Advanced protein glycation has been proposed as a major factor in the development of diabetic nephropathy. Advanced glycation end products (AGEs) have altered the structure of extracellular matrix component and impaired self association in vitro. To elucidate the role of AGEs in the progression of diabetic nephropathy, the present study was undertaken to localize glomerular AGEs immunohistochemically. Ultrastructural changes of the mesangial matrix were analyzed with high resolution scanning electron microscopy. No glomerular AGEs staining was noted in normal control kidney specimens, or in tissue from glomerulonephritis patients without diabetes mellitus. The mesangium showed a positive AGEs staining in advanced stages of diabetic nephropathy, and the most characteristic finding was the strong AGEs staining in nodular lesions. By high resolution scanning electron microscopy, control and diabetic mesangial matrices revealed a meshwork structure composed of fine fibrils (10 nm in width) and numerous pores (12 to 13 nm in diameter). In the nodular lesions, however, loosening of the meshwork was significant, and the diameter of the pores was enlarged (approximately 24 nm). This study provides the first immunohistochemical evidence that AGEs are localized in diabetic glomeruli, most notably to nodular lesions. Advanced glycation might play a role in the progression of diabetic nephropathy through impairment of the assembly of matrix proteins in vivo.

Three-dimensional ultrastructures of basement membranes of the rat kidney were investigated with an ultrahigh resolution scanning electron microscope (HSEM) equipped with a resolving power of 0.5 nm. All cellular components were extracted from renal cortical tissues by sequential-detergent treatment. Four types of acellular basement membranes were observed after tannin-osmium conductive staining: the glomerular basement membrane (GBM) associated with the mesangial matrix, the tubular basement membrane (TBM), the Bowman's capsule basement membrane (BCBM), and the peritubular capillary basement membrane (PTCBM). We could demonstrate the polygonal meshwork structures composed of strands in the respective basement membranes. The strands averaged 6 to 7 nm wide, whereas the pore sizes within the meshworks were variable and differed according to the basement membrane type. Moreover, we confirmed the presence of the heterogeneity of the GBM suggested by several approaches. Present data support the proposition that a polygonal meshwork structure may represent the basic structure of basement membrane. Some of the observed architectural dissimilarities in basement membrane types may reflect their different functional properties, which in turn may reflect the heterogeneous distribution of major basement membrane components as demonstrated by immunohistochemical and biochemical studies.

Background/Aims: Glomerular basement membranes (GBM) and tubular basement membranes (TBM) consist of a fine meshwork composed mainly of type IV collagen. Each segment of tubules has specialized physiologic functions, and thus we investigated the ultrastructure of various basement membranes in rat kidneys. Methods: Since purifying basement membranes from different tubule segments is technically challenging, we employed tissue negative staining rather than conventional negative staining to compare the ultrastructures of proximal and distal TBM and GBM in normal rats. We also assessed the distribution of extracellular matrix components including type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin in the basement membranes by immunohistochemistry. Results: TBM and GBM of normal rats showed a fine meshwork structure consisting of fibrils forming small round to oval pores. Short- and long-pore diameters in proximal tubules were 3.3 ± 0.5 and 3.9 ± 0.6 nm, respectively, and in distal tubules 3.5 ± 0.7 and 4.3 ± 0.8 nm, respectively. For GBM the respective diameters were 2.5 ± 0.5 and 3.0 ± 0.5 nm. Immunohistochemical analysis showed no significant difference in distribution of extracellular matrix components between proximal and distal TBM. However, immunofluorescence scores of α1 chain of type IV collagen, fibronectin, and laminin were higher in the TBM than in the GBM. On the other hand, heparan sulfate proteoglycan was higher in the GBM. Conclusion: Ultrastructural differences in renal basement membranes may be related to differences in physiologic function in each segment.

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