Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1983 Feb 1;157(2):667–686. doi: 10.1084/jem.157.2.667

Immunocytochemical localization of the Heymann nephritis antigen (GP330) in glomerular epithelial cells of normal Lewis rats

D Kerjaschki, MG Farquhar
PMCID: PMC2186920  PMID: 6337231

Abstract

The nephritogenic antigen of Heymann's nephritis (HN) was previously purified from tubular brush-border fractions of rat kidney and found to be a 330,000- mol-wt glycoprotein (gp330). This study was conducted to determine whether gp330 is also present in the rat glomerulus, and, if so, to establish where in the glomerulus it is located. Rabbit polyclonal and mouse monoclonal antibodies were raised against purified gp330, which specifically immunoprecipitated gp330 from solubilized brush-border fractions and specifically stained microvilli and coated invaginations (located at the base of the microvilli) of proximal tubule cells. Accordingly, they were used to localize gp330 by immunoprecipitation and immunocytochemistry in glomeruli of normal Lewis rats. For immunoprecipitation, purified glomerular fractions were prepared from [(35)S]-methionine-labeled kidneys, extracted with Triton X-100, and the extract was used for immunoprecipitation with affinity-purified rabbit polyclonal, or mouse monoclonal, anti-gp330 IgG. Analysis of immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography indicated that a band corresponding in mobility to gp330 was specifically precipitated. When unfixed cryostat sections were incubated for indirect immunofluorescence with monoclonal or affinity-purified polyclonal IgG, a fine granular fluorescent staining was seen throughout the glomerulus. When aldehyde-fixed cryostat sections were incubated for indirect immunoperoxidase, reaction product was detected only in the epithelial cells and was not seen in the GBM, endothelium, or mesangium. Within the epithelium it was localized to the endoplasmic reticulum, occasional Golgi elements, multivesicular bodies, and coated pits at the cell surface. The reactive coated pits were distributed all along the cell membrane, including the sides and base of the foot processes. Reaction product was detected in the latter location only in sections that had been digested with neuraminidase before antibody incubation. When rats were given rabbit anti-gp330 IgG by intravenous injection and their kidneys stained for direct immunoperoxidase 3 d later, rabbit IgG was seen to be deposited beneath the slit diaphragms and in the coated pits at the base of the foot processes. The immunocytochemical and immunoprecipitation data indicate, in confirmation of the results of others, that the nephritogenic HN antigen is present in renal glomeruli as well as in proximal tubular brush borders. The immunocytochemical results further demonstrate that gp330 is an epithelial, rather than a glomerular basement membrane, antigen. It appears to be synthesized by glomerular epithelial cells and subsequently becomes concentrated in coated pits. As both the endogenous antigen (gp330) and exogenously administered anti-gp330 antibody were localized to coated pits, it seems likely that coated pits located at the base of the foot processes are the sites where the HN antigen (gp330) and circulating antibodies directed against gp330 meet and where immune complexes are formed.

Full Text

The Full Text of this article is available as a PDF (4.9 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barabas A. Z., Lannigan R. Induction of an autologous immune-complex glomerulonephritis in the rat by intravenous injection of heterologous anti-rat kidney tubular antibody. I. Production of chronic progressive immune-complex glomerulonephritis. Br J Exp Pathol. 1974 Feb;55(1):47–55. [PMC free article] [PubMed] [Google Scholar]
  2. Bertani T., Nolin L., Foidart J., Vandewalle A., Verroust P. The effect of puromycin on subepithelial deposits induced by antibodies directed against tubular antigens: a quantitative study. Eur J Clin Invest. 1979 Dec;9(6):465–472. doi: 10.1111/j.1365-2362.1979.tb00914.x. [DOI] [PubMed] [Google Scholar]
  3. Collett M. S., Erikson R. L. Protein kinase activity associated with the avian sarcoma virus src gene product. Proc Natl Acad Sci U S A. 1978 Apr;75(4):2021–2024. doi: 10.1073/pnas.75.4.2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Courtoy P. J., Kanwar Y. S., Hynes R. O., Farquhar M. G. Fibronectin localization in the rat glomerulus. J Cell Biol. 1980 Dec;87(3 Pt 1):691–696. doi: 10.1083/jcb.87.3.691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Courtoy P. J., Timpl R., Farquhar M. G. Comparative distribution of laminin, type IV collagen, and fibronectin in the rat glomerulus. J Histochem Cytochem. 1982 Sep;30(9):874–886. doi: 10.1177/30.9.7130672. [DOI] [PubMed] [Google Scholar]
  6. Couser W. G., Steinmuller D. R., Stilmant M. M., Salant D. J., Lowenstein L. M. Experimental glomerulonephritis in the isolated perfused rat kidney. J Clin Invest. 1978 Dec;62(6):1275–1287. doi: 10.1172/JCI109248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. DIXON F. J., FELDMAN J. D., VAZQUEZ J. J. Experimental glomerulonephritis. The pathogenesis of a laboratory model resembling the spectrum of human glomerulonephritis. J Exp Med. 1961 May 1;113:899–920. doi: 10.1084/jem.113.5.899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Edgington T. S., Glassock R. J., Dixon F. J. Autologous immune complex nephritis induced with renal tubular antigen. I. Identification and isolation of the pathogenetic antigen. J Exp Med. 1968 Mar 1;127(3):555–572. doi: 10.1084/jem.127.3.555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ehrlich P. H., Moyle W. R., Moustafa Z. A., Canfield R. E. Mixing two monoclonal antibodies yields enhanced affinity for antigen. J Immunol. 1982 Jun;128(6):2709–2713. [PubMed] [Google Scholar]
  10. Fleuren G. J., Grond J., Hoedemaeker P. J. The pathogenetic role of free-circulating antibody in autologous immune complex glomerulonephritis. Clin Exp Immunol. 1980 Aug;41(2):205–217. [PMC free article] [PubMed] [Google Scholar]
  11. Glassock R. J., Edgington T. S., Watson J. I., Dixon F. J. Autologous immune complex nephritis induced with renal tubular antigen. II. The pathogenetic mechanism. J Exp Med. 1968 Mar 1;127(3):573–588. doi: 10.1084/jem.127.3.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goldstein J. L., Anderson R. G., Brown M. S. Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature. 1979 Jun 21;279(5715):679–685. doi: 10.1038/279679a0. [DOI] [PubMed] [Google Scholar]
  13. HEYMANN W., HACKEL D. B., HARWOOD S., WILSON S. G., HUNTER J. L. Production of nephrotic syndrome in rats by Freund's adjuvants and rat kidney suspensions. Proc Soc Exp Biol Med. 1959 Apr;100(4):660–664. doi: 10.3181/00379727-100-24736. [DOI] [PubMed] [Google Scholar]
  14. Hubbard A. L., Cohn Z. A. Externally disposed plasma membrane proteins. II. Metabolic fate of iodinated polypeptides of mouse L cells. J Cell Biol. 1975 Feb;64(2):461–479. doi: 10.1083/jcb.64.2.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kerjaschki D., Farquhar M. G. The pathogenic antigen of Heymann nephritis is a membrane glycoprotein of the renal proximal tubule brush border. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5557–5561. doi: 10.1073/pnas.79.18.5557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Louvard D., Reggio H., Warren G. Antibodies to the Golgi complex and the rough endoplasmic reticulum. J Cell Biol. 1982 Jan;92(1):92–107. doi: 10.1083/jcb.92.1.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Makker S. P., Moorthy B. In situ immune complex formation in isolated perfused kidney using homologous antibody. Lab Invest. 1981 Jan;44(1):1–5. [PubMed] [Google Scholar]
  18. Malathi P., Preiser H., Fairclough P., Mallett P., Crane R. K. A rapid method for the isolation of kidney brush border membranes. Biochim Biophys Acta. 1979 Jun 13;554(1):259–263. doi: 10.1016/0005-2736(79)90023-3. [DOI] [PubMed] [Google Scholar]
  19. Maunsbach A. B. The influence of different fixatives and fixation methods on the ultrastructure of rat kidney proximal tubule cells. I. Comparison of different perfusion fixation methods and of glutaraldehyde, formaldehyde and osmium tetroxide fixatives. J Ultrastruct Res. 1966 Jun;15(3):242–282. doi: 10.1016/s0022-5320(66)80109-0. [DOI] [PubMed] [Google Scholar]
  20. Michael A. F., Blau E., Vernier R. L. Glomerular polyanion. Alteration in aminonucleoside nephrosis. Lab Invest. 1970 Dec;23(6):649–657. [PubMed] [Google Scholar]
  21. Miettinen A., Törnroth T., Tikkanen I., Virtanen I., Linder E. Heymann nephritis induced by kidney brush border glycoproteins. Lab Invest. 1980 Dec;43(6):547–555. [PubMed] [Google Scholar]
  22. Naruse T., Fukasawa T., Hirokawa N., Oike S., Miyakawa Y. The pathogenesis of experimental membranous glomerulonephritis induced with homologous nephritogenic tubular antigen. J Exp Med. 1976 Nov 2;144(5):1347–1362. doi: 10.1084/jem.144.5.1347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Naruse T., Fukasawa T., Miyakawa Y. Laboratory model of membranous glomerulonephritis in rats induced by pronase-digested homologous renal tubular epithelial antigen. Lab Invest. 1975 Aug;33(2):141–146. [PubMed] [Google Scholar]
  24. Neale T. J., Couser W. G., Salant D. J., Lowenstein L. M., Wilson C. B. Specific uptake of Heymann's nephritic kidney eluate by rat kidney: studies in vivo and in isolated perfused kidneys. Lab Invest. 1982 Apr;46(4):450–453. [PubMed] [Google Scholar]
  25. Neale T. J., Wilson C. B. Glomerular antigens in Heymann's nephritis: reactivity of eluted and circulating antibody. J Immunol. 1982 Jan;128(1):323–330. [PubMed] [Google Scholar]
  26. Pearse B. M., Bretscher M. S. Membrane recycling by coated vesicles. Annu Rev Biochem. 1981;50:85–101. doi: 10.1146/annurev.bi.50.070181.000505. [DOI] [PubMed] [Google Scholar]
  27. Pearse B. M. Coated vesicles from human placenta carry ferritin, transferrin, and immunoglobulin G. Proc Natl Acad Sci U S A. 1982 Jan;79(2):451–455. doi: 10.1073/pnas.79.2.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. ROTH T. F., PORTER K. R. YOLK PROTEIN UPTAKE IN THE OOCYTE OF THE MOSQUITO AEDES AEGYPTI. L. J Cell Biol. 1964 Feb;20:313–332. doi: 10.1083/jcb.20.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sugisaki T., Klassen J., Andres G. A., Milgrom F., McCluskey R. T. Passive transfer of Heymann nephritis with serum. Kidney Int. 1973 Feb;3(2):66–73. doi: 10.1038/ki.1973.13. [DOI] [PubMed] [Google Scholar]
  30. Van Damme B. J., Fleuren G. J., Bakker W. W., Vernier R. L., Hoedemaeker P. J. Experimental glomerulonephritis in the rat induced by antibodies directed against tubular antigens. V. Fixed glomerular antigens in the pathogenesis of heterologous immune complex glomerulonephritis. Lab Invest. 1978 Apr;38(4):502–510. [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES