurrence of collagen type III in late diffuse glomerulosclerosis could be interpreted as an irreversible change in glomerular structure¹⁶⁾.

Fibronectin also increases in the GBM and mesangial matrix in human diabetic nephropathy¹⁷⁾ and STZ-induced diabetic rats^18,19). Fibronectin is a multifunctional matrix protein important in wound healing, and is markedly increased in glomerular crescents²⁰⁾. There are two phases of fibronectin metabolism in anti-GBM models of rapidly progressive glomerulonephritis (RPGN) in the rabbit²⁰⁾. Phase I is associated with increased glomerular fibronectin content from plasma, and phase II with increased fibronectin mRNA in glomeruli at days 7 to 14. This finding suggests that fibronectin synthesis may take place in glomeruli and that it may also be a molecule regulating glomerular cell proliferation and fibrosis.

Two types of proteoglycan, HSPG and basement membrane-specific CSPG, have been studied in human diabetes²¹⁾ and diabetic rats²²⁾. The expression of HSPG decreases in the GBM^16,23) and other capillaries²⁴⁾ in diabetes, while CSPG increases in STZ-diabetic rats²²⁾. A mildly increased expression of CSPG was noted only after 3 to 7 days of Vacor administration in this experiment. Other studies suggest that changes of CSPG displace the capillary endothelial cells from the GBM causing loss of fenestrae, while loss of the HSPG results in alteration of GBM charge-selectivity²³⁾. The abnormal levels of type IV collagen, laminin, fibronectin and proteoglycan may, in part, be the cause of diabetic nephropathy induced by Vacor administration.

To elucidate the biochemical component changes in the GBM in Vacor-induced diabetic glomerulopathy, immunohistochemical analyses of type IV collagen, laminin, fibronectin and CSPG were performed in adult male Wistar rats.

Mild thickening of the GBM was evident 7 days after Vacor administration, and the width of the GBM was more than twice that of normal controls at 28 and 56 days. Significantly increased expressions of type IV collagen, laminin, fibronectin and neutral polysaccharide in the thickened GBM was noted 14 to 56 days after the administration, and the mildly increased expression of CSPG was present after 3 to 7 days. These abnormal levels of GBM components might, in part, be the cause of diabetic nephropathy induced by Vacor administration.