Jon I. Scheinman

Polyantigenic Expansion of Basement Membrane Constituents in Diabetic Nephropathy

The immunohistopathology of the intrinsic basement membrane-associated antigens were examined in diabetic nephropathy. In early and moderate stages of disease there was polyantigenic expansion of all the intrinsic components of mesangium, glomerular basement membrane (GBM), and tubular basement membrane (TBM) assessed by polyclonal antisera to collagen types IV and V, laminin, and by monoclonal antibodies to type IV collagen and fibronectin and to four other intrinsic components of normal renal extracellular matrices (MBM10, 11, 12, and 15). In the mesangium the first intrinsic antigens to increase were fibronectin and type V collagen. In late stages of disease, there was a diminution in the mesangium of all of these antigens with the exception of type V collagen, which persisted. Additionally, antigens appeared in the mesangium, recognized by MBM11 and MBM15, which are normally present in fetal but not adult mesangial regions. Similarly, in the GBM in late stages of disease, there was a decrease in all of the antigens, except for a persistence of the antigen recognized by MBM15. However, in TBM all of the antigens assessed increased in early, moderate, and severe disease. These studies document the complexity of polyantigenic alterations in the development of diabetic nephropathy.

The immunohistology of glomerular antigens. IV. Laminin, a defined noncollagen basement membrane glycoprotein.

Abstract This study describes the detailed immunohistologic localization of laminin, a defined noncollagen basement membrane glycoprotein and its relationship to fibronectin and the antigens localized by the Goodpasture antibody (GPaGBM) and heterologous anti-GBM antibody (RaGBM) in normal human kidney. The bilaminar GBM staining pattern for laminin reveals an outer epithelial line continuous with Bowmans capsule and the TBM and an inner subendothelial line continuous with the mesangium and vascular smooth muscle wall. This suggests a previously unrecognized role for the mesangial cell in the normal structure of the peripheral GBM. Fibronectin, predominantly mesangial, also has a weak localization in the subendothelial space, but no epithelial component. These defined glycoprotein antigens differ from those localized by GP antibody and RaGBM. The role of noncollagen glycoproteins in the maintenance of GBM filtration barrier remains to be elucidated. The adhesive or repulsive properties of molecules such as fibronectin may be required for the maintenance of normal glomerular cellular relationships.

The Immunohistopathology of Glomerular Antigens THE GLOMERULAR BASEMENT MEMBRANE, COLLAGEN, AND ACTOMYOSIN ANTIGENS IN NORMAL AND DISEASED KIDNEYS

The immunofluorescent localization of antisera to human glomerular basement membrane (GBM), collagen, and smooth muscle actomyosin was examined in 15 specimens of normal renal tissue and 98 specimens from patients with renal disease. The anti-GBM and anticollagen antisera normally localize to GBM, while antiactomyosin localizes to the mesangium. Diabetic nephropathy revealed a striking expansion of mesangial material reacting with antiactomyosin. In contrast, the expanded mesangium in membranoproliferative glomerulonephritis did not react with antiactomyosin, and the GBM localization of anti-GBM and anticollagen sera was similarly lost. The thickened GBM in diabetes mellitus and membranous nephropathy reacted with anti-GBM and anticollagen, but with accentuation of staining on the inner aspect of the GBM. In proliferative glomerulonephritis there was a moderate increase in the distribution of actomyosin. Glomerular sclerosis and hyalinization in all diseases studied was accompanied by a loss of immunofluorescent staining for all glomerular antigens, including collagen.

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

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--Goodpastures 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.

Collagen synthesis by human glomerular cells in culture.

Collagen synthesis was studied in three subcultured human glomerular cell types, by radiolabeled incorporation of [14C]proline and [3H]lysine. The epithelioid circular glomerular cells secrete a collagen with a single size of chain (possibly type IV) with a high ratio of hydroxyproline to proline, hydroxylysine to lysine, and 11--17% of hydroxyproline as the 3-isomer. The smooth muscle-like rhomboid glomerular cells secrete collagen with a chain pattern suggesting types III and I collagen, distinct from that found in the media of fibroblasts. Small ovoid glomerular cells are morphologically and biochemically intermediate between circular glomerular cells and rhomboid glomerular cells, and may represent an in vitro modification of either circular glomerular cells or rhomboid glomerular cells.

Immunopathological studies of the ruptured human renal allograft

SUMMARY The immunopathology of five cases of spontaneous allograft rupture has been studies. All kidneys were edematous on exploration and routine histological sections showed interstitial edema and mononuclear cell infiltration characteristic of acute rejection. Immunofluorescence revealed, at most, scattered vascular deposition of IgM and mild mesangial C3 deposition. These findings are compared with findings in normal kidneys and kidneys which had been hyperacutely rejected. The normal kidney showed focal afferent arteriolar and proximal mesangial stalk deposition of C3 without IgM. The kidneys of patients with hyperacute rejection showed brilliant staining for fibrin and IgM in all arterial and arteriolar walls with lesser amounts of C3 and IgG; IgM and C3 were prominent in the glomerulus. These findings suggest that mechanisms other than circulating preformed antibodies are responsible for the pathogenesis of spontaneous allograft rupture.

The Immunohistopathology of Glomerular Antigens: III. Increased Mesangial Actomyosin in Experimental Diabetes in the Rat

Antisera to rat smooth muscle actomyosin (AMY) and myosin localize in the rat glomerular mesangium. The width of mesangial staining for AMY is increased in rats diabetic for four months (p < 0.01) and seven months (p < 0.0005) compared with age-matched controls. Mesangial AMY staining of unilaterally nephrectomized control animals was moderately increased after seven months, whereas unilaterally nephrectomized diabetic rats had prominently increased AMY mesangial width at four months, when they were compared with intact diabetic animals (p < 0.05). Thus, a distinctive alteration that is found in human diabetic nephropathy also occurs in experimental (streptozotocin) diabetes in the rat. Further, this alteration appears to be accelerated by the changes in nephron hemodynamics resulting from unilateral nephrectomy. While the function of mesangial AMY is unknown, it may be related to intrarenal regulation of glomerular ultrafiltration, which appears to be altered in diabetic nephropathy in man.

Immunochemical and Biochemical Studies of Human Glomerular Cells in Culture

Glomerular culture makes it possible to determine the contributions of the different cells of the glomerulus to normal and abnormal in situ glomerular structure and function. The confident identification of glomerular cells in vitro with the differentiated cells of the glomerulus requires more certain cell markers than are currently available.

The Effects of Lathyrism on Renal Chemistry, Anatomy and Function

Summary A significant increase was induced by lathyrism in the small fraction of collagen-like material solubilized from rat glomeruli. An increased affinity and staining by PTA of fibrils within the GBM of lathyritic rats was found by electron microscopy, and also probably reflects an alteration in the collagen-like moiety of the GBM by APN, a material known to affect the cross-linking of collagens. No change in total proteinuria was found in lathyritic rats, nor other major changes in renal structure by conventional microscopy, electron microscopy, or specialized stains of the sialoprotein layer of the glomerulus. Further study of physicochemical alterations induced in the GBM combined with methods to evaluate glomerular permeability, may reveal the location and nature of the filtration barrier.