Clin. exp. Immunol. (1979) 38, 235-242.

Antibodies directed against renal collecting ducts in sera of human renal allograft recipients L. C. PAUL, L. A. VAN ES, M. W. KALFF & G. J. FLEUREN* Department of Nephrology, University Hospital, Leiden, and *Department of Pathology, Westeinde Hospital, The Hague, The Netherlands

(Accepted for publication 4 May 1979)

SUMMARY

Antibodies directed against the plasma membrane of renal collecting ducts were found in the sera of two out of 101 renal allograft recipients. The sera also reacted with the epithelium of the renal pelvis, the ureter, the ductus deferens, the skin, the oesophagus, the trachea and the bile ducts of random individuals, indicating that the antibodies were directed against a widespread epithelial antigen. The antibody activity against these structures was removed by absorption of the sera with homogenized renal medullary tissue; absorption with plasma proteins, erythrocytes, spleen leucocytes or platelets did not influence the antibody titers. In the two patients with circulating antibodies, the graft function was unremarkable eight and fifteen months after transplantation, respectively. From this study we conclude that circulating epithelial antibodies directed against the collecting ducts of the graft are of little or no clinical importance. INTRODUCTION Circulating antibodies directed against antigens in the graft may be found in renal allograft recipients in association with graft rejection (Williams et al., 1968; Cochrum & Kountz, 1969; Jeannet et al., 1970; Perkins et al., 1975; Paul et al., 1978, 1979a, 1979b, 1979c). This report concerns two renal allograft recipients with circulating antibodies directed against the collecting ducts of the graft but without signs of rejection. The specificity of the antibodies and their clinical significance for renal allografts are discussed.

MATERIALS AND METHODS Patients. The two patients of this study were found among 101 patients who had received one or more renal allografts between January, 1976, and May, 1978. Case histories. (a) P., a 56 year old male, received a first graft in October, 1977. He had required haemodialysis since 1974 because ofchronic glomerulonephritis and in 1977 he developed chronic prostatitis. The graft started to function after 10 days; rejection treatments were initiated on the 7th, 17th and 32nd day because rejection was suspected. At present, 15 months after transplantation, the endogenous creatinine clearance is 70-75 ml/min and neither proteinuria nor glucosuria is present. The minimal pH in a random urine sample is 5. The urinary sediment contains many leucocytes per high power field, presumably due to chronic prostatitis. (b) G., a 42 year old female, received a first cadaveric renal allograft in April, 1978. She had been on chronic haemodialysis since 1974 because of end-stage pyelonephritis. The graft started to function after 15 days; on the 4th, 20th and 33rd day :reatment of rejection was started. At present, 8 months after transplantation, the endogenous creatinine clearance is 60-65 nl/min and neither proteinuria nor glucosuria is present. The marginal pH in a random urine sample is 5; the urinary ;ediment shows no abnormalities. Serology. The recipients' serum samples were studied for the presence of antibodies against the donor kidney using a tandard indirect immunofluorescence technique, on biopsies taken from the graft before transplantation, as previously

Correspondence: Dr L. C. Paul, Department of Nephrology, University Hospital 2333 AA Leiden, The Netherlands. 0099-9104/79/1100-0235 $02.00 @) 1979 Blackwell Scientific Publications

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described (Paul et al., 1979a). In addition to the standard technique which is performed at 20'C, the sera were also tested at

40C and 370C. Furthermore, sections were pretreated with neuraminidase (500 mU/ml phosphate-buffered saline (PBS) for 30 min at 370C) or papain (1 mg/ml PBS for 30 min at 37QC). The enzyme preparations were purchased from Worthington

Biochemical Corporation (Freehold, New Jersey, USA). Sera from patient P. were tested against a panel of pretransplant graft biopsies obtained from forty-three donors; those from patient G. were tested against twenty-six biopsies. In order to establish the intrarenal localization of the antigens, sections taken from the cortex and the medulla of four kidneys were used. In addition, sections were used from four ureters, three vasectomy preparations, and four skin biopsies; two of these skin biopsies were taken from the allograft recipients. Furthermore, sections taken from kidneys, livers, oesophagus, tracheae, hearts and skin were obtained at autopsy from two individuals. To localize accurately the binding of the antibodies to the kidney, the sera were studied using the indirect immunoperoxidase method, in which the sections were counterstained with haematoxylin and then fixed in formalin (Paul, Fleuren & van Es, 1979d). Since the immunofluorescence technique does not discriminate between the binding of monomeric antibodies and that of complexed immunoglobulins to Fc receptors, the sera were fractionated by ultracentrifugation on a calibrated linear gradient of 10%-30% w/v sucrose in borate-buffered saline, as published elsewhere (Paul et al., 1979b). To study the specificity of the antibodies, the sera were examined for the presence of lymphocytotoxic antibodies with the two-colour fluorescence technique (Van Rood, Van Leeuwen & Ploem, 1976) using peripheral leucocytes from forty random donors. The presence of erythrocyte antibodies was assayed with a direct and indirect Coomb's technique using an erythrocyte panel which included the Rhesus, Duffy, Kidd, Xg, Lewis, M.N., P. and Lutheran antigens (Reagent Red Blood Cells Identigen, Ortho Diagnostic Incorporated, Beerse, Belgium). In addition, agglutination titers were determined at 4°C, 20°C and 37°C using erythrocytes from the serum donors, random adults and random newborns (umbilical cord erythrocytes). To exclude the possibility that the antibodies were directed against heterophile antigens, agglutinating titers were determined for the erythrocytes from sheep, guinea-pigs (Rapaport, Kano & Milgrom, 1968) and Wistar strain rats (McDonald & Mukherjee, 1973). The sera were absorbed with pooled plasma proteins coupled to agarose (March, Parikh & Cuatrecasas, 1974), erythrocytes with A and B blood group antigens from five normal adult donors, the recipients's own erythrocytes, erythrocytes from three newborns (blood groups A and 0), pooled platelets and spleen leucocytes from two selected kidney donors. The leucocyte preparations contained up to 45% B cells and monocytes according to the criteria described elsewhere (Paul et al., 1979c). In addition, absorption was carried out with erythrocytes from sheep, guinea-pigs and Wistar rats. Absorption was performed at 4°C, 20°C and 37°C during three 45-minute periods using one volume of serum and three volumes of packed cells or immunoabsorbens. Absorption with leucocytes took place at room temperature for 2 hr at a ratio of 3 x 108 packed cells/ml of serum. The sera were also absorbed with renal medullary tissue, prepared by homogenizing the renal papillae of four kidneys that could not be used for transplantation. After homogenization in PBS, the suspension was centrifuged at 27,000 g for 50 min at 4°C and washed repeatedly in PBS at 4°C for 24 hr. The sera were absorbed twice for 90 min each at room temperature; 1 vol. serum was absorbed with 2 vol. packed homogenate each time (dry weight of 1 ml packed homogenate 150 mg). 1251I labelled normal IgG was added to the sera to be able to correct for the dilution that occurred during absorption experiments. Graft biopsies taken after transplantation were studied by light microscopy after staining with haematoxylin, eosin and silver methenamine. Immunofluorescence studies of these biopsies were performed as previously described (Paul et al., 1978, 1979a). Control biopsies were taken from seven recipients, who were matched for graft function and time after trans-

plantation.

RESULTS The sera taken from both patients before and after transplantation contained IgG which bound in vitro to the epithelium of the collecting ducts in the donor kidney (Fig. 1) and all other kidneys that were tested. When the sera were fractionated by ultracentrifugation, only the 7S fractions bound to the sections therefore indicating that monomeric IgG antibodies were detected by the immunofluorescence test. To localize the antigen-positive cells in relation to the surrounding antigen-negative cells, the sera were studied by means of the indirect immunoperoxidase method. As shown (Fig. 2), the antibodies bound only to the collecting duct epithelium and not to the epithelium of the glomeruli, the proximal tubules, the loops of Henle or the distal tubules. When the sera were applied to sections taken from the renal cortex, the antibodies bound to approximately 10% of the tubules. In sections of the papilla it was obvious that the antibodies bound to the collecting ducts, since the columnar epithelial cells contrasted clearly with the flat epithelial cells of Henle's loops (Fig. 3) that did not bind the antibodies. The fluorescence of the collecting duct was continuous with that of the multilayer epithelium of the renal pelvis. The antibodies also bound to the lining epithelium of the ureter and the ductus deferens and the cells of the basal layers stained more

Renal collecting duct antibodies

FIG. 1. Immunofluorescence micrograph of a pretransplant graft biopsy incubated with serum from patient G. and stained with fluorescein-conjugated swine antihuman IgG. Binding of IgG is present along the epithelial cells of cortical collecting ducts. Magnification x 360.

FIG. 2. Photomicrograph of a pretransplant graft biopsy incubated with serum from patient P., rabbit antihuman IgG and peroxidase-conjugated swine anti-rabbit Ig. The sections were stained with 3-3'-diaminobenzidine-tetrahydrochloride and hydrogen peroxide; the counterstain was haematoxylin. Specific staining is seen along the plasma membranes of the epithelium of a cortical collecting duct, whereas the tubules are not specifically stained. Magnification x 630.

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FIG. 3. Immunofluorescence micrograph of a renal medullary section incubated with serum from patient G. and stained with fluorescein-conjugated swine anti-human IgG. Binding of IgG can be seen along the epithelium of a collecting duct but not along Henle's loops (H). Magnification x 630.

intensely than the cells of the luminal layers. Furthermore, the antibodies bound to the epithelial cells of the intrahepatic bile ducts, the oesophagus, the trachea, the submucosal glands, the Malpighian layer of the skin, the sebaceous glands and the hair follicles. No binding was observed to sections taken from heart tissue. Absorption of the sera with homogenized renal papillae eliminated the antibody activity against the kidneys and all other epithelial structures, indicating that the antibodies were directed against a common, widespread epithelial antigen. The in vitro antibody fixation was not temperature-dependent, since incubation of kidney sections with sera at different temperatures (40C, 20'C, 370C) always resulted in binding of the antibodies. The effect of enzymes on the antigen was studied by pretreatment of the sections with papain and neuraminidase (Lenhard et al., 1978). After pretreatment no binding of the antibodies was observed, indicating that the antigen is sensitive to papain and neuraminidase.

Serology To study the specificity of the antibodies, sera from both patients were tested for the presence of erythrocyte antibodies. Various haemagglutinating antibodies were found which are summarized in Table 1. Absorption of the sera with the erythrocytes listed in Table 1 (except the RBC panel) resulted in the disappearance of the haemagglutinating antibodies when present, whereas the epithelial antibody titers were not influenced. The leucocyte panel revealed weak antibodies without HLA specificity. To exclude the possibility that the antibodies were directed against HLA or other leucocyte antigens, the sera were absorbed with spleen leucocytes from two donors whose kidneys were available for study. Leucocyte absorption had no influence on the epithelial antibody titer, nor did absorption with platelets or insolubilized plasma proteins. The results of the absorption experiments are summarized in Table 2. Clinical significance In both patients the antibodies were present in pretransplant serum samples and remained detectable in all samples taken up to 8 and 15 months respectively after transplantation. At present, both grafts are functioning normally.

Renal collecting duct antibodies

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TABLE 2. Titers of epithelial antibody-positive sera from patient P. determined by immunofluorescence against kidney sections before and after absorption with various tissues*.

Titer of epithelial antibodies

Absorbed withS

Renal medullary tissue Plasma proteins Spleen leucocytes Platelets Adult erythrocytes Autologous erythrocytes Newborn erythrocytes Sheep erythrocytes Guinea-pig erythrocytes Wistar strain rat erythrocytes

Before absorption

After absorptiont

32 32 32 32 32 32 32 32 32 32

0 32 16 16 32 32 32 16 16 32

Comparable data were obtained with serum G; the antibody titer before absorption was 16. § See Materials and Methods Section. t Titers not corrected for the dilution, which varied between 2% and 53%. + Pooled adult red blood cells. *

Immunofluorescence studies of the biopsies of both recipients taken 30 min after transplantation and during rejection episodes did not show specific binding of IgG in the grafts. A biopsy was taken from recipient P. 15 months after transplantation. Although focal lymphocytic and histiocytic infiltrates as well as focal fibrosis were seen in the cortex, the medulla did not show abnormalities. Cortical infiltrates were, however, not observed in the biopsies from seven recipients who were matched for graft function and time after transplantation. The significance of this finding in relation to the circulating antibodies is not clear since specific staining for IgG was not seen along the collecting ducts. A biopsy from patient G. could not be taken for technical reasons. DISCUSSION This report concerns two renal allograft recipients with circulating antibodies directed against the collecting ducts of the donor kidney (Figs 1, 2 and 3) and all other kidneys tested. These antibodies have, to the best of our knowledge, not been described previously. Since the antibodies bound in vitro to the epithelium of the collecting ducts, the pyelum, the ureter, the ductus deferens, the bile ducts, the oesophagus, the trachea and to the skin, we conclude that the antibodies are directed against a general epithelial antigen which is selectively absent from the epithelium of the nephron per se. Since the antibodies reacted in vitro with the recipient's own epithelium, these antibodies are auto-antibodies. We cannot explain the presence of these antibodies. Since absorption of the sera with erythrocytes did not influence the epithelial antibody titers, we conclude that the antibodies were not directed against ABO blood group antigens which are present on the renal endothelium, the transitional epithelium of the urinary tract, and only in secretors, on the luminal aspect of the renal collecting ducts (Szulman, 1960). These experiments also exclude the possibility that the antibodies were directed against the I/i-erythrocyte antigens on the epithelium of Henle's loops and distal tubules (Lenhard et al., 1978). The observation that the antibodies bound to the plasma membranes of the collecting duct epithelium and not to intracellular antigens of the cells of Henle's loops means that the antibodies are not the same as those described by Ford (1973). Furthermore, absorption experiments showed that the antibodies were not directed against antigens present on

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platelets, such as the HLA-A, B and C antigens. In addition, the HLA-A and B antigens have been localized along the endothelium of the renal arteries and glomerular basal membranes (Sybesma et 4l., 1974). Absorption with spleen leucocytes ruled out that the antibodies were directed against HLA-Dr antigens. Since antibodies directed against antigens on erythrocytes from various species are frequently encountered in the sera of human renal allograft recipients (Rapaport et al., 1968; McDonald & Mukherjee, 1973), absorption was carried out with erythrocytes carrying heterophile antigens. A decrease in the epithelial antibody titers was, however, not observed, indicating that the antibodies were not directed against HT-A (McDonald & Mukherjee, 1973) or other heterophile antigens present on rat, sheep or guinea-pig erythrocytes. Screening of the sera for the presence of anti-nuclear factor activity was also negative (data not shown). In contrast to antibodies directed against antigens on the plasma membranes of endothelial cells in the graft (Paul et al., 1978, 1979a, 1979b, 1979c), epithelial antibodies are not causally related to graft rejection, possibly because of the relative inaccessibility of the antigen for the circulating antibodies. Both patients had epithelial antibodies in the circulation before transplantation and throughout the post-transplant period but graft function was not obviously disturbed in either case. Focal cellular interstitial infiltrates and fibrosis were present in the biopsies taken from both patients during rejection episodes. These changes could, however, also be due to the rejection process itself. The biopsy from patient P. taken 15 months after transplantation when there were no signs of rejection showed focal interstitial cellular infiltrates in the cortex. These changes were not observed in control biopsies. None of the biopsies showed, however, a specific pattern of IgG binding, which makes it difficult to relate the histologic findings with the circulating antibodies. From this study we conclude that circulating antibodies directed against epithelial cells of the collecting ducts, the pyelum and the ureter of the graft are not associated with clinical rejection or extensive graft damage, although we cannot exclude the possibility that humoral and/or cellular immunity against these antigens is associated with low grade tissue destruction. We would, however, not consider the presence of the antibodies to be a contra-indication for renal transplantation. We are indebted to Dr G. Brutel de la Riviere for the histological examination of the biopsies, to Mrs Heleen van Schie and Mrs Marianne Berends for their technical assistance, to Eurotransplant for providing kidneys that could not be used for transplantation and to Mrs Henny de Vries for typing the manuscript. REFERENCES an ABO-incompatible recipient Transplantation, 26, 268. COCHRUM, K.C., & KOUNTZ, S.L. (1969) Cytotoxic antibodies following human renal transplantation. Surg. PAUL, L.C., VAN Es, L.A., VAN ROOD, J.J., VAN LEEuwEN, Forum, 20, 302. A., BRUTEL DE LA RIVIERE, G. & DE GRAEFF, J. (1979a) Antibodies directed against antigens on the endothelium FoRD, P.M. (1973) A naturally occurring human antibody to of peritubular capillaries in patients with rejecting renal loops of Henle. Clin. exp. Immunol. 14, 569. JEANNET, M., PINN, V.W., FLAX, M.H., WirNN, H.J. & allografts. Transplantation, 27, 175. RUSSELL, P.S. (1970) Humoral antibodies in renal allo- PAUL, L.C. VAN Es L.A., KALFF, M.W. & DE GRAEFF, J. transplantation in man. New Engl. J. Med. 282, 111. (1979b) Intrarenal distribution of endothelial antigens LENHARD, V., SEELIG, H.P., GEISEN, H.P. & ROELCKE, D. recognized by antibodies from renal allograft recipients. (1978) Identification of I/i, Prl-3 and Gd antigens in the Transplant. Proc. 11, 427. human kidney: possible relevance to hyperacute graft PAUL, L.C., CLAws, F.H.J., VAN Es, L.A., KALFF, M.W. & rejection induced by cold agglutinins. Clin. exp. Immunol. DE GRAEFF J. (1979c) Accelerated rejection of a renal 33, 276. allograft associated with pretransplant antibodies directed MARCH, S.C., PARIKH, J. & CUATRECASAS, P. (1974) A against donor antigens on endothelium and monocytes. simplified method for cyanogen bromide activation of New Engl. 5. Med. 300, 1258. agarose for affinity chromatography. Anal. Biochem. 60, PAUL, L.C., FLEuREN, G.J. & VAN Es L.A. (1979d) Demon149. stration of transplantation antigens on the endothelium of MCDONALD, J.C. & MUKHERJEE, G.N. (1973) A heterophile peritubular capillaries in renal allografts by the immunosystem in human renal transplantation. III. The HT-A peroxidase method. Transplantation, 28, 72. specificities. Transplant. Proc. 5, 481. PERKINS, H.A., GANTAN, Z., SIEGEL, S., HOWELL, E., PAUL, L.C., VAN Es, L.A., BRUTEL DE LA RIvI1RE, G., BELZER, F.O. & KOUNTZ, S.L. (1975) Reactions of kidney EERNIssE, G. & DE GRAEFF, J. (1978) Blood group B cells with cytotoxic antisera: possible evidence for kidney antigen on renal endothelium as the target for rejection in specific antigens. Tissue Antigens, 5, 88.

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RAPAPORT, F.T., KANO, K. & MILGROM, F. (1968) Heterophile antibodies in human transplantation. 3. clin. Invest. 47, 633. SYBESMA, J.PH., KATER, L., BORST-EILERS, E., DE PLANQUE, B.A., VAN SOELEN, T. & TUIT, G. (1974) HL-A antigens in kidney tissue. Localization by means of an immunofluorescence technique. Transplantation, 17, 576. SZULMAN, A.E. (1960) The histological distribution of blood group substances A and B in man. 3. exp. Med. 111, 785.

VAN ROOD, J.J., VAN LEEuwFN, A. & PLOEM, J.S. (1976) Simultaneous detection of two cell populations by twocolour fluorescence and application to the recognition of B-cell determinants. Nature, 262, 795. WILLIAMS, G.M., HUME, D.M., HUDSON, JR, R.P., MomIs, P.J., KANo, K. & MILGROM, F. (1968) 'Hyperacute' renal-homograft rejection in man. New Engl. J. Med. 279, 611.

Antibodies directed against renal collecting ducts in sera of human renal allograft recipients.

Clin. exp. Immunol. (1979) 38, 235-242. Antibodies directed against renal collecting ducts in sera of human renal allograft recipients L. C. PAUL, L...
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