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Some metabolic and structural characteristics of experimental nephrosis
AHERICA~
HEART JOUR
October, 1959 Volume
stme
58, No. 4
dE
Mary Ellen Havtnean, M.D., Philadelphia, Pa. Nephrosis was first experimentally reproduced by Masugi,’ in 1933, by means of an antiserum against rat kidney produced in the rabbit. When the antiserum is injected into the rat its nephrotoxic effect results from an antigen-a&body reaction, as evidenced by a fall in complement2 and a localization of antibody in the renal glomerulus.’ The specific site of glomerular localization, and therefore by inference the localization of the specific antigen or antigens, is in or on the glomerular basement membrane.‘Jj There are multiple glomerular antigens! which are identical with, or closely related to, antigens present in extragknnerruiar tissues. These antigens are present in various tissues in propoxtion to the degree of vascularity of the tissue and are presumed to be associated with the basement membrane of precapillary, capillary, and postcapillary vessels5~ Since the dinical picture of hyperlipemia, hypoproteinemia, proteinuria, and anasarca resulting from the antibody localization is referable to the kidney, it may be inferred that the basement membrane subserves its most vital role in that location. A similar type of diseasecan be produced in the rat by the daily subcutaneous injection of 6-dimethylamino-9-(3’-amino-3’-deoxy-b-D-ribofuranosyl) purine, the aminonucleoside derivative of the antibiotic, Puromycin.s The aminonucleoside disease occurs after the latent period of 5 to 15 days, depending on dosage.s Following the daily administration of 0.015 mg. of aminonucleoside per gram of rat the animals usually die on about the sixteenth day. With the same dosage it has been found that the disease regresses if the injections are terminated on about the fourteenth day.rO In addition to the in vivo toxicity of this compound both the aminonucleoside and the antiserum against rat kidney have cytotoxic effects on tissue cultures of rat kidney.“J2 Examination of kidney sections with the light microscope reveals that in the lipoid nephrosis of children, and in both varieties of experimental nephrosis discussed here, a thickening of the basement membrane, an increased number of From the Department of Anatomy,’ Temple delphia, Pa. Received for publication June 19, 1959.
University
483
School
of Medicine
and
Hospital,
Phita-
484
HARTMAN
Am. Heart J. October. 1959
PAS positive droplets in the proximal convoluted tubules, and a decrease in studies of alkaline phosphatase in the tubules occur.13-15 Electron microscopic kidney sections also show identical changes in the lipoid nephrosis of children and in the aminonucleoside nephrosis. These changes consist of swelling and coaIescence of the foot processes of the epitheliai cells covering the glomerular basement membrane, and an increase in the number and size of the cytoplasmic vacuoles within these cells.1”J6J7 In animals recovering from the aminonucleoside nephrosis the pathologic changes observable with the electron microscope show beginning regression.lO This may be compared with the observation that in the human nephrotic syndrome, steroid therapy reverses the changes in the foot processes.18~1s The electron microscopic studies both in the lipoid nephrosis of children16J7 and in the aminonucleoside diseasei indicate that the first morphologic changes occur in the epithelial celis at the same time as the onset of the severe proteinuria. Later in the development of the disease there is an increase in the amount of the basement membrane material and an increased number of endothelial cell nuclei. It has also been reported that defects measuring several hundred to 1,000 A were found in the glomerular basement membrane of a patient with nephrosis. 2o The primary change in the morphology of the epithelial cell component of the renal corpuscle is also reflected in the abnormal presence of alkaline phosphatase in these cells in the antibody-induced nephrosis.21 Later in the course of the disease the basement membrane and the endothelial cells also become alkaline-phosphatase positive, whereas the tubular cells have a decreased concentration of this enzyme. The latter event is probably coincidental to the loss of the brush border.” The ability of the aminonucleoside to reproduce so exactly the same pathologic picture found in the nephrotic disease of children and in the antigenantibody disease produced in rats has provided a convenient tool with which to study the possible mechanism by which such a disease process might occur. Since there is a marked structural resemblance between the aminonucleoside and adenosine, the possibility was considered that the aminonucleoside might act as an inhibitor in the formation of, or in reactions involving, ATP, or in some other phase of nucleotide metabolism. If such were the case, administration of various purine derivatives might prevent the inhibition. Adenine and a number of synthetic purine derivatives were known to reverse the antitrypanosomal action of the aminonucleoside in mice,22 and more recently it was established that the aminonucleoside inhibits the formation of ATP from inorganic phosphate and adenosine by brewer’s yeast.2s The data obtained .to date24 support the idea that the aminonucleoside acts as an antimetabolite during the production of the nephrotic syndrome in rats. When given simuItaneousIy with the aminonucleoside, adenine partially inhibited the development of the nephrosis, whereas adenosine, in the doses given, did not. The failure of adenosine was probably due to the high level of adenosine deaminase present in mammalian tissues.z5 The picture is complicated by the fact that adenine is toxic and can produce thickening of the basement membrane.24 The possible practical implications of these experimental observations revolve on either or both of two premises: (1) In the human patient with nephrosis
yaLl~r
“,”
CHARACTERISTICS
OF EXPERIMENTAL
NEPHROSIS
485
there may exist at some time during the onset of his disease either an abnormal nucleotide or nucleoside which functions as an antimetabolite, or a normal nucleotide or nucleoside which is present in abnormally high concentrations and serves as an inhibitor of sensitive enzyme systems. (2) The morphologic common denominator of nephrosis is injury to a specific component of the renal corpuscle. At the present time there is no evidence for the existence of a specific antimetabolite which may give rise either to the allergic experimental nephrosis or to human nephrosis. However, because of the marked resemblance between the antibodyinduced nephrosis and the aminonucleoside nephrosis the possibility cannot be discounted that some substance which functions as an antimetabolite may be formed as a by-product of the antigen-antibody reaction. Currently, the only link between renal disease and aberrant purine metabolism is a preliminary report showing that the blood of azotemic patients contains an abnormally high level of nucleotides. In this study the greatest increase was in ATP, but increases were also found in DPN, TPN, and GTP .26 There is a gradually accumulating body of evidence that the epithelial cell is the primary site of damage in both human and experimental nephrosis.10j16a17 That a chemical agent causing this damage might exert its effect in some phase of nucleotide or nucleic acid metabolism is an enticing concept which deserves further consideration and investigation. REFERENCES
Masugi, M.:
Beitr. path. Anat. u. allg. Path. 91:82, 1933. Proc. Sot. Exner. Biol. & Med. 85:593. 1934. Pressman, D., Hill, R. F., and Foote, F. W.: Science 109~65, 1949. Krakower, C. A., and Greenspon, S. A.: A.M.A. Arch. Path. 51:629, 1951. Mellors, R. C., Siegel, M., and Pressman, D.: Lab. Invest. 4:69, 1955. Yagi, Y., and Pressman, D.: J. Immunol. 81:7, 1958. Krakower, C. A., and Greenspon, S. A.: A.M.A. Arch. Path. 64:364, 1958. Frenk, S., Antonowicz, I., Craig, J. M., and Metcoff, J.: Proc. Sot. Exper. Biol. & Med. 59:424, 195.5. Wilson, S.: Proc. Ninth Ann. Conf. on the Nephrotic Syndrome, October, 1957, New York, 1958, National Nephrosis Foundation, p. 213. 1959. Vernier, R. L., Papermaster, B. W:, and Good, R. A.: J. Exper. Med. ltWrl15, Liu, C., McCrory, W. W., and Flick, J. A.: Proc. Sot. Exper. Biol. Br Med. %r331, 1957. McCrory, W. W.: Proc. Ninth Ann. Conf. on the Nephrotic Syndrome, October, 1957, New York, 1958, National Nephrosis Foundation, p. 217. Spater. H. W.: Proc. Ninth Ann. Conf. on the Nephrotic Svndrome. October. 1957. New York, 1958, National Nephrosis Foundation, p. 220. _ Heymann, W., and Lund, H. 2.: Pediatrics 7:691, 1951. Ehrich, W. E., Forman, C. W., and Seiier, J.: A.M.A. Arch. Path. 54,463, 1952. Farquhar, M., Vernier, R. L., and Good, R. A.: J. Exper. Med. 1@&649, 1957. Farquhar, M., Vernier, R. L., and Good, R. A.: Am. J. Path. 35:791, 19.57. FoIli, G., Pollack, V. E., Retd, R. T. W., Pirani, C., and Kark, R.: Ann. Int. Med. 49:775, 1958. Vernier, R. L., Farquhar, M., Brunson, J. G., and Good, R. A.: A.M.A. Am. J. Dis. Child. %:306, 1958. Spiro, D.: Am. J. Path. 35:47, 1959. Hartman, M. E.: A.M.A. Arch. Path. 64:679, 1957. Hewitt, R. I., Gumble, A. R., Wallace, W. S., and Williams, J. H.: Antibiotics & Chemother. 4:1222, 1954. Kessner, D. M., Borowsky, B. A., and Recant, L.: Proc. .Soc. Exper. Biol. & Med. %r766, 1958. Hartman, M. E., Hartman, J. D., and Baldridge, R. C.: Proc. Sot. Exper. BioI. & Med. 100:152, 1959. Kalcker, H. M.: J. Biol. Chem. 167:429, 1947. Bishop, C., Rankine, D., and Talbott, J. H.: Fed. Proc. 18:11, 1959.
:: Stavitskv, A. B.. Hackel. D. B.. and Hevmann. W.: 3. 4. i: 7. 8. 9. ::: 12. 13.
19. 20. 21. 22. 23. 24. E:
HEART JOUR
October, 1959 Volume
stme
58, No. 4
dE
Mary Ellen Havtnean, M.D., Philadelphia, Pa. Nephrosis was first experimentally reproduced by Masugi,’ in 1933, by means of an antiserum against rat kidney produced in the rabbit. When the antiserum is injected into the rat its nephrotoxic effect results from an antigen-a&body reaction, as evidenced by a fall in complement2 and a localization of antibody in the renal glomerulus.’ The specific site of glomerular localization, and therefore by inference the localization of the specific antigen or antigens, is in or on the glomerular basement membrane.‘Jj There are multiple glomerular antigens! which are identical with, or closely related to, antigens present in extragknnerruiar tissues. These antigens are present in various tissues in propoxtion to the degree of vascularity of the tissue and are presumed to be associated with the basement membrane of precapillary, capillary, and postcapillary vessels5~ Since the dinical picture of hyperlipemia, hypoproteinemia, proteinuria, and anasarca resulting from the antibody localization is referable to the kidney, it may be inferred that the basement membrane subserves its most vital role in that location. A similar type of diseasecan be produced in the rat by the daily subcutaneous injection of 6-dimethylamino-9-(3’-amino-3’-deoxy-b-D-ribofuranosyl) purine, the aminonucleoside derivative of the antibiotic, Puromycin.s The aminonucleoside disease occurs after the latent period of 5 to 15 days, depending on dosage.s Following the daily administration of 0.015 mg. of aminonucleoside per gram of rat the animals usually die on about the sixteenth day. With the same dosage it has been found that the disease regresses if the injections are terminated on about the fourteenth day.rO In addition to the in vivo toxicity of this compound both the aminonucleoside and the antiserum against rat kidney have cytotoxic effects on tissue cultures of rat kidney.“J2 Examination of kidney sections with the light microscope reveals that in the lipoid nephrosis of children, and in both varieties of experimental nephrosis discussed here, a thickening of the basement membrane, an increased number of From the Department of Anatomy,’ Temple delphia, Pa. Received for publication June 19, 1959.
University
483
School
of Medicine
and
Hospital,
Phita-
484
HARTMAN
Am. Heart J. October. 1959
PAS positive droplets in the proximal convoluted tubules, and a decrease in studies of alkaline phosphatase in the tubules occur.13-15 Electron microscopic kidney sections also show identical changes in the lipoid nephrosis of children and in the aminonucleoside nephrosis. These changes consist of swelling and coaIescence of the foot processes of the epitheliai cells covering the glomerular basement membrane, and an increase in the number and size of the cytoplasmic vacuoles within these cells.1”J6J7 In animals recovering from the aminonucleoside nephrosis the pathologic changes observable with the electron microscope show beginning regression.lO This may be compared with the observation that in the human nephrotic syndrome, steroid therapy reverses the changes in the foot processes.18~1s The electron microscopic studies both in the lipoid nephrosis of children16J7 and in the aminonucleoside diseasei indicate that the first morphologic changes occur in the epithelial celis at the same time as the onset of the severe proteinuria. Later in the development of the disease there is an increase in the amount of the basement membrane material and an increased number of endothelial cell nuclei. It has also been reported that defects measuring several hundred to 1,000 A were found in the glomerular basement membrane of a patient with nephrosis. 2o The primary change in the morphology of the epithelial cell component of the renal corpuscle is also reflected in the abnormal presence of alkaline phosphatase in these cells in the antibody-induced nephrosis.21 Later in the course of the disease the basement membrane and the endothelial cells also become alkaline-phosphatase positive, whereas the tubular cells have a decreased concentration of this enzyme. The latter event is probably coincidental to the loss of the brush border.” The ability of the aminonucleoside to reproduce so exactly the same pathologic picture found in the nephrotic disease of children and in the antigenantibody disease produced in rats has provided a convenient tool with which to study the possible mechanism by which such a disease process might occur. Since there is a marked structural resemblance between the aminonucleoside and adenosine, the possibility was considered that the aminonucleoside might act as an inhibitor in the formation of, or in reactions involving, ATP, or in some other phase of nucleotide metabolism. If such were the case, administration of various purine derivatives might prevent the inhibition. Adenine and a number of synthetic purine derivatives were known to reverse the antitrypanosomal action of the aminonucleoside in mice,22 and more recently it was established that the aminonucleoside inhibits the formation of ATP from inorganic phosphate and adenosine by brewer’s yeast.2s The data obtained .to date24 support the idea that the aminonucleoside acts as an antimetabolite during the production of the nephrotic syndrome in rats. When given simuItaneousIy with the aminonucleoside, adenine partially inhibited the development of the nephrosis, whereas adenosine, in the doses given, did not. The failure of adenosine was probably due to the high level of adenosine deaminase present in mammalian tissues.z5 The picture is complicated by the fact that adenine is toxic and can produce thickening of the basement membrane.24 The possible practical implications of these experimental observations revolve on either or both of two premises: (1) In the human patient with nephrosis
yaLl~r
“,”
CHARACTERISTICS
OF EXPERIMENTAL
NEPHROSIS
485
there may exist at some time during the onset of his disease either an abnormal nucleotide or nucleoside which functions as an antimetabolite, or a normal nucleotide or nucleoside which is present in abnormally high concentrations and serves as an inhibitor of sensitive enzyme systems. (2) The morphologic common denominator of nephrosis is injury to a specific component of the renal corpuscle. At the present time there is no evidence for the existence of a specific antimetabolite which may give rise either to the allergic experimental nephrosis or to human nephrosis. However, because of the marked resemblance between the antibodyinduced nephrosis and the aminonucleoside nephrosis the possibility cannot be discounted that some substance which functions as an antimetabolite may be formed as a by-product of the antigen-antibody reaction. Currently, the only link between renal disease and aberrant purine metabolism is a preliminary report showing that the blood of azotemic patients contains an abnormally high level of nucleotides. In this study the greatest increase was in ATP, but increases were also found in DPN, TPN, and GTP .26 There is a gradually accumulating body of evidence that the epithelial cell is the primary site of damage in both human and experimental nephrosis.10j16a17 That a chemical agent causing this damage might exert its effect in some phase of nucleotide or nucleic acid metabolism is an enticing concept which deserves further consideration and investigation. REFERENCES
Masugi, M.:
Beitr. path. Anat. u. allg. Path. 91:82, 1933. Proc. Sot. Exner. Biol. & Med. 85:593. 1934. Pressman, D., Hill, R. F., and Foote, F. W.: Science 109~65, 1949. Krakower, C. A., and Greenspon, S. A.: A.M.A. Arch. Path. 51:629, 1951. Mellors, R. C., Siegel, M., and Pressman, D.: Lab. Invest. 4:69, 1955. Yagi, Y., and Pressman, D.: J. Immunol. 81:7, 1958. Krakower, C. A., and Greenspon, S. A.: A.M.A. Arch. Path. 64:364, 1958. Frenk, S., Antonowicz, I., Craig, J. M., and Metcoff, J.: Proc. Sot. Exper. Biol. & Med. 59:424, 195.5. Wilson, S.: Proc. Ninth Ann. Conf. on the Nephrotic Syndrome, October, 1957, New York, 1958, National Nephrosis Foundation, p. 213. 1959. Vernier, R. L., Papermaster, B. W:, and Good, R. A.: J. Exper. Med. ltWrl15, Liu, C., McCrory, W. W., and Flick, J. A.: Proc. Sot. Exper. Biol. Br Med. %r331, 1957. McCrory, W. W.: Proc. Ninth Ann. Conf. on the Nephrotic Syndrome, October, 1957, New York, 1958, National Nephrosis Foundation, p. 217. Spater. H. W.: Proc. Ninth Ann. Conf. on the Nephrotic Svndrome. October. 1957. New York, 1958, National Nephrosis Foundation, p. 220. _ Heymann, W., and Lund, H. 2.: Pediatrics 7:691, 1951. Ehrich, W. E., Forman, C. W., and Seiier, J.: A.M.A. Arch. Path. 54,463, 1952. Farquhar, M., Vernier, R. L., and Good, R. A.: J. Exper. Med. 1@&649, 1957. Farquhar, M., Vernier, R. L., and Good, R. A.: Am. J. Path. 35:791, 19.57. FoIli, G., Pollack, V. E., Retd, R. T. W., Pirani, C., and Kark, R.: Ann. Int. Med. 49:775, 1958. Vernier, R. L., Farquhar, M., Brunson, J. G., and Good, R. A.: A.M.A. Am. J. Dis. Child. %:306, 1958. Spiro, D.: Am. J. Path. 35:47, 1959. Hartman, M. E.: A.M.A. Arch. Path. 64:679, 1957. Hewitt, R. I., Gumble, A. R., Wallace, W. S., and Williams, J. H.: Antibiotics & Chemother. 4:1222, 1954. Kessner, D. M., Borowsky, B. A., and Recant, L.: Proc. .Soc. Exper. Biol. & Med. %r766, 1958. Hartman, M. E., Hartman, J. D., and Baldridge, R. C.: Proc. Sot. Exper. BioI. & Med. 100:152, 1959. Kalcker, H. M.: J. Biol. Chem. 167:429, 1947. Bishop, C., Rankine, D., and Talbott, J. H.: Fed. Proc. 18:11, 1959.
:: Stavitskv, A. B.. Hackel. D. B.. and Hevmann. W.: 3. 4. i: 7. 8. 9. ::: 12. 13.
19. 20. 21. 22. 23. 24. E: