Laboratory Investigation

Kidney International (1983) 24, 74–86; doi:10.1038/ki.1983.128

Monoclonal antibodies to human renal basement membranes: Heterogenic and ontogenic changes

Alfred F Michael1, Ji-Yun Yang1, Ronald J Falk1, Martha J Bennington1, Jon I Scheinman1, Robert L Vernier1 and Alfred J Fish1

1Departments of Pediatrics, Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota

Correspondence: Dr A F Michael, Department of Pediatrics, P.O. Box 491 Mayo Memorial Building, University of Minnesota Medical Center, 515 Delaware Street, S.E., Minneapolis, Minnesota 55455, USA

Received 23 August 1982; Revised 28 December 1982.

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Abstract

Monoclonal antibodies to human renal basement membranes: Heterogenic and ontogenic changes. Monoclonal antibodies reactive with rensssssal basement membranes have been developed following immunization of mice, subsequent hybridization of spleen cells with the NS1 cell line, and appropriate cloning techniques. The antibody reactivity and specificity of clonal supernatants and ascites fluid were evaluated by indirect epifluorescence-phase contrast microscopy on sections of human kidney and other tissues. This permitted characterization of clones based on the immunohistochemical phenotype of the antibody in binding to anatomically defined basement membranes as well as to site-specific loci or regions within the membrane. Based upon unique immunohistochemical reactivity, nine différent antibodies were developed: two reacting with known components of basement membrane by ELISA (MBM4-type IV collagen and MBM20-fibronectin) and seven failing to react with these or other defined antigens (fibronectin, laminin, or types IV and V collagen). Among the latter group, five immunohistochemical phenotypes were identified in mature human kidney: (1) MBM7 and MBM15 reacted with the lamina densa, Bowman's capsule, tubular basement membrane, and vascular walls. (2) MBM10 and MBM14 fixed in a double-linear granular pattern to each side of the tubular basement membrane and Bowman's capsule as well as within the mesangium and in vascular basement membranes but not the glomerular basement membrane (GBM). (3) MBM11 identified sites along the internal aspect of Bowman's capsule and the tubular basement membrane but not within the glomerulus. It also stained type I but not type II skeletal muscle. (4) MBM12 reacted with the full thickness of Bowman's capsule, the mesangium and stalk region, the tubular basement membrane, but not the GBM. (5) MBM21, which had the most restricted binding in tissue, identified an antigen in the tubular basement membrane of the proximal tubules. During nephron formation type IV collagen and fibronectin were redistributed from the undifferentiated mesenchyme to appear with induction within the "S" shaped anlage, in latter stages in the region of the primitive GBM, and in the mature glomerulus in the mesangium and the GBM. This pattern seen in the human kidney is similar to that described in the mouse. Changes in the distribution of antigens identified by other monoclonal antibodies were observed during ontogenesis. Eight monoclonal antibodies recognized antigens in fetal GBM, but only four of these reacted with mature GBM. Certain antibodies (MBM4, MBM10, MBM14, MBM12) had a broad range of reactivity with basement membranes in different tissues, whereas others (MBM7, MBM15, MBM11) demonstrated more limited reactivity; MBM21 was the most restrictive of all reacting only with the proximal tubular basement membrane. Specific basement membranes in different tissues shared the same antigens as evidenced by similar profiles of reactivity with various antibodies. Basement membranes in human kidney are heterogeneous with respect to constituent antigens and undergo changes during ontogenesis. These studies provide the foundation for further studies that will permit mapping and characterization of immunoreactive basement membrane components.

Anticorps monoclonaux à les membranes basales rénales humains: Modifications des hétérogénique et ontogénique. Des anticorps monoclonaux réagissant contre des membranes basales rénales ont été développés après immunisation de souris, puis hybridation de cellules spéniques avec la lignée cellulaire NS1, et techniques de clonage appropriées. La réactivité et la spécificité des anticorps des surnageants de clonage et de liquides d'ascite ont été évaluées par microscopie à contraste de phase avec épifluorescence indirecte sur des coupes de rein et d'autres tissus humains. Cela a permis la caractérisation de clones fondée sur le phénotype immunohistochimique des anticorps se liant aux membranes définies anatomiquement, et à la spécificité de site pour certains lieux ou certaines régions de la membrane. En utilisant uniquement la réactivité immunohistochimique, neuf anticorps différents ont été développés: deux réagissant avec des constituants connus de la membrane basale par ELISA (MBM4-collagène de type IV et MBM20-fibronectine) et sept ne réagissant pas avec ces antigènes ou d'autres (fibronectine, laminine, ou collagène de type IV et V). Dans ce dernier groupe, cinq phénotypes immunohistochimiques ont été identifiés dans du rein humain mature: (1) MBM7 et MBM15 réagissaient avec la lamina densa, la capsule de Bowman, la membrane basale tubulaire, et les parois vasculaires. (2) MBMIO et MBM14 se fixaient avec un aspect granulaire en double contour à chaque coté de la membrane basale tubulaire et de la capsule de Bowman, ainsi que dans le mésangium et sur les membranes basales vasculaires, mais non sur la membrane basale glomérulaire (GBM). (3) MBM11 a permis l'identification de sites le long de la partie interne de la capsule de Bowman et de la membrane basale tubulaire mais non dans le glomérule. Il colorait également le muscle squelettique de type I mais non de type II. (4) MBM12 réagissait avec la totalité de l'épaisseur de la capsule de Bowman, le mésangium et la région des tiges, la membrane basale tubulaire mais non la GBM. (5) MBM21, qui avait la liaison tissulaire la plus réduite permettait la localisation d'un antigène dans la membrane basale tubulaire des tubules proximaux. Pendant la formation néphronique, le collagène de type IV et la fibronectine étaient redistribués à partir du mésenchyme non différencié pour apparaître lors de l'induction à l'intérieur des structures en forme de "S" aux stades plus tardifs dans la région de la GBM primitive, et dans le glomérule mature, dans le mésangium et la GBM. Cet aspect vu dans le rein humain est identique à celui décrit chez la souris. Des modifications dans la distribution d'antigènes identifiés par d'autres anticorps monoclonaux ont été observées pendant l'ontogenèse. Huit anticorps monoclonaux ont reconnu des antigènes dans la GBM foetale, mais seulement quatre d'entre eux ont réagi avec des GBM matures. Certains anticorps (MBM4, MBM10, MBM14, MBM12) avaient un large éventail de réactivité avec les membranes basales dans différents tissus, tandis que d'autres (MBM7, MBM15, MBM11) ont démontré une réactivité plus limitée; MBM21 était le plus restreint de tous, réagissant seulement avec la membrane tubulaire proximale. Des membranes basales spécifiques dans différents tissus partageaient les mêmes antigènes, comme cela été montré par des profils de réactivités identiques avec des anticorps variés. Les membranes basales du rein humain sont hétérogènes en ce qui concerne les antigènes les constituant et subissent des modifications pendant l'ontogenèse. Ces études constituent la base pour des travaux ultérieurs qui permettront de localiser et de caractériser définitivement biochimiquement les constituants immuno-réactifs des membranes basales.

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