Preservation of mesangium and immunohistochemically defined antigens in glomerular basement membrane isolated by detergent extraction.

Abstract

A B S T R A C T To define the characteristics of isolated glomerular basement membrane (GBM), immunohistochemical and morphometric analyses have been carried out on rat and human tissues. Site-specific arrays of antigens were identified in detergent-isolated GBM in a distribution similar to that observed in intact kidney. In the human, fibronectin, procollagen IV, and collagen V were observed along the internal aspect of GBM continuous with antigenic sites in the mesangium. Another array of antigens was identified in the GBM but not within the mesangium-Goodpasture's antigen, bovine lens capsule type IV collagen, and amyloid P component. In addition, sites reactive with rabbit antiserum to laminin were present on both sides of the lamina densa as well as within the mesangial region. Actomyosin, a presumed mesangial cell antigen persisted in the mesangium of isolated GBM. Mesangial matrix was identified in detergent-isolated GBM in an amount equivalent to that present in intact glomeruli. Sonicated GBM contained the same antigens but it was not possible to quantitate the amount of mesangial material by immunofluorescence or morphometric analysis. The thickness of the lamina densa was greater in sonicated and detergent-treated rat GBM preparations than in native rat kidney. These studies demonstrated that isolated GBM is heterogeneous with respect to its antigenic constituents and in addition contains mesangial matrix, which is morphologically and immunohistochemically distinct from peripheral GBM.

Neoantigen of the polymerized ninth component of complement. Characterization of a monoclonal antibody and immunohistochemical localization in renal disease.

“…However, the GBM preparation is only partially purified and is known to consist of a variety of renal extracellular matrix and plasma antigenic determinants (42,43). That Poly C9-MA did not react with the preparation used for immunization reflects the scarcity of the antigen in this preparation, as well as the capacity to develop monoclonal antibodies to trace amounts of an antigen.…”

Identification of autoantibody clusters that best predict lupus disease activity using glomerular proteome arrays

“…The literature was first surveyed to identify Ags that were known to be expressed in the glomerular milieu and/or GBM (43)(44)(45). Total glomerular sonicates were obtained by harvesting glomeruli from B6 mouse kidneys and sonicating them as described previously (46,50).…”

“…The purpose of the present study was to adopt a similar multiplexed approach to define the spectrum of autoantibodies reactive with different glomerular or glomerular basement membrane (GBM) Ags, which best correlate with disease activity and may be present in lupus. To this end, a panel of Ags documented to be present in the glomeruli/GBM (43)(44)(45) was spotted onto specially precoated glass slides; these arrays have been termed glomerular proteome arrays. These novel proteome microarrays were then used to analyze the nephrophilic autoantibody profiles in murine and human lupus.…”

Occurrence of Type VI Collagen in Extracellular Matrix of Renal Glomeruli and Its Increase in Diabetes

“…Because GBM as isolated by available procedures, whether sonication (6) or detergent treatment (7), contains both the peripheral basement membrane and the basement membrane-like material of the mesangium (8), biochemical alterations observed could reflect changes occurring in either of these distinct but closely integrated regions of the glomerular extracellular matrix, which are indeed synthesized by different cell types (9). To evaluate whether diabetes may differentially affect the cells of these two regions, it would be valuable to have a biochemical marker that is not, like type IV collagen (9), distributed throughout the glomerular extracellular material.…”