Original Papers Nephron 24: 10 16(1979)

Membranous Nephropathy: A Radioimmunologie Search for Anti-Renal Tubular Epithelial Antibodies and Circulating Immune Complexes Richard A.Zager, William G. Couser, Brian S. Andrews, W. Kline Bolton and Marc A. Pohl Departments of Medicine, West Roxbury Veterans Administration Hospital, Harvard Medical School: Boston University Medical Center, Boston, Mass.: University of Virginia School of Medicine, Charlottesville, Va., and the Cleveland Clinic Foundation, Cleveland, Ohio

Key Words. Membranous nephropathy • RTE antigens • Immune complexes • Raji cell assay

Introduction Membranous nephropathy (MN) is the most common cause of idiopathic nephrotic syndrome in the adult [I], It is a well-characterized glomerular disease in which finely granular immune deposits are localized exclusively along the subepithelial surface of the glomerular basement membrane (GBM) [2], These immunopathologic features, as well as the similarity of this lesion to that found in some rabbits with chronic serum sickness [3], support the hypo­ thesis that the pathogenesis of MN involves chronic glomerular deposition of circulating low molecular weight immune complexes (ICs). A lesion indistinguishable from MN in man can be in­ duced in rats by immunization with a proximal renal tubular epithelial (RTE) (brush border) antigen with re­ sultant immune complexes containing RTE developing on the GBM (autologous immune complex nephritis, AICN. Heymann nephritis) [4,5]. Demonstrations that RTE antigens circulate in normal human serum [6,7] and that antibody with RTE specificity may develop in man [8-10] suggest that some cases of human MN may be mediated by a mechanism analogous to the AICN model. Clinical and experimental support for this hypothesis exists. Naruse et al. [11.12] have reported RTE antigen in glomerular

immune deposits in 4 of 9 patients with idiopathic MN. although others have not confirmed this finding [13]. RTE antigens have also been described in glomerular immune deposits in patients with nephrotic syndrome associated with sickle cell anemia, renal cell carcinoma and renal vein thrombosis [8.9,14]. An RTE antigen present in normal human urine (URTE) has been shown to be capable of inducing AICN [15]. Furthermore, it has recently been de­ monstrated that the AICN model can be produced in the absence of circulating ICs by the reaction of anti-RTE antibodies with antigens apparently localized on the epi­ thelial surface of the normal glomerular capillary wall [16. 17]. This suggests that some cases of MN might result from in situ IC formation mediated by anti-RTE antibody alone. The above studies have raised controversy concerning the immunopathogenic significance of autologous antiRTE antibodies and circulating ICs in MN. Circulating antibody to one RTE antigen (URTE) has not been found in patients with MN by indirect immunofluorescence (IF) [13]. However, the relative insensitivity of this technique is well documented [18], and antibodies with specificity for other RTE antigens have not been sought. Assays for circulating ICs in MN have produced conflicting results. Most studies have been carried out on MN sera after pro­ longed storage, and ICs have been either undetectable [19]

Downloaded by: King's College London 137.73.144.138 - 1/16/2019 6:52:28 PM

Abstract. In an effort to elucidate immunopathogenic mechanisms of membranous nephropathy (MN). freshly col­ lected sera from patients with biopsy proven MN were assayed for circulating immune complexes (ICs) by the Raji cell method and for anti-renal tubular epithelial (RTE) antibodies by a newly established radioimmunoassay (RIA) and by indirect immunofluorescence. 6 of 26 MN patients tested by the Raji cell assay had detectable circulating ICs. However. 5 of these 6 patients had other medical conditions which might also explain the IC reactivity. 29 MN patients and 11 pa­ tients with other glomerular diseases had no demonstrable circulating anti-RTE antibodies. This study suggests that if RTE antigens possess a nephritogenic potential for man it is probably only rarely expressed. The inconstant detection of circulating immune complexes in idiopathic MN raises speculation as to their immunopathogenic significance.

Membranous Nephropathy

M aterials and Methods Renal Tabular Epithelial Fractions A crude tubular fraction, known as FxIA, was prepared from normal kidney by a technique adapted from that of Krakower and Greenspan [22] as previously described [7], Antiserum to RTE Antipens (Anti-FxlA) Antiserum to human RTE antigens was raised in New Zealand white rabbits by immunization with FxIA and incomplete Freund's adjuvant and then extensively absorbed with normal human serum (NHS) as previously described [7]. Indirect immunofluorescence was then performed, according to the technique of Coons el at. [23], to localize intrarenal sites of reactivity. Controls included phosphatebuffered saline pH 7.5 (PBS), nonimmune rabbit serum, and anti­ serum absorbed X2 with solubilized FxIA, 4 mg,ml of antiserum. Antiserum was diluted 1:8 in PBS prior to use. Immunolopic Identification of RTE Antigens Human RTE antigens were identified during isolation techniques by double immunodiffusion in 1% agarose. Precipitin reactions were allowed to develop for 3 days. Solubilization o f RTE Antipens from FxIA RTE antigens w'ere solubilized from FxIA using 1% sodium deoxycholate and the resultant solution was then brought to 20% (NH i)>SO.i saturation, as previously described [7], The supernatant was then concentrated by osmotic equilibrium dialysis against sucrose, and then extensively dialyzed against 0.0175 M sodium phosphate pH 6.5. DEA E-Cellulose Chromatography DEAE-cellulose was equilibrated with 0.0175 M sodium phos­ phate pH 6.5 and packed to a height of 25 cm in a 1.5-cm column. 20 mg of the solubilized RTE proteins, previously dialyzed against the starting buffer, was applied to the column. A salt gradient for elution of RTE antigens was formed using 2 M NaCI (70 ml in a reservoir beaker) and starting buffer (70 ml in a mixing beaker). The column was washed with 50 ml of starting buffer and elution was begun with 2-ml fractions being collected. Fractions were selectively pooled, concentrated, and assayed by immunodiffusion for RTE antigens.

Radioiodination o f RTE Antigens RTE antigens recovered front DEAE-cellulose were concentrated to 200 |zl and radiolabelled with 125l by the chloramine-T method [24]. The reaction mixture (containing RTE antigens, non-RTE protein fragments, unreacted iodine, and low molecular weight reactants) was applied to a 30 x 1.5 cm Sephadex G200 column equilibrated with PBS. One ml fractions were collected and assayed for radioactivity with a gammacounter. The protein content of the fractions was as­ sessed by precipitation of radioactivity with 10% trichloracetic acid (TCA). The human RTE antigens, previously shown to be excluded by Sephadex G-200 [7,15, unpubl. observations, R. /.] were recovered in the exclusion peak, extensively dialyzed against PBS, diluted to an approximate protein concentration of 0.50 ¡zg/ml in RIA buffer (0.02 M Tris, 0.13 M NaCI. bovine serum albumin 0.02%. Na azide 0.05%, pH 7.8) and then stored at - 2 0 C. Radioimmunoassay for RTE Antibodies An assay by direct protein binding was performed by adding to polyethylene lubes 0.2 ml of RIA buffer. 0.1 ml of the radiolabelled RTE solution (containing approximately 50 ngof protein, or approxi­ mately 10,000 cpm of radioactivity) and 0.1 ml of the serum sample to be tested for anti-RTE antibodies. All human serum samples were tested at 1:40, I :80.and 1:160dilutions to circumvent prozone reactions (dilutions greater than 1:40 were performed using 1:40NHS-RIA buffer). Rabbit anti-FxlA antiserum was used as a positive control and tested at 2-fold serial dilutions of 1:10 1:656,000. (All antiserum dilutions greater than 1:40 were performed using 1:40 normal rabbit serum-RIA buffer.) The reactions were allowed to incubate at 4 Cfor 18 h. Then. 0.1 ml a 1:5 dilution of goat antihuman IgG (Cappel Labs, Cochranville, Pa) was added to tubes containing human test serum. A 1:5 dilution of goat antirabbit IgG (Cappel) was added to samples containing rabbit test serum. The reactions were again in­ cubated at 4 C for 18 h. Finally, I ml of RIA buffer was added, the tubes centrifuged at 1.400 g for 40 min, and the supernatant was decanted. All tubes were counted in a gammacounter and the percent of precipitated (PCP) radioactivity was determined. Nonspecific pre­ cipitation was determined by substituting 0.1 ml of RIA buffer for 0.1 ml of test serum. Percent specific PCPN for clinical samples was then calculated by the formula: Observed PCPN (cpm) - nonspecific PCPN (cpm) Maximum PCPN (cpm) - nonspecific PCPN (cpm) = specific precipitation. To determine assay specificity for RTE antigens. 0.1 ml of rabbit anti-FxlA antiserum was absorbed with either 0.1 ml of solubilized Fxl A, 40 ¡xg/ml, or with 0.1 ml of a Sephadex G-200 exclusion peak of concentrated normal human urine, known to contain URTE [13] in addition to other RTE antigens [unpubl. observations, R.Z.\. These absorbed antisera were then used in the assay as described above. Additional controls included the following: (I) goat antihuman IgG without prior addition of NHS; (2) goat antirabbit IgG without prior addition of rabbit anti-Fxl A antiserum: (3) rabbit anti-NHS followed in 18 h by goat antirabbit IgG. Immuno/lttorescenl Procedures Indirect IF was carried out on heat-inactivated (56 C, 30 min) aliquots of scrum (undiluted and 1:4 dilutions) as described elsewhere [25].

Downloaded by: King's College London 137.73.144.138 - 1/16/2019 6:52:28 PM

or present less frequently than in other nephrotic glom­ erular diseases [20,21], The present study was undertaken to further explore roles of human anti-RTE antibodies and circulating ICs in the pathogenesis of MN. We have developed a highly sen­ sitive radioimmunoassay (R1A) capable of detecting anti­ body with specificity to multiple human RTE antigens. The establishment of this assay and the results of a prospective search for anti-RTE antibodies by RIA and indirect IF in freshly collected sera from 29 patients with biopsy proven MN is presented. These sera were also assayed for circulat­ ing ICs by the Raji cell RIA, the most sensitive currently available method for their detection [18].

II

Zager Couser Andrews/Bolton/Pohl

12

-i— I----1— I— I— I— r5 25 45 65

A

Fig. I. A Separation of RTE antigens from other FxIA proteins on DEAE-cellulosc. B Recovery of radiolabelled RTE antigens in a Sephadex G-200 exclusion peak followed by free 1251 and uncharac­ terized radiolabelled proteins.

Measurement o f Circulating Immune Complexes Human sera were tested for circulating immune complexes by the Raji cell RIA performed as described by Theofilopoulos er at. [26]. Results are expressed as microgram equivalents of heat aggregated normal human IgG (p.g equiv. AHG/ml). Positive values are those which exceed the mean plus 2 SD obtained from a group of 50 normal sera. Clinical Material Serum was collected prospectively on 29 patients with MN specifically for the present study. All patients had the diagnosis of MN confirmed by renal biopsy with characteristic findings present by light, IF and EM [2]. Additional sera were collected from 11 patients with a variety of other glomerular diseases including 3 with high titer anti-GBM anti­ bodies confirmed by indirect IF and RIA [18. 25]. 12 normal subjects were studied as negative controls. Positive controls included rabbit and guinea pig antisera to human RTE prepared as described else­ where [27]. All serum samples were coded blindly and stored at - 7 0 C without addition of preservatives until the time of study. The samples studied as well as the age. sex, duration of disease, renal function, treatment status, and associated clinical findings are listed in table I.

Isolation o f RTE Antigens Approximately 50% of the starting FxIA (150 mg) was solubilized with 1% sodium deoxycholate. with the RTE antigens being recovered in the supernatant. 20% (NH-OaSOi fractionation precipitated approximately 60% of the solubilized protein, with the RTE antigens again being recovered in the supernatant. Elution of this material from DEAE-cellulose produced 3 protein peaks (fig. IA) with all 3 RTE antigens being recovered in the 2nd peak. The first peak contained an unknown quantity of IgG as delineated by immunodiffusion using rabbit antihuman IgG, while the third peak contained uncharacterized proteins. Establishment o f RIA for Anti-RTE Antibodies Radiolabelling o f RTE Antigens. The RTE solution, containing all 3 previously defined antigens, was radiolabelled by the chloramine-T method to a specific activity of approximately 100 ¡xCi/mg protein. RTE antigens were recovered from the reaction mixture in the Sephadex G-200 exclusion peak as described above (fig. IB). Radioactivity contained within this peak was pooled, and 85% was 10% TCA precipitable (which increased to 96% after dialysis against PBS). The remaining Sephadex fractions were pooled. Without prior dialysis 28% was TCA precipitable. Rabbit Anti-FxIA Antiserum Titration Curve. Two-fold serial dilutions of rabbit anti-FxIA absorbed with NHS was used to test the percent precipitability of l23I-labelled RTE antigens (fig.2). Dilutions of 1:10-1:656.000 were tested, followed by the addition of the 1:5 dilution of goal antirabbit IgG to help separate bound from unbound radiolabelled antigens. A maximum precipitation of 70% was achieved with the 1:40 dilution of NHS-absorbed anti-FxIA. Anti-RTE antibodies were detected at a maxi­ mum dilution of 1:326,000. Nonspecific precipitation ranged between 12 and 14%.

Rabbit Antiserum to Tubular Antigens (Anti-FxIA) By immunofluorescence microscopy, anti-FxIA. ab­ sorbed with NHS, stained exclusively the luminal surface of the proximal tubule in the region of the brush border as previously depicted [7]. Immunofluorescence was inhibited by prior absorption of anti-FxIA with solubilized FxIA. NHS and PBS controls were negative. By double immunodiffusion, anti-FxIA absorbed with NHS identified 3 RTE antigens in solubilized FxIA. Two major precipitin lines and one minor precipitin line de­ veloped, as previously reported [7,15].

Fig. 2. Radioimmunoassay by direct protein binding for detection of anti-RTE antibodies. Rabbit anti-FxIA (absorbed with NFIS) titration curve demonstrating percent actual precipitation at 2-fold serial antiserum dilutions.

Downloaded by: King's College London 137.73.144.138 - 1/16/2019 6:52:28 PM

Results

Membranous Nephropathy

Testing o f Clinical Samples for Anti-RTE Antibody by R1A 54 serum samples (29 MN, 3 anti-GBM disease, 8 other glomerular diseases, 12 NHS. I rabbit anti-Fx 1A. different from the one used in the above antiserum titration curve, and I guinea pig anti-FxlA) were assayed in a blinded fashion for anti-RTE antibodies at 1:40, 1:80, 1:160 di­ lutions (table I). Of the 54 serum samples tested, only the 2 anti-Fx IA control sera resulted in specific precipitation (rabbit 21%. guinea pig 37% at 1:80 dilutions). A higher degree of specific precipitation could be expected from the rabbit and guinea pig control sera if the second precipitat­ ing antibody had consisted of antirabbit IgG and anti­ guinea pig IgG. respectively, instead of the antihuman IgG used in the clinical assay.) Indirect IF None of the 29 test samples tested from patients with MN had demonstrable reactivity with RTE antigens in normal human kidney by indirect IF. Sera from 2 patients (No.4,26, table I) who later developed clinical evidence of systemic lupus erythematosis were reactive with nuclear antigens in human kidney. 3 patients with rapidly pro­ gressive glomerulonephritis associated with linear GBM deposits of IgG had anti-GBM antibody present in di­ lutions up to 1:256. Rabbit and guinea pig antisera to human RTE reacted with the proximal tubular brush border in dilutions up to 1:256. Circulating Immune Complexes Reactivity in 50 samples of NHS tested in the Raji cell assay (mean ± 2 SD) was 6 ± 6 jug equiv. AHG/ml. No in­ creased reactivity was demonstrable in the 3 patients with anti-GBM disease. 26 of 29 MN patients had serum assayed by the Raji cell technique with 6 patients showing increased reactivity ranging from 25 to 64 ¡¿g equiv. AHG/ml (table I). 5 of these patients had other conditions which may

have accounted for the circulating ICs. I patient had mela­ noma (N.6), one had prostatic carcinoma (No. 14). 2 pa­ tients had MN in transplanted kidneys (No. 13,16) and 1 patient had SLE (N o.26). Thus, only I of 20 patients (No. I) with apparently idiopathic MN had circulating ICs detectable by the Raji cell assay.

Discussion

Although MN is considered to be a classic example of an lC-mediated glomerulopathy [28], measurements of circulating IC levels in patients with MN have failed to convincingly demonstrate an association between cir­ culating ICs and this lesion [19-21], Our demonstration of circulating ICs in only 6 of 26 MN patients whose sera were freshly collected and assayed by the Raji cell technique again emphasizes that such complexes can be demon­ strated in only a minority of patients with this disease. Furthermore, 5 of our 6 patients who had IC reactivity had other medical conditions known to be independently associated with circulating ICs such as malignancy [29], systemic lupus erythematosis [30] and renal transplanta­ tion [31]. There are several possible explanations for this failure to consistently detect circulating ICs in this purportedly IC mediated renal disease. These include the following: that ICs are present below the sensitivity of the Raji cell assay: that they are present only episodically; that the Raji cell only detects complement fixing ICs; or that patients were studied either too late in the course of their disease or while on therapy [32]. However, experimental data suggest that the Raji cell technique is sufficiently sensitive to detect complement-fixing circulating ICs when present in nephritogenic quantities [26]. Moreover, of our 20 MN patients without demonstrable circulating ICs by the Raji cell assay. 12 were tested relatively early in their clinical course (< 2 years), 6 had never received immunosuppression, and 12 were receiving no immunosuppression at the time of assay. An alternate explanation for the difficulty demonstrat­ ing circulating ICs in idiopathic MN is that this lesion may be mediated by an immunologic mechanism other than deposition of circulating ICs. Strong support for this pos­ sibility is provided by the experimental observations that infusions of preformed ICs fail to produce subepithelial IC localization [33-35], and that a lesion pathologically iden­ tical to human MN can be produced in rat kidney by per­ fusion with anti-RTE antibodies alone [16,17]. These find­ ings, as well as the reports of deposition of human RTE antigens and anti-RTE antibodies in glomeruli from pa-

Downloaded by: King's College London 137.73.144.138 - 1/16/2019 6:52:28 PM

Controls. Absorption of 1:40. 1:80, 1:160 dilutions of rabbit anti-Fx IA with either solubilized Fx IA or with the Sephadex G-200 exclusion peak of concentrated normal human urine completely inhibited specific precipitation of RTE-labelled antigens. Similarly, goat antirabbit IgG and goat antihuman IgG (without prior addition ofanti-FxIA or NHS, respectively) failed to demonstrate specific pre­ cipitation of labelled antigens. However, rabbit anti-NHS produced 7% specific precipitation. (Since NHS contains RTE antigens, this may reflect either anti-RTE antibodies in rabbit anti-NHS, or an uncharacterized serum protein contaminant in the antigen preparation.)

13

14

Zager Couser Andrews/ Bolton/Pohl

Table I. Clinical/laboratory data for 29 MN patients Patient No.

Age/sex

Disease duration years

Serum creatinine, mg%

1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19

19 M 69 M 69 F 52 M 44 F 25 F 51 M 64 F 52 M 66 F 59 M 45 M 46 F 71 M 60 M 29 M 60 M 48 M 44 M

6 0.16 2.5 2 6 1 4 2 2.5 3.5 0.2 2.5 7 0.2 0.3 3 5 0.6 1

0.9 1.3 0.7 0.8 0.6 0.5 1.4 4.1 1.0 3.0 1.0 1.4 8.0 1.3 l.l 1.5 4.5 1.3 5.5

20 21 22 23 24 25 26 27 28 29

40 51 43 39 47 57 62 28 40 24

2 2 1.5 1.5 5 0.3 2.5 2.5 1

1.4 1.0 1.2 0.9 1.7 1.5 1.5 1.5 1.8

3

1 .0

M M M F F M F M M M

Treatment status previous _

p p p 6MP -

P -

P

current

_ P P P 6MP P -

P

-

-

-

-

P/AZA -

P/AZA P/AZA

-

P/AZA P P/Gold P P

-

P 2.5 mg/day -

P P P P P P -

-

-

-

Anti-RTE antibody RIA/IF

Raji cell (ICs)1

Other medical conditions

31** 8 8 7 5 55* 6 4 7 8 5 9 36* 32* 6 25* 4 4 5

RVT RVT — SLE — melanoma

5 10 6 2 ND ND 64* 8 2 ND

-

Felly's syndrome —

MN in Tx kidney MN in Tx; rejection prostatic Ca colonic Ca M N: recurred in Tx -

rheumatoid arthritis allergic interstitial nephritis — -

RVT SLE -

thyroiditis -

tients with MN [8,11.12], suggest that humoral immunity to RTE antigens may play a role in the pathogenesis of some cases of this disease. Our RIA for anti-RTE anti­ bodies, as described above, represents an attempt to con­ firm whether RTE antigens possess such a potential, either via the formation of circulating ICs, or by the induction of anti-RTE antibodies which then might react directly with ill-defined antigens located on the epithelial surface of the glomerular capillary wall. The detection of circulating anti-RTE antibodies would strongly suggest that one of these pathogenetic mechanisms is operative in at least some cases of MN.

Since there are no data to suggest that any single RTE antigen may have an exclusive nephritogenic potential, a radiolabelled mixture of all 3 presently defined RTE anti­ gens was used in this RIA, thereby permitting a more thorough screening for antibodies with brush border re­ activity. The precipitation of 70% of this radiolabelled antigenic mixture with NHS-absorbed-rabbit-anti-Fxl A attests to its highly enriched nature. Detection of anti-RTE antibody at a rabbit anti-Fxl A dilution of 1:326,000 con­ firms assay sensitivity. The complete inhibition of precipi­ tation of radiolabelled antigens by prior incubation of rabbit anti-FxlA antiserum with trace amounts of Fxl A

Downloaded by: King's College London 137.73.144.138 - 1/16/2019 6:52:28 PM

Tx = Transplant kidneys: RVT = renal vein thrombosis: Ca = carcinoma: SLE=systemic lupus erythematosis; P = prednisone; 6M P=6mercaptopurine: AZA = azathioprine; ND = not done. 1 ¡jtgequiv. AHG/ml serum. * Positive for circulating ICs.

or with a human urine fraction known to contain RTE antigens supports assay specificity. The positive identifi­ cation of 2 animal control sera with known anti-RTE specificity during the course of a blinded assay for human anti-RTE antibodies further attests to the utility of this technique. Our inability to detect anti-RTE antibodies in sera of all 29 patients with MN strongly suggest that if immunity to RTE antigens does exist in this disease it is probably an unusual event. The failure of Whitworth et al. [13] to docu­ ment an RTE antigen (URTE) in biopsies of 24 patients with MN by IF supports this conclusion. Nevertheless, reports by different investigators of glomerular RTE depo­ sition in human glomerulonephritis [8,9,11,12,14] con­ tinue to suggest that the AICN model may have a clinical counterpart in man. We believe, however, that further substantiation of the pathogenetic significance of RTE antigens in such cases would be very helpful. In a situation analogous to the radioimmunologic detection ofanti-GBM antibodies in sera of patients with linear immunofluores­ cence [18], the detection of circulating anti-RTE antibodies in patients with glomerular immunofluorescent localization of RTE antigen would help to further substantiate a role for RTE antigens in the immunopathogenesis of human glomerular disease. We believe that such studies would be a useful application of this newly established assay in the future.

Acknowledgements Portions of this work were supported by research grant No. AMI 7722 from the cnited States Public Health Service. Dr. Conner is the recipient of a Research Career Development Award (AM00I02) from the National Institutes of Health. Christine Darby. Attrdry Levine. Timothy Conlan, and Betty Gillant provided valuable technical assistance.

References 1 Coggins, C.H .: An interhospital study of the adult idiopathic nephrotic syndrome and its response to treatment. Abstract. Kidney int. 8: 408 (1975). 2 Ehrenreich, T. and Churg, J.: Pathology of membranous nephro­ pathy. Path. Annu. 3: 145-188(1968). 3 Germuth. F.G., jr. and Rodriquez, E.: Immunopathology of the renal glomerulus (Little, Brown, Boston 1973). 4 Heymann, W .; Hackel, D. B.: Harwood, J.; Wilson, S.G .F., and Hunter, J.L .P .: Production of nephrotic syndrome in rats by Freund's adjuvants and kidney suspensions. Proc. Soc. exp. Biol. Med. ¡00: 660-666 ( 1959). 5 Edgington, T.S.: Cilassock. R.J., and Dixon. R.J.: Autologous

15

immune complex pathogenesis of experimental allergic glomerulo­ nephritis. Science 155: 1432-1434(1967). 6 Miyakawa, Y.: Kitamura, K .: Shibata, S., and Naruse, T .: Dem­ onstration of human nephritogenic tubular antigen in the serum and organs by radioimmunoassay. J. Imniun. 117: 1203-1210 (1976) . 7 Zager, R. A. and Carpenter, C. B.: Radioimmunoassay for urinary renal tubular antigen. A potential marker of tubular injury. Kidney int. 13: 505-512(1978). 8 Ozawa. T.: Pluss. R.; Sacher, J.; Boedecker, E.; Guggenheim, S.t Hammond, W.. and McIntosh, R. W.: Endogenous immune com­ plex nephropathy associated with malignancy. 1. Studies on the nature and immunopathogenic significance of glomerular bound antigen and antibody, isolation and characterization of tumor specific antigen and circulating immune complexes. Q. J1 Med. 44: 523-541 (1975). 9 Strauss, T .; Pardo, V.: Koss, M.t Griswold, W., and McIntosh, R. W.: Nephropathy associated with sickle cell anemia. An auto­ logous immune complex nephritis. I. Studies on the nature of the glomerular bound antibody and antigen identification in a patient with sickle cell disease and immune deposit glomerulonephritis. Am. J. Med.58: 382-387(1975). 10 Makker, S. P.: Brush border antibodies of Heymann nephritis of rats in normal human serum. Proc. Soc. exp. Biol. Med. 154:9-13 (1977) . 11 Naruse, T.; Kitamura, D.: Miyakawa, Y., and Shibata, S.: Deposition of renal tubular epithelial antigen along the glomerular capillary walls of patients with membranous glomerulonephritis. J.lmmun. 110: 1163-1166(1973). 12 Naruse, T.; Miyakawa, Y.; Kitamura, K., and Shibata, S .: Membranous glomerulonephritis mediated by renal tubular epi­ thelial antigen-antibody complexes. J. Allergy clin. Immunol. 54: 311-318(1974). 13 Whitworth, J. A.; Leibowitz, S.; Kennedy, M .C.; Cameron, J.S.; Evans, D.J.; Glassock, R.J., and Schoenfeld, L.S.: Absence of glomerular renal tubular epithelial antigen in membranous glom­ erulonephritis. Clin. Nephrol.5: 159-162(1976). 14 Pardo. W.: Strauss, J.: Krarrer, H.; Ozawa, T., and McIntosh, R. W.: Nephropathy associated with sickle cell anemia. An auto­ logous immune complex nephritis. II. Clinicopathologic study of seven patients. Am. J. Med. 59: 650-659 (1975). 15 Schoenfeld, L.S. and Glassock, R.J.: Renal tubular antigen ex­ cretion in normal urine. I. Immunochemical identification. Kidney int. 3: 309-314(1973). 16 Van Damme, B.: Fleuren, G .J.; Bakker, W.W.; Hoedemacker, P.J., and Vernier, R.L.: Fixed glomerular antigens in the patho­ genesis of heterologous immune complex glomerulonephritis. Kidney int. !0 : 511 (1976). 17 Steinmuller, D. R.: Bclok,S.;Stilmant, M. M.; Lowenstein, L. M., and Couser, W .G.: Experimental glomerulonephritis in the iso­ lated perfused rat kidney (IPK). Kidney int. 12: 519 (1977). 18 Wilson, C. B.: Recent advances in the immunological aspects of renal disease. Fed. Proc. Fed. Am. Socsexp. Biol. 36:2171 (1977). 19 Ooi, T. M .: Ooi. B.S., and Poliak, V. E .: Relationship of level of circulating immune complexes to histologic patterns of nephritis. A comparative study of membranous glomerulonephropathy and diffuse proliferative glomerulonephritis. J. Lab. clin. Med. 90: 819-898(1977). 20 Woodroffe, J.A .: Border, W.A.; Theofilopoulos, M.A.; Gotze, O.: Glassock, R.J.: Dixon, F.J., and Wilson, C.B.: Detection of

Downloaded by: King's College London 137.73.144.138 - 1/16/2019 6:52:28 PM

Membranous Nephropathy

16

30 Nydegger, U.E.; Lambert, P. N.: Gerber. H., and Miescker. P.A.: Circulating immune complexes in the serum in systemic lupus erythematosis and in carriers of hepatitis B antigen. Quan­ titation by binding to radiolabelled Clq. J. clin. Invest. 54:297-309 (1974). 31 Ooi, Y.M.: Ooi. B.S.; Vallota, E.N.: First, M.R., and Poliak, V. E.: Circulating immune complexes after renal transplantation. Correlation of increased l25I-Clq binding activity with acute rejec­ tion characterized by fibrin deposition in the kidney. J. clin. Invest. 60: 611-619 (1977). 32 Dreisin, R.B.; Schwarz, M.I.; Theofilopoulos, A.N., and Stan­ ford, R.E.: Circulating immune complexes in idiopathic inter­ stitial pneumonias. New Engl. J. Med. 298: 353-357 (1978). 33 Cochrane. C.G .: Mechanisms involved in the deposition of im­ mune complexes in tissues. J. exp. Med. 134: 75-89 (1971). 34 Okumura, K.; Kondo, Y., and Tada, T.: Studies on passive serum sickness. I. The glomerular fine structure of serum sickness nephritis induced by preformed antigen-antibody complexes in the mouse. Lab. Invest. 23: 383-391 (1971). 35 O'Regan, S.: Smith, M.,and Drummond, K. N .: Immune complex infusion in the rat. Renal functional and morphological changes. Clin. exp. Immunol. 24: 110-115 (1976).

Received: April 11, 1978: Accepted: July 26, 1978. Richard A.Zager, MD, Department of Medicine, Veterans Administration Hospital, 1400 VFW Parkway, West Roxbury, MA 02132 (USA)

Downloaded by: King's College London 137.73.144.138 - 1/16/2019 6:52:28 PM

circulating immune complexes in patients with glomerulonephritis. Kidney int. 12: 268-278 (1977). 21 Border. W.; Abrass. C.; Hall, C.; Brown, C.: Glassock, R.. and Coggins, C.: Detection of circulating immune complexes (CIC) in adult idiopathic nephrotic syndrome (AINS). Abstract. Kid­ ney int. 12: 510(1977). 22 Krakower, C. A. and Greenspon, S. A .: Localization of the neph­ rotoxic antigen within the isolated renal glomerulus. Am.med. Ass.Archs Path. 51 : 629-637(1951). 23 Coons, A. H.: LeDuc, E. H., and Connelly, J. M .: Studies on anti­ body production. LA method for the histochemical demonstration of specific antibody and its application to a study of the hyper­ immune rabbit. J.exp. Med. 102: 49-60 (1955). 24 Greenwood, F.C.: Hunter, W. M.. and Glover. J.S.: The prepa­ ration of 1311 labelled human growth hormone of high specific radioactivity. Biochem.3.89: 114(1963). 25 Couser, W .G.; Wallace, C.A.; Monaco, A.P., and Lewis, E.J.: Successful renal transplantation in patients with circulating anti­ body to glomerular basement membrane. Clin. Nephrol. I: 381 388 0974). 26 Theofilopoulos, A .N .; Wilson, C.B., and Dixon. F.J.: The Raji cell radioimmunoassay for detecting immune complexes in human sera. J.clin. Invest.57. 169-182 (1976). 27 Couser. W .G .: Hoyer, J. R .; Stilmant, M. M .; Jermanovich. N. B.. and Belok, S.: The effect of aminonucleoside nephrosis on im­ mune complex localization in autologous immune complex neph­ ropathy in rats. J. clin. Invest. 61 : 561-572 (1978). 28 Brenner, B. M. and Rector, F. C .: The kidney, p. 1005 (Saunders, Philadelphia 1976). 29 Theofilopoulos, A.N.: Andrews, B.S.; Urist, M.M.. et al.: The nature of immune complexes in human cancer sera. J. Immun. 119: 657-663 (1977).

Zager/Couser/Andrews/ Bollon/Pohl

Membranous nephropathy: a radioimmunologic search for anti-renal tubular epithelial antibodies and circulating immune complexes.

Original Papers Nephron 24: 10 16(1979) Membranous Nephropathy: A Radioimmunologie Search for Anti-Renal Tubular Epithelial Antibodies and Circulatin...
1MB Sizes 0 Downloads 0 Views