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Autoimmune interstitial disease of the kidney and associated antigen purification and characterization of a soluble tubular basement membrane antigen
CLINICAL
IMMUNOLOGY
Autoimmune Antigen
AND
Department
19, 360-371 (1981)
Interstitial Disease of the Kidney and Associated Purification and Characterization of a Soluble Tubular Basement Membrane Antigen
YOKO WAKASHIN,* First
IMMUNOPATHOLOGY
IZUMI TAKEI, SHIRO UEDA, YOSHIO MORI, MASAFUMI WAKASHIN, AND KUNIO OKUDA
of Internal Medicine, School Health Sciences Center, Chihu
of Medicine, University,
and Chiba
*Department (280). Japan
KENJI IESATO, of Health
Care,
Received November 17, 1980 A soluble tubular basement membrane (TBM) antigen has been purified from human kidneys by a combination of immunochemical and physicochemical procedures. Its chemical properties were entirely different from those of glomerular basement membrane (GBM) antigen as studied by DEAE-cellulose chromatography and electrophoresis. The molecular weight of TBM antigen was estimated to be 30,008 daltons and that of GBM 40,060 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified TBM antigen did not cross-react with GBM antigen and cross-reacted with the TBM antigen similarly purified from other animals such as goats, guinea pigs, and mice in immunodiffusion plates. Goats and some mice immunized with the purified human TBM antigen developed a typical interstitial nephritis. The goat sera stained the basement membrane of normal tubules and Bowmann’s capsules, and no anti-GBM activity was detected in the serum by immunofluorescence. The system provides a simple and useful model of interstitial nephritis produced with a purified TBM antigen.
INTRODUCTION
Recently, attention has been focused on autoimmune tubulointerstitial nephritis (l-3), the etiology of which is poorly understood. To elucidate the immunopathogenesis of this type of renal disease, it is necessary to determine the immunological properties of the tubular basement membrane (TBM) antigen which seems to play an important role in the stimulation of immune systems of the diseased host. Much less work has been done on this antigen in contrast to the glomerular basememt membrane (GBM), whose structure (4-6) chemical (7-9), and immunochemical (lo- 13) characteristics, and etiologic role in glomerulonephritis (14- 18) have been fairly well documented. After Steblay described a new tubular disease of the renal cortex in experimental animals in 1971 (19), it has been demonstrated that TBM antigen is a causal or contributing factors in tubulointerstitial nephritis, and that it is compositionally different from GBM antigen (20-21). These models for experimental autoimmune renal disease are important because they are immunologically similar to several recently described cases of human interstitial disease (22-27), and will help clarify the mechanisms of immune responsiveness. To study the pathogenesis of these experimental and human autoimmune renal disease, it is mandatory to use purified antigens. In this study, therefore, efforts were made to purify these antigens immunochemically and produce an experimental model which is simpler and less complicated in pathogenetic analysis and 360 0090-1229/81/060360-12$01.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
PURIFICATION
OF
TBM
ANTIGEN
361
using several successive procedures, we have been able to purify TBM antigen in a soluble form. The antigen possessed an immunological determinant which is not species specific and it is quite different from that of GBM antigen. MATERIALS
AND METHODS
Preparation of TBM$bers. A total of 2000 g of normal kidney was collected at autopsy from human cadavers with no renal disease. The cortex tissue from 200 g kidney at a time was minced into pieces of approximately 5 mm and were passed first through a rough mesh and then a IOO-mesh stainless steel screen. The fiber rich material which remained on the 100 mesh was suspended in saline and centrifuged at 4°C for 30 min at 1SOOg on a Ficoll (Pharmacia Fine Chemicals, Uppsala, Sweden) discontinuous gradient (10, 20, 30, 40, and 50%). After centrifugation the layer most rich in fibers on 20% Ficoll was obtained. The material was washed with saline several times and sonicated to exclude fragments of renal tubular epitheliums attached to fibers. Trypsinization of TBMfibers. Isolated TBM fibers were solubilized by adding trypsin (Sigma Chemical Co., St Louis, MO.), 25 mg/lO g TBM/S ml, in 0.1 M Tris-HCl buffer containing 0.02 M CaCl,. The mixture was incubated while stirring at 37°C for 18 hr, and the digestion was stopped by adding excess amounts of soybean trypsin inhibitor (Sigma). This was centrifuged at 30,OOOg for 60 min according to Cole et al. (28). The supernatant containing crude solubilized TBM antigen was processed further. Block electrophoresis. The crude solubilized TBM antigen was subjected to block electrophoresis in 0.85% agar (Difco Laboratories, Detroit, Mich.) in a barbital NaOH buffer, pH 8.6, ionic strength 0.1, under an electric potential of 12 V cm-’ across a 1.0 x 10 x 24-cm agar for 36 hr at 4°C. The gel was cut into 5-mm segments and the protein content of each was determined by the Folin-Ciocalteu method (29). Preparation of rabbit antisera. The crude preparation of solubilized TBM antigen and protein peaks separated by block electrophoresis were injected with Freund’s complete adjuvant into the footpads and back muscles of rabbits three times at 2-week intervals. The antisera were absorbed with normal human whole serum, and with renal epithelial antigen purified according to Edgington et al. (30). The IgG fractions of sera were conjugated with fluorescein isothiocyanate by the method of Riggs et al. (31). The specificity of the antisera was confirmed by immunodiffusion and immunofluorescence. Chromatography. The y fractions which had reacted with rabbit serum antiTBM antibody, were pooled and futher purified by DEAE-cellulose column chromatography. DEAE-cellulose, equilibrated wth 0.005 M sodium phosphate buffer of pH 8.0, was packed into a 20 x l-cm column. The sample was first dialyzed against the same buffer, and eluted at the same pH with a salt gradient from 0 to 1 M NaCl. The protein peak in 0.12 M salt concentration which reacted with antiserum, was then applied to a Sephadex G-200 column in 0.005 M borate saline. Purification of GBM antigen. GBM antigen was purified by the method of Krakower et al. (32) and its properties were compared with those of TBM antigen.
362
WAKASHIN
ET AL.
Like in the purification process for TBM, normal human kidney cortices were minced into pieces approximately 5 mm in size, and forced through a lOO-mesh stainless steel sieve, and then through a 180-mesh sieve, leaving glomeruli on the sieve. The glomeruli were disrupted with sonication and digested by a combined immunochemical and physicochemical technique (33). Determination of molecular weights. Purified TBM and GBM antigens were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE (34) to determine the molecular weight. This was carried out with an indirect precipitation method using radioiodinated TBM and GBM antigen, respective rabbit antisera, and goat IgG against rabbit IgG. One milligram of antigen was incubated with 500 &i of a carrier-free Na1251 and 1% chloramine-T in 0.01 M phosphate buffer, pH 7.2, for 5 min. The reaction was stopped after 1 min of incubation by adding 1% sodium bisulfate (Koso Chemical Co. Ltd., Tokyo, Japan). The reaction mixture was filtered with Sephadex G-25 and the eluted protein peak was used as the iodinated antigen. The iodinated antigen and the specific antiserum was incubated at room temperature for 30 min and then at 0°C overnight. After addition of goat anti-rabbit IgG, the precipitate was pelleted at 15OOg for 30 min at 4”C, washed with 0.01 M phosphate-buffered saline (PBS) of pH 7.4, and dissolved with a small amount of PBS. Each sample was electrophoresed in 7.5% polyacrylamide gel which was reduced with 2mercaptoethanol in SDS at 8 mA/gel for 5 hr. The gel was frozen, sliced into l-mm-wide sections with a special cutter, and counted in a scintillation counter. The molecular weight (MW) of purified antigen was estimated using y-globulin (MW 160,000), bovine serum albumin (MW 67,000), and lyzozyme (MW 14,000) as references. Purification of TBM antigen from animals. Kidneys were obtained from goats, guinea pigs, and mice. These kidneys were perfused in isotonic saline, minced, and passed through a mesh selected according to the size of the glomeruli of each species. The successive procedures were the same as those for purification of human TBM. The TBM fibers were collected on a Ficoll discontinuous gradient, sonicated, trypsinized, and the solubilyzed TBM antigen was separated first by block electrophoresis and then by DEAE-cellulose column chromatography. A specific antiserum against human TBM was used in these preparatory procedures. Immunodiffusion study. Antigenicity of the purified TBM antigen was compared with that of GBM antigen by the double immunodiffision method. The reaction was carried out in a 1% agarose (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in sodium barbital buffer, pH 8.6, ionic strength 0.05, at room temperature for 12 hr. In the Ouchterlony plate, peripheral wells were filled with GBM and TBM antigen, and a mixture of antiserum against TBM and GBM was placed in the central well. Another Ouchterlony plate was used to determine the cross-reactivity among TBM antigens of these animal species. In this plate, TBM antigens from the goat, guinea pig, mouse, and man were placed in the peripheral wells. Immunization study. Three goats were injected with the amount of purified human TBM antigen, equivalent to 150 pg of protein, together with an equivalent volume of Freund’s complete adjuvant. The injection was given three times at
PURIFICATION
OF
TBM
ANTIGEN
363
2-week intervals. Another goat was injected with Freund’s adjuvant alone for the control. Renal biopsies were performed on these goats at the time of the first immunization and then at 2-week intervals, and routine morphological examinations were made of the biopsied kidney. Sera were also obtained at the same time, and examined for the presence of anti-TBM antibody by the direct immunofluorescence techniques using immunized goat serum. The purified human TBM antigen was also injected to five mice each of the three inbred strains: BALB/c, C3H/He, and C57BL/6. The antigen equivalent to 10 pg of protein was injected with the adjuvant to each mouse two times at a 2-week interval. The sera and the kidneys were examined to detect anti-TBM antibody and the development of interstitial nephritis. RESULTS
Tubular fibers were isolated on a 20% Ficoll layer by centrifugation. These materials were almost devoid of glomeruli, being approximately 99% pure as examined by projective microscopy. The cellular components adherent to the fibers were removed by sonication. On electrophoretic analysis, one main protein was observed in the y region as determined by the Folin-Ciocalteu method and other small peaks in the (Y and p regions (Fig. 1). Rabbit antisera were prepared against these individual fractions and the specificity was tested using the immunoflourescence technique on normal human kidney sections. Among these rabbit antisera, the antiserum against the y-fractions stained only the TBM. while other antisera did not stain the TBM (Table 1). Therefore, the y fractions were further purified. On DEAE-cellulose column chromatography, the y fractions were separated into two protein peaks. The first peak was eluted at 0.12 M salt concentration, and the second at about 0.2 M (Fig. 1). The protein eluted at 0.12 M reacted with the rabbit antiserum against the y fractions. By gel filtration with Sephadex G-200, the peak separated by DEAE was further separated into two peaks, and the second peak contained TBM antigen as determined in our Ouchterlony plate using antiserum against y fractions absorbed with normal human serum (Fig. 1). When run on SDS-PAGE and determined by the indirect precipitin methods, the molecular weight of this antigen was estimated to be 30,000 daltons (Fig. 2). Thus, we were able to obtain 1 mg of protein of the purified TBM antigen from 2000 g kidney processed in 10 procedures and reproducible results. The glomerulus preparation collected on a 180-mesh stainless steel sieve did not contain tubular fibers and tubular cells when examined by microscopy, and the GBM separated from the sediment by sonication had a purity of approximately 9%. Electrophoretically, the GBM antigen was in the /3 zone. Concentrated extract from the /3 zone was further purified by DEAE-cellulose chromatography and a sharp elution peak containing pure GBM antigen was detected at 0.3 M salt concentration. The estimated molecular weight of GBM antigen was 40,000 daltons as determined by the same procedures for the TBM antigen (Fig. 3). The antigenicity of these two purified antigens was tested against an antiserum which reacted with both TBM and GBM on immunodiffision plates. On the plate, precipitin lines forming between TBM and antiserum and between GBM and
WAKASHIN
364
2M) Zone
ET AL.
Eiectrophoresis
TBM anugen /
1
O.D.
DEAE
CehiOSe
TBM p P N
COhTWI
antI@ - 0.5M
lcm-
10
23
40
0.0. Sephadex
60
100 (tube
No)
G-200 Column
2001
20
40
60
80
loo
120
140 (effluent
volume)
1. Purification procedures for TBM antigen in sequence: block electrophoresis (upper), DEAE-cellulose column (middle), and Sephadex G-200 column (lower). The pattern of protein content of each fractions is shown in this figure. FIG.
TABLE SPECIFICITY
OF ANTISERA
I
AS DETERMINED
BY IMMUNOFLUORESCENCE
Target constituents
Crude TBM a Fractions p Fractions y Fractions DEAE peak 1 (0.12 MY DEAE peak 2 (0.2 M)” Sephadex peak 1 Sephadex peak 2
TBM
GBM
RTE
+ + + + -
+ ++ +-
+++-
Nofe. TBM, renal tubular basement membrane; GBM, renal glomerular RTE, renal tubular epithelium. (+) Reacted; (-) not reacted. (’ Eluted at the said salt concentration.
Other tubular constituents + + + +basement membrane;
PURIFICATION
OF TBM
365
ANTIGEN
SDS-PAGE Tt3M
40
20
60
ant,gen
60
100
Mlgratmn D&axe (mm) FK. 2. The pattern of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of TBM antigen as determined by indirect precipitation method employing ‘2sI-TBM antigen rabbit anti-TBM and goat IgG against rabbit IgG.
antiserum crossed over each other, indicating clearly the difference in the antigenicity between the two antigens (Fig. 4). The antigenicity of TBM antigens prepared from the four animal species were then examined in an immunodiffision plate. All these antigens cross-reacted each other against anti-human TBM antibody (Fig. 5). Zone
Electrophoresis
100-
II,,, (+),
6
DEAE
4
2
G;“-ay4
cellulose
6
*C-J a
Column
O.D. m
100
“a j7
0
loo
ZcaJ (affluent
volume)
SDS-PAGE 1.0.
J
20
40 Migratmn
60 Distance
80 (nn)
3. Purification procedures for GBM antigen block electrophoresis column (middle), and in sequence: SDS-PAGE (lower). FIG.
,
(upper), DEAE-cellulose
366
WAKASHIN
ET AL.
FI(3. 4. Double immunodiffusion plate demonstrating the antigenicity of TBM antigen and GBM antig en. Central well, antiserum against both GBM and TBM; top and upper right wells, GBM antigen; w-e :r left well, TBM antigen. Precipitin lines between both antigens and antiserum crosses over eacn -, indicating the difference in antigenicity of each antigen.
e human, goat, guinea pig, and mouse. Central well, antiserum against purified TBM antigen; 1, human TBM antigen; 2, guinea pig TBM antigen; 3, mouse TBM antigen; 4, goat TBM antigen. A precipitin line is seen between each antigen and the antiserum, and the line is identical among them; human TBM has made a spur over the goat TBM.
PURIFICATION
OF TBM
ANTIGEN
367
-FIG. 6. Photomicrograph of goat kidney, demonstrating typical interstitial nephritis which consisted of areas of mononuclear cell infiltration, interstitial fibrosis, and tubular cell damage (H & E, X200).
Routine microscopic study of kidney biopsies from the goat immunized with TBM antigen revealed severe interstitial changes with focal intense infiltration of mononuclear cells, irregular interstitial fibrosis, and tubular cell damage, whereas the glomeruli showed only minimal cellular proliferations (Fig. 6). These changes were demonstrable in all three goats after 4 weeks and severest around 8 weeks after immunization. IgG from goat serum reacted specifically with the TBM of normal human kidney in li-ozen sections, but there was no reaction with GBM (Fig. 7). No such changes occurred in the control goat receiving adjuvant only. Morphological examination of the kidney of the mice immunized with TBM antigen revealed a typical interstitial nephritis in three of the five BALB/c mice. Anti-TBM antibody was demonstrated by gel diffusion in all of the immunized BALB/c mice. In C3H/He mice, antibody against TBM was detected in four of the live, but no nephritis had occurred. In C57BL/6 mice, no antibody activity or nephritis was demonstrated. DISCUSSION
It is now gradually accepted that a tubular basement membrane antigen of the kidney plays an important role in the immunopathologic mechanism responsible for interstitial nephritis. In 1971, Steblay et al. (19) described a type of renal tubular disease which developed in guinea pigs after immunization with rabbit renal cortical basement membranes. In this model, glomerular injury was usually mild but tubulointerstitial injury was severe with extensive mononuclear cell infiltration, giant cell formation, and tubular cell destruction. The smm reacted to both TBM and GBM, but the antibody to the TBM was regarded as a causal factor of tubular injury.
368
FIG. 7. Immunofluorescent (direct staining, x200).
WAKASHIN
ET AL.
staining of normal human kidney using fluorescinated
goat serum
Subsequently, the role of anti-TBM antibody in the tubular injury has been discussed. Lehman et al. (20) also described an association between anti-TBM antibody and tubular injury in their investigation which was made in guinea pigs immunized with bovine TBM preparations. In their system, anti-TBM antibody was thought to be nephritogenic. Another model of tubulointerstitial nephritis was developed with a modified method for producing the so-called Heymann’s nephritis (35) by Sugisaki ef al. (36). In their study a suspension of homologous kidney was injected to Brown-Norway and Lewis (Le) x BN Fl hybrid rats and the developed antibodies reacted to tubular cells as well as to TBM. Makker et al. (37) and Klassen et al. (38) also reported on experimental tubulointerstitial nephritis, and all these studies were focused on autologous immune complex nephritis. Further investigations attempted to elucidate the mechanisms for antibody formation and development of the disease. Lehman et al. (21) suggested that the development of the disease depended upon the animal, and that only BN and LeBN hybrid deposited sufficient amounts of antibody along the TBM. They discussed that the most important factor was whether or not the particular strain possessed an immunogenic TBM antigen. Subsequently Hyman et al. (39, 40) published another paper in which they used inbred systems of guinea pigs and suggested that anti-TBM antibody formation and tubular injury as a response might be genetically controlled. Recently, Neilson et al. (1, 2) speculated that the immune reaction leading to interstitial nephritis was mediated by cellular immunity. In another paper, Browner al. (3) reported that the production of disease was specifically inhibited by the injection of antisera raised to the autoantibodies to TBM, suggesting further that it was probably due to idiotype suppression. Thus, the immune mechanisms involved in interstitial nephritis seem to be
PURIFICATION
OF
TBM
369
ANTIGEN
gradually unravelled. To study the mechanisms and/or role of autoantibody formation in this disease and discuss the important factors in the production of tubular injury, a purified antigen is to be used to eliminate complications in the experimental systems. And rigorous purification of TBM antigen which will stimulate the immune systems is an absolute necesity. Mahieu et al. (9) isolated human TBM by rather simple procedures, but the TBM prepared was apparently contaminated by interstitial collagen, as pointed out by Krisko ef al. (41). Subsequently, bovine TBM, rabbit TBM, and rat TBM were also isolated by Ferwerda et al. (42), Meezan et al. (43), and Krisko et al. (41), respectively. Their procedures are good for isolation of tubular fibers and for chemical analysis of the fibers. However, such preparation are not adequate for the study of the immunogenic properties of TBM antigen, and a TBM antigen preparation which possesses. only one immunological determinant ought to be employed. Our fiber rich samples which served as the starting material were completely free of glomeruli, cytoplasm, and other kidney tissue fragments, after Ficoll discontinuous centrifugation separation and sonical disruption. But fibers obtained after these procedures had antigenic substances which were cross-reactive with GBM. After trypsinization, antiserum against the material was reproducibly bound to the TBM, Bowmann’s basement membrane, and GBM as studied by direct immunofluorescence. Therefore, we attempted to isolate the original antigenic element of the TBM from the digested crude TBM, and succeeded in purifying a TBM antigen in a soluble form. The purification procedures were repeated more than 10 times and the results were always reproducible. Many different immunochemical properties were observed with TBM antigen, GBM antigen, and renal tubular epithelial (RTE) antigen. Electrical mobility was different among them; TBM antigen was in the y region, GBM antigen in the p region, and RTE antigen in the (Yregion as described by Edgington (30). In elution on a DEAE-cellulose column, the adequate pH for TBM, GBM, and RTE antigens differed with each other. The molecular weights of the antigens were not the same. The TBM antigen had an immunological determinant as studied by the Ouchter-
TABLE
2
RESULTSOF~MMUNIZATION
Goat Case Case Case Case
1 2 3 4
Mouse BALBlc C3H/He C57BLl6
STUDY
Interstitial nephritis
Anti-TBM antibody
Immunization
+ + + -
+ + + -
150 Ng of TBM antigen 150 pg of TBM antigen 150 fig of TBM antigen Adjuvant alone
+(3/5) -(O/5) -(O/5)
+(5/5) +(4/5) -(O/S)
10 pg of TBM antigen 10 pg of TBM antigen 10 pg of TBM antigen
-
370
WAKASHIN
ET AL.
lony plate using specific antiserum. The antigenicity lacked species specificity among the human, guinea pig, mouse, and goat. The antibody to the purified TBM antigen was highly specific to the tubular basement membrane of kidney as studied by immunofluorescence. The typical interstitial nephritis produced in three goats after immunization with the purified human TBM antigen, with demonstrable anti-TBM activity in the absence of anti-GBM antibody and/or anti-RTE antibody formation, is good evidence for the role of this antigen in the pathogenesis of the disease. Some mouse strains showed a similar response. We, therefore, believe that our purified TBM antigen possessed the nephritogenic potency in goats and BALB/c mice, and that the produced nephritis was closely related to anti-TBM antibody. Immunological study of interstitial nephritis in animals and humans may be made much easier and more definitive with the use of a purified TBM antigen. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.
Neilson, E. G., and Phillips, S. M., J. Immune/. 123, 2373, 1979. Neilson, E. G., and Phillips, S. M., J. Zmmunol. 123, 2381, 1979. Brown, C. A., Carey, K., and Colhn, R. B., J. Immunol. 123, 2102, 1979. Kurz, S. M., and McManus, J. F. A., .I. Ulfrasrruct. Res. 4, 81, 1960. Fartquhar, M. G., Wissing, S. L., and Palade, G. E., J. Exp. Med. 113, 47, 1960. Walker, F., J. Parhol. 110, 233, 1973. Spiro, R. G., J. Biol. Chem. 242, 1915, 1967. Kefalides, N. A., Biochemistry 7, 3103, 1968. Mahieu, P., and Winand, R. J., Eur. J. Biochem. 12, 410, 1970. Cruickshank, B., and Hill, G. S., J. Pathol. Bacterial. 96, 283, 1953. Skoza, L., and Mohos, S. C., Proc. Sot. Exp. Biol. Med. 131, 1189, 1969. Myers, C., and Bartlett, P., Biochem. Biophys. Acta 290, 150, 1972. Misra, R. P., Immunology 25, 967, 1973. Steblay, R. W., J. Exp. Med. 116, 253, 1962. Shibata, S., Naruse. T., and Nagasawa, T., J. Immunol. 99, 454, 1967. Lerner, R. A., Glassock, R. J., and Dixon, F. J., J. Exp. Med. 126, 989, 1967. Lerner, R. A., and Dixon, F. J., J. Immunol, 100, 1277, 1968. Wilson, C. B., and Dixon, F. J., Kidney Int. 3, 74, 1973. Steblay, R. W., and Rudofsky, U., J. Immunol. 107, 589, 1971. Lehman, D. H., Marquardt, H., and Wilson, C. B., J. Immune/. 112, 241, 1974. Lehman, D. H., Wilson, C. B.. and Dixon, F. J., Kidney Int. 5, 187. 1974. Baldwin, D. S., Levine, B. B., and McClusky. R. J., N. En& J. Med. 279, 1245, 1968. Morel-Merger, L., Kourilsky, O., and Mignon, F., C&z. fmmunoi. Immunopathot. 2, 185. 1974. Border, W. A., Lehman, D. H., and Egan, J. D., N. Engl. J. Med. 291, 381. 1974. Bergstein, J.. and Litman, N., N. Engl. J. Med. 292, 875. 1975. Swayder, M., Ozawa, T., and Boedecker, E.. Ann. intern. Med. 84, 433, 1976. Mancilla-Jimenez, R., and Katzenstein. A.-LA., Transplantation 24, 39, 1977. Cole, L. R.. Cromatie, W. J., and Watson, D. W., Proc. Sot. Exp. Biol. Med. 77, 498, 1951. Lowry, 0. H., Rosenbrough, N. J., and Farr, A. L., .I. Biol. Chem. 193, 265, 1951. Edgington. T. S., Glassock, R. J., and Dixon. F. J.. J. Exp. Med. 127, 555. 1968. Riggs, J. L., Seiwald. R. J.. and Burckhalter, J. H., Amer. J. Pathol. 34, 1081, 1958. Krakower, C. A., and Greenspon, S. A.. Arch. Pathol. 51, 629. 1951. Wakashin, Y., Wakashin, M., and Narita, M., Contrib. Nephrol. 6, 124. 1977. Maizel, F. J. V., Science 151, 988, 1966. Heymann, W., Hackel, D. B., and Harwood, J., Proc. Sot. Exp. Biol. Med. 100, 660, 1959. Sugisaki, T., Klassen, 3.. and Milgrom. F., Lab. Invest. 28, 658, 1973. Makker, S. P., and Heymann, W., Proc. Sot. Exp. Biol. Med. 143, 995, 1973. Klassen, J., McCluskey, R. T.. and Milgrom, F., Amer. J. Pathol. 63, 333, 1971.
PURIFICATION
39. 40. 41. 42.
OF
TBM
Hyman, L. R., Colvin, R. B., and Steinberg, A. D., Hyman, L. R., Steinberg, A. D., and Calvin. R. B., Krisko, I., DeBernardo, E., and Sato, C. S., Kidney Ferwerda, W., Meijer, J. F. M., and Eijnden, D. H., 1974. 43. Meezan, E., Hjelle, J. T., and Brendal, K.. Life SC;.
371
ANTIGEN J. Immunol. 116, 327, 1976. J. fmmunol. 117, 1894, 1976. ht. 12, 238, 1977. Hoppe-Seyler’s
Z. Physio/.
17, 1721. 1975.
Chem.
335,976.
IMMUNOLOGY
Autoimmune Antigen
AND
Department
19, 360-371 (1981)
Interstitial Disease of the Kidney and Associated Purification and Characterization of a Soluble Tubular Basement Membrane Antigen
YOKO WAKASHIN,* First
IMMUNOPATHOLOGY
IZUMI TAKEI, SHIRO UEDA, YOSHIO MORI, MASAFUMI WAKASHIN, AND KUNIO OKUDA
of Internal Medicine, School Health Sciences Center, Chihu
of Medicine, University,
and Chiba
*Department (280). Japan
KENJI IESATO, of Health
Care,
Received November 17, 1980 A soluble tubular basement membrane (TBM) antigen has been purified from human kidneys by a combination of immunochemical and physicochemical procedures. Its chemical properties were entirely different from those of glomerular basement membrane (GBM) antigen as studied by DEAE-cellulose chromatography and electrophoresis. The molecular weight of TBM antigen was estimated to be 30,008 daltons and that of GBM 40,060 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified TBM antigen did not cross-react with GBM antigen and cross-reacted with the TBM antigen similarly purified from other animals such as goats, guinea pigs, and mice in immunodiffusion plates. Goats and some mice immunized with the purified human TBM antigen developed a typical interstitial nephritis. The goat sera stained the basement membrane of normal tubules and Bowmann’s capsules, and no anti-GBM activity was detected in the serum by immunofluorescence. The system provides a simple and useful model of interstitial nephritis produced with a purified TBM antigen.
INTRODUCTION
Recently, attention has been focused on autoimmune tubulointerstitial nephritis (l-3), the etiology of which is poorly understood. To elucidate the immunopathogenesis of this type of renal disease, it is necessary to determine the immunological properties of the tubular basement membrane (TBM) antigen which seems to play an important role in the stimulation of immune systems of the diseased host. Much less work has been done on this antigen in contrast to the glomerular basememt membrane (GBM), whose structure (4-6) chemical (7-9), and immunochemical (lo- 13) characteristics, and etiologic role in glomerulonephritis (14- 18) have been fairly well documented. After Steblay described a new tubular disease of the renal cortex in experimental animals in 1971 (19), it has been demonstrated that TBM antigen is a causal or contributing factors in tubulointerstitial nephritis, and that it is compositionally different from GBM antigen (20-21). These models for experimental autoimmune renal disease are important because they are immunologically similar to several recently described cases of human interstitial disease (22-27), and will help clarify the mechanisms of immune responsiveness. To study the pathogenesis of these experimental and human autoimmune renal disease, it is mandatory to use purified antigens. In this study, therefore, efforts were made to purify these antigens immunochemically and produce an experimental model which is simpler and less complicated in pathogenetic analysis and 360 0090-1229/81/060360-12$01.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
PURIFICATION
OF
TBM
ANTIGEN
361
using several successive procedures, we have been able to purify TBM antigen in a soluble form. The antigen possessed an immunological determinant which is not species specific and it is quite different from that of GBM antigen. MATERIALS
AND METHODS
Preparation of TBM$bers. A total of 2000 g of normal kidney was collected at autopsy from human cadavers with no renal disease. The cortex tissue from 200 g kidney at a time was minced into pieces of approximately 5 mm and were passed first through a rough mesh and then a IOO-mesh stainless steel screen. The fiber rich material which remained on the 100 mesh was suspended in saline and centrifuged at 4°C for 30 min at 1SOOg on a Ficoll (Pharmacia Fine Chemicals, Uppsala, Sweden) discontinuous gradient (10, 20, 30, 40, and 50%). After centrifugation the layer most rich in fibers on 20% Ficoll was obtained. The material was washed with saline several times and sonicated to exclude fragments of renal tubular epitheliums attached to fibers. Trypsinization of TBMfibers. Isolated TBM fibers were solubilized by adding trypsin (Sigma Chemical Co., St Louis, MO.), 25 mg/lO g TBM/S ml, in 0.1 M Tris-HCl buffer containing 0.02 M CaCl,. The mixture was incubated while stirring at 37°C for 18 hr, and the digestion was stopped by adding excess amounts of soybean trypsin inhibitor (Sigma). This was centrifuged at 30,OOOg for 60 min according to Cole et al. (28). The supernatant containing crude solubilized TBM antigen was processed further. Block electrophoresis. The crude solubilized TBM antigen was subjected to block electrophoresis in 0.85% agar (Difco Laboratories, Detroit, Mich.) in a barbital NaOH buffer, pH 8.6, ionic strength 0.1, under an electric potential of 12 V cm-’ across a 1.0 x 10 x 24-cm agar for 36 hr at 4°C. The gel was cut into 5-mm segments and the protein content of each was determined by the Folin-Ciocalteu method (29). Preparation of rabbit antisera. The crude preparation of solubilized TBM antigen and protein peaks separated by block electrophoresis were injected with Freund’s complete adjuvant into the footpads and back muscles of rabbits three times at 2-week intervals. The antisera were absorbed with normal human whole serum, and with renal epithelial antigen purified according to Edgington et al. (30). The IgG fractions of sera were conjugated with fluorescein isothiocyanate by the method of Riggs et al. (31). The specificity of the antisera was confirmed by immunodiffusion and immunofluorescence. Chromatography. The y fractions which had reacted with rabbit serum antiTBM antibody, were pooled and futher purified by DEAE-cellulose column chromatography. DEAE-cellulose, equilibrated wth 0.005 M sodium phosphate buffer of pH 8.0, was packed into a 20 x l-cm column. The sample was first dialyzed against the same buffer, and eluted at the same pH with a salt gradient from 0 to 1 M NaCl. The protein peak in 0.12 M salt concentration which reacted with antiserum, was then applied to a Sephadex G-200 column in 0.005 M borate saline. Purification of GBM antigen. GBM antigen was purified by the method of Krakower et al. (32) and its properties were compared with those of TBM antigen.
362
WAKASHIN
ET AL.
Like in the purification process for TBM, normal human kidney cortices were minced into pieces approximately 5 mm in size, and forced through a lOO-mesh stainless steel sieve, and then through a 180-mesh sieve, leaving glomeruli on the sieve. The glomeruli were disrupted with sonication and digested by a combined immunochemical and physicochemical technique (33). Determination of molecular weights. Purified TBM and GBM antigens were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE (34) to determine the molecular weight. This was carried out with an indirect precipitation method using radioiodinated TBM and GBM antigen, respective rabbit antisera, and goat IgG against rabbit IgG. One milligram of antigen was incubated with 500 &i of a carrier-free Na1251 and 1% chloramine-T in 0.01 M phosphate buffer, pH 7.2, for 5 min. The reaction was stopped after 1 min of incubation by adding 1% sodium bisulfate (Koso Chemical Co. Ltd., Tokyo, Japan). The reaction mixture was filtered with Sephadex G-25 and the eluted protein peak was used as the iodinated antigen. The iodinated antigen and the specific antiserum was incubated at room temperature for 30 min and then at 0°C overnight. After addition of goat anti-rabbit IgG, the precipitate was pelleted at 15OOg for 30 min at 4”C, washed with 0.01 M phosphate-buffered saline (PBS) of pH 7.4, and dissolved with a small amount of PBS. Each sample was electrophoresed in 7.5% polyacrylamide gel which was reduced with 2mercaptoethanol in SDS at 8 mA/gel for 5 hr. The gel was frozen, sliced into l-mm-wide sections with a special cutter, and counted in a scintillation counter. The molecular weight (MW) of purified antigen was estimated using y-globulin (MW 160,000), bovine serum albumin (MW 67,000), and lyzozyme (MW 14,000) as references. Purification of TBM antigen from animals. Kidneys were obtained from goats, guinea pigs, and mice. These kidneys were perfused in isotonic saline, minced, and passed through a mesh selected according to the size of the glomeruli of each species. The successive procedures were the same as those for purification of human TBM. The TBM fibers were collected on a Ficoll discontinuous gradient, sonicated, trypsinized, and the solubilyzed TBM antigen was separated first by block electrophoresis and then by DEAE-cellulose column chromatography. A specific antiserum against human TBM was used in these preparatory procedures. Immunodiffusion study. Antigenicity of the purified TBM antigen was compared with that of GBM antigen by the double immunodiffision method. The reaction was carried out in a 1% agarose (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in sodium barbital buffer, pH 8.6, ionic strength 0.05, at room temperature for 12 hr. In the Ouchterlony plate, peripheral wells were filled with GBM and TBM antigen, and a mixture of antiserum against TBM and GBM was placed in the central well. Another Ouchterlony plate was used to determine the cross-reactivity among TBM antigens of these animal species. In this plate, TBM antigens from the goat, guinea pig, mouse, and man were placed in the peripheral wells. Immunization study. Three goats were injected with the amount of purified human TBM antigen, equivalent to 150 pg of protein, together with an equivalent volume of Freund’s complete adjuvant. The injection was given three times at
PURIFICATION
OF
TBM
ANTIGEN
363
2-week intervals. Another goat was injected with Freund’s adjuvant alone for the control. Renal biopsies were performed on these goats at the time of the first immunization and then at 2-week intervals, and routine morphological examinations were made of the biopsied kidney. Sera were also obtained at the same time, and examined for the presence of anti-TBM antibody by the direct immunofluorescence techniques using immunized goat serum. The purified human TBM antigen was also injected to five mice each of the three inbred strains: BALB/c, C3H/He, and C57BL/6. The antigen equivalent to 10 pg of protein was injected with the adjuvant to each mouse two times at a 2-week interval. The sera and the kidneys were examined to detect anti-TBM antibody and the development of interstitial nephritis. RESULTS
Tubular fibers were isolated on a 20% Ficoll layer by centrifugation. These materials were almost devoid of glomeruli, being approximately 99% pure as examined by projective microscopy. The cellular components adherent to the fibers were removed by sonication. On electrophoretic analysis, one main protein was observed in the y region as determined by the Folin-Ciocalteu method and other small peaks in the (Y and p regions (Fig. 1). Rabbit antisera were prepared against these individual fractions and the specificity was tested using the immunoflourescence technique on normal human kidney sections. Among these rabbit antisera, the antiserum against the y-fractions stained only the TBM. while other antisera did not stain the TBM (Table 1). Therefore, the y fractions were further purified. On DEAE-cellulose column chromatography, the y fractions were separated into two protein peaks. The first peak was eluted at 0.12 M salt concentration, and the second at about 0.2 M (Fig. 1). The protein eluted at 0.12 M reacted with the rabbit antiserum against the y fractions. By gel filtration with Sephadex G-200, the peak separated by DEAE was further separated into two peaks, and the second peak contained TBM antigen as determined in our Ouchterlony plate using antiserum against y fractions absorbed with normal human serum (Fig. 1). When run on SDS-PAGE and determined by the indirect precipitin methods, the molecular weight of this antigen was estimated to be 30,000 daltons (Fig. 2). Thus, we were able to obtain 1 mg of protein of the purified TBM antigen from 2000 g kidney processed in 10 procedures and reproducible results. The glomerulus preparation collected on a 180-mesh stainless steel sieve did not contain tubular fibers and tubular cells when examined by microscopy, and the GBM separated from the sediment by sonication had a purity of approximately 9%. Electrophoretically, the GBM antigen was in the /3 zone. Concentrated extract from the /3 zone was further purified by DEAE-cellulose chromatography and a sharp elution peak containing pure GBM antigen was detected at 0.3 M salt concentration. The estimated molecular weight of GBM antigen was 40,000 daltons as determined by the same procedures for the TBM antigen (Fig. 3). The antigenicity of these two purified antigens was tested against an antiserum which reacted with both TBM and GBM on immunodiffision plates. On the plate, precipitin lines forming between TBM and antiserum and between GBM and
WAKASHIN
364
2M) Zone
ET AL.
Eiectrophoresis
TBM anugen /
1
O.D.
DEAE
CehiOSe
TBM p P N
COhTWI
antI@ - 0.5M
lcm-
10
23
40
0.0. Sephadex
60
100 (tube
No)
G-200 Column
2001
20
40
60
80
loo
120
140 (effluent
volume)
1. Purification procedures for TBM antigen in sequence: block electrophoresis (upper), DEAE-cellulose column (middle), and Sephadex G-200 column (lower). The pattern of protein content of each fractions is shown in this figure. FIG.
TABLE SPECIFICITY
OF ANTISERA
I
AS DETERMINED
BY IMMUNOFLUORESCENCE
Target constituents
Crude TBM a Fractions p Fractions y Fractions DEAE peak 1 (0.12 MY DEAE peak 2 (0.2 M)” Sephadex peak 1 Sephadex peak 2
TBM
GBM
RTE
+ + + + -
+ ++ +-
+++-
Nofe. TBM, renal tubular basement membrane; GBM, renal glomerular RTE, renal tubular epithelium. (+) Reacted; (-) not reacted. (’ Eluted at the said salt concentration.
Other tubular constituents + + + +basement membrane;
PURIFICATION
OF TBM
365
ANTIGEN
SDS-PAGE Tt3M
40
20
60
ant,gen
60
100
Mlgratmn D&axe (mm) FK. 2. The pattern of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of TBM antigen as determined by indirect precipitation method employing ‘2sI-TBM antigen rabbit anti-TBM and goat IgG against rabbit IgG.
antiserum crossed over each other, indicating clearly the difference in the antigenicity between the two antigens (Fig. 4). The antigenicity of TBM antigens prepared from the four animal species were then examined in an immunodiffision plate. All these antigens cross-reacted each other against anti-human TBM antibody (Fig. 5). Zone
Electrophoresis
100-
II,,, (+),
6
DEAE
4
2
G;“-ay4
cellulose
6
*C-J a
Column
O.D. m
100
“a j7
0
loo
ZcaJ (affluent
volume)
SDS-PAGE 1.0.
J
20
40 Migratmn
60 Distance
80 (nn)
3. Purification procedures for GBM antigen block electrophoresis column (middle), and in sequence: SDS-PAGE (lower). FIG.
,
(upper), DEAE-cellulose
366
WAKASHIN
ET AL.
FI(3. 4. Double immunodiffusion plate demonstrating the antigenicity of TBM antigen and GBM antig en. Central well, antiserum against both GBM and TBM; top and upper right wells, GBM antigen; w-e :r left well, TBM antigen. Precipitin lines between both antigens and antiserum crosses over eacn -, indicating the difference in antigenicity of each antigen.
e human, goat, guinea pig, and mouse. Central well, antiserum against purified TBM antigen; 1, human TBM antigen; 2, guinea pig TBM antigen; 3, mouse TBM antigen; 4, goat TBM antigen. A precipitin line is seen between each antigen and the antiserum, and the line is identical among them; human TBM has made a spur over the goat TBM.
PURIFICATION
OF TBM
ANTIGEN
367
-FIG. 6. Photomicrograph of goat kidney, demonstrating typical interstitial nephritis which consisted of areas of mononuclear cell infiltration, interstitial fibrosis, and tubular cell damage (H & E, X200).
Routine microscopic study of kidney biopsies from the goat immunized with TBM antigen revealed severe interstitial changes with focal intense infiltration of mononuclear cells, irregular interstitial fibrosis, and tubular cell damage, whereas the glomeruli showed only minimal cellular proliferations (Fig. 6). These changes were demonstrable in all three goats after 4 weeks and severest around 8 weeks after immunization. IgG from goat serum reacted specifically with the TBM of normal human kidney in li-ozen sections, but there was no reaction with GBM (Fig. 7). No such changes occurred in the control goat receiving adjuvant only. Morphological examination of the kidney of the mice immunized with TBM antigen revealed a typical interstitial nephritis in three of the five BALB/c mice. Anti-TBM antibody was demonstrated by gel diffusion in all of the immunized BALB/c mice. In C3H/He mice, antibody against TBM was detected in four of the live, but no nephritis had occurred. In C57BL/6 mice, no antibody activity or nephritis was demonstrated. DISCUSSION
It is now gradually accepted that a tubular basement membrane antigen of the kidney plays an important role in the immunopathologic mechanism responsible for interstitial nephritis. In 1971, Steblay et al. (19) described a type of renal tubular disease which developed in guinea pigs after immunization with rabbit renal cortical basement membranes. In this model, glomerular injury was usually mild but tubulointerstitial injury was severe with extensive mononuclear cell infiltration, giant cell formation, and tubular cell destruction. The smm reacted to both TBM and GBM, but the antibody to the TBM was regarded as a causal factor of tubular injury.
368
FIG. 7. Immunofluorescent (direct staining, x200).
WAKASHIN
ET AL.
staining of normal human kidney using fluorescinated
goat serum
Subsequently, the role of anti-TBM antibody in the tubular injury has been discussed. Lehman et al. (20) also described an association between anti-TBM antibody and tubular injury in their investigation which was made in guinea pigs immunized with bovine TBM preparations. In their system, anti-TBM antibody was thought to be nephritogenic. Another model of tubulointerstitial nephritis was developed with a modified method for producing the so-called Heymann’s nephritis (35) by Sugisaki ef al. (36). In their study a suspension of homologous kidney was injected to Brown-Norway and Lewis (Le) x BN Fl hybrid rats and the developed antibodies reacted to tubular cells as well as to TBM. Makker et al. (37) and Klassen et al. (38) also reported on experimental tubulointerstitial nephritis, and all these studies were focused on autologous immune complex nephritis. Further investigations attempted to elucidate the mechanisms for antibody formation and development of the disease. Lehman et al. (21) suggested that the development of the disease depended upon the animal, and that only BN and LeBN hybrid deposited sufficient amounts of antibody along the TBM. They discussed that the most important factor was whether or not the particular strain possessed an immunogenic TBM antigen. Subsequently Hyman et al. (39, 40) published another paper in which they used inbred systems of guinea pigs and suggested that anti-TBM antibody formation and tubular injury as a response might be genetically controlled. Recently, Neilson et al. (1, 2) speculated that the immune reaction leading to interstitial nephritis was mediated by cellular immunity. In another paper, Browner al. (3) reported that the production of disease was specifically inhibited by the injection of antisera raised to the autoantibodies to TBM, suggesting further that it was probably due to idiotype suppression. Thus, the immune mechanisms involved in interstitial nephritis seem to be
PURIFICATION
OF
TBM
369
ANTIGEN
gradually unravelled. To study the mechanisms and/or role of autoantibody formation in this disease and discuss the important factors in the production of tubular injury, a purified antigen is to be used to eliminate complications in the experimental systems. And rigorous purification of TBM antigen which will stimulate the immune systems is an absolute necesity. Mahieu et al. (9) isolated human TBM by rather simple procedures, but the TBM prepared was apparently contaminated by interstitial collagen, as pointed out by Krisko ef al. (41). Subsequently, bovine TBM, rabbit TBM, and rat TBM were also isolated by Ferwerda et al. (42), Meezan et al. (43), and Krisko et al. (41), respectively. Their procedures are good for isolation of tubular fibers and for chemical analysis of the fibers. However, such preparation are not adequate for the study of the immunogenic properties of TBM antigen, and a TBM antigen preparation which possesses. only one immunological determinant ought to be employed. Our fiber rich samples which served as the starting material were completely free of glomeruli, cytoplasm, and other kidney tissue fragments, after Ficoll discontinuous centrifugation separation and sonical disruption. But fibers obtained after these procedures had antigenic substances which were cross-reactive with GBM. After trypsinization, antiserum against the material was reproducibly bound to the TBM, Bowmann’s basement membrane, and GBM as studied by direct immunofluorescence. Therefore, we attempted to isolate the original antigenic element of the TBM from the digested crude TBM, and succeeded in purifying a TBM antigen in a soluble form. The purification procedures were repeated more than 10 times and the results were always reproducible. Many different immunochemical properties were observed with TBM antigen, GBM antigen, and renal tubular epithelial (RTE) antigen. Electrical mobility was different among them; TBM antigen was in the y region, GBM antigen in the p region, and RTE antigen in the (Yregion as described by Edgington (30). In elution on a DEAE-cellulose column, the adequate pH for TBM, GBM, and RTE antigens differed with each other. The molecular weights of the antigens were not the same. The TBM antigen had an immunological determinant as studied by the Ouchter-
TABLE
2
RESULTSOF~MMUNIZATION
Goat Case Case Case Case
1 2 3 4
Mouse BALBlc C3H/He C57BLl6
STUDY
Interstitial nephritis
Anti-TBM antibody
Immunization
+ + + -
+ + + -
150 Ng of TBM antigen 150 pg of TBM antigen 150 fig of TBM antigen Adjuvant alone
+(3/5) -(O/5) -(O/5)
+(5/5) +(4/5) -(O/S)
10 pg of TBM antigen 10 pg of TBM antigen 10 pg of TBM antigen
-
370
WAKASHIN
ET AL.
lony plate using specific antiserum. The antigenicity lacked species specificity among the human, guinea pig, mouse, and goat. The antibody to the purified TBM antigen was highly specific to the tubular basement membrane of kidney as studied by immunofluorescence. The typical interstitial nephritis produced in three goats after immunization with the purified human TBM antigen, with demonstrable anti-TBM activity in the absence of anti-GBM antibody and/or anti-RTE antibody formation, is good evidence for the role of this antigen in the pathogenesis of the disease. Some mouse strains showed a similar response. We, therefore, believe that our purified TBM antigen possessed the nephritogenic potency in goats and BALB/c mice, and that the produced nephritis was closely related to anti-TBM antibody. Immunological study of interstitial nephritis in animals and humans may be made much easier and more definitive with the use of a purified TBM antigen. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.
Neilson, E. G., and Phillips, S. M., J. Immune/. 123, 2373, 1979. Neilson, E. G., and Phillips, S. M., J. Zmmunol. 123, 2381, 1979. Brown, C. A., Carey, K., and Colhn, R. B., J. Immunol. 123, 2102, 1979. Kurz, S. M., and McManus, J. F. A., .I. Ulfrasrruct. Res. 4, 81, 1960. Fartquhar, M. G., Wissing, S. L., and Palade, G. E., J. Exp. Med. 113, 47, 1960. Walker, F., J. Parhol. 110, 233, 1973. Spiro, R. G., J. Biol. Chem. 242, 1915, 1967. Kefalides, N. A., Biochemistry 7, 3103, 1968. Mahieu, P., and Winand, R. J., Eur. J. Biochem. 12, 410, 1970. Cruickshank, B., and Hill, G. S., J. Pathol. Bacterial. 96, 283, 1953. Skoza, L., and Mohos, S. C., Proc. Sot. Exp. Biol. Med. 131, 1189, 1969. Myers, C., and Bartlett, P., Biochem. Biophys. Acta 290, 150, 1972. Misra, R. P., Immunology 25, 967, 1973. Steblay, R. W., J. Exp. Med. 116, 253, 1962. Shibata, S., Naruse. T., and Nagasawa, T., J. Immunol. 99, 454, 1967. Lerner, R. A., Glassock, R. J., and Dixon, F. J., J. Exp. Med. 126, 989, 1967. Lerner, R. A., and Dixon, F. J., J. Immunol, 100, 1277, 1968. Wilson, C. B., and Dixon, F. J., Kidney Int. 3, 74, 1973. Steblay, R. W., and Rudofsky, U., J. Immunol. 107, 589, 1971. Lehman, D. H., Marquardt, H., and Wilson, C. B., J. Immune/. 112, 241, 1974. Lehman, D. H., Wilson, C. B.. and Dixon, F. J., Kidney Int. 5, 187. 1974. Baldwin, D. S., Levine, B. B., and McClusky. R. J., N. En& J. Med. 279, 1245, 1968. Morel-Merger, L., Kourilsky, O., and Mignon, F., C&z. fmmunoi. Immunopathot. 2, 185. 1974. Border, W. A., Lehman, D. H., and Egan, J. D., N. Engl. J. Med. 291, 381. 1974. Bergstein, J.. and Litman, N., N. Engl. J. Med. 292, 875. 1975. Swayder, M., Ozawa, T., and Boedecker, E.. Ann. intern. Med. 84, 433, 1976. Mancilla-Jimenez, R., and Katzenstein. A.-LA., Transplantation 24, 39, 1977. Cole, L. R.. Cromatie, W. J., and Watson, D. W., Proc. Sot. Exp. Biol. Med. 77, 498, 1951. Lowry, 0. H., Rosenbrough, N. J., and Farr, A. L., .I. Biol. Chem. 193, 265, 1951. Edgington. T. S., Glassock, R. J., and Dixon. F. J.. J. Exp. Med. 127, 555. 1968. Riggs, J. L., Seiwald. R. J.. and Burckhalter, J. H., Amer. J. Pathol. 34, 1081, 1958. Krakower, C. A., and Greenspon, S. A.. Arch. Pathol. 51, 629. 1951. Wakashin, Y., Wakashin, M., and Narita, M., Contrib. Nephrol. 6, 124. 1977. Maizel, F. J. V., Science 151, 988, 1966. Heymann, W., Hackel, D. B., and Harwood, J., Proc. Sot. Exp. Biol. Med. 100, 660, 1959. Sugisaki, T., Klassen, 3.. and Milgrom. F., Lab. Invest. 28, 658, 1973. Makker, S. P., and Heymann, W., Proc. Sot. Exp. Biol. Med. 143, 995, 1973. Klassen, J., McCluskey, R. T.. and Milgrom, F., Amer. J. Pathol. 63, 333, 1971.
PURIFICATION
39. 40. 41. 42.
OF
TBM
Hyman, L. R., Colvin, R. B., and Steinberg, A. D., Hyman, L. R., Steinberg, A. D., and Calvin. R. B., Krisko, I., DeBernardo, E., and Sato, C. S., Kidney Ferwerda, W., Meijer, J. F. M., and Eijnden, D. H., 1974. 43. Meezan, E., Hjelle, J. T., and Brendal, K.. Life SC;.
371
ANTIGEN J. Immunol. 116, 327, 1976. J. fmmunol. 117, 1894, 1976. ht. 12, 238, 1977. Hoppe-Seyler’s
Z. Physio/.
17, 1721. 1975.
Chem.
335,976.