DEPOSITS OF IMMUNOGLOBULIN AND FIBRIN IN HUMAN ALLOGRAFTED KIDNEYS

1313 was 2800 g., the liver-copper concentration was 160-6 g. per g. fresh tissue, and the duration of cholestasis was 5 years. Assuming a daily copper intake of 3 mg., that up to 40% of this is absorbed (Sternlieb 1967), and that all the absorbed copper is retained in the liver, it can be calculated that it would have taken approximately a year for the excess copper found to have accumulated. This is a much shorter time than the actual duration of cholestasis, and, even allowing for large errors in the assumptions made, the high copper concentrations appear to be accounted for simply by failure of biliary excretion. Similar calculations in patients with primary biliary cirrhosis and other forms of cholestasis led to the same conclusion, which seems reasonable as copper is mainly excreted in the bile (Bearn and Kunkel 1955, Mahoney et al. 1955, Neumann et al. 1962, Sass-Kortsak 1965). If the high copper content of the liver in Wilson’s disease is instrumental in producing cirrhosis and liver-cell failure, then copper could play a role in the pathogenesis of livercell failure in biliary cirrhosis.

liver-weight

Hunt et al. (1963) treated two cases of primary biliary cirrhosis with penicillamine without noticeable benefit. They were advanced cases with presumably irreversible liver damage, and these workers considered that a more extensive trial of penicillamine was warranted. Any therapeutic trial of penicillamine would need to be carefully controlled, particularly in view of the variable course of primary biliary cirrhosis, and the different stages at which it is diagnosed. Unless the therapeutic effect were dramatic, the comparative rarity of this disease might trial extending over many years to allow any definite conclusions to be drawn. Moreover, penicillamine therapy is not without hazard (Walshe 1963, Scheinberg and Sternlieb 1965, Bearn 1966). However, experience with penicillamine and Wilson’s disease in this unit suggests that side-effects are much less prominent when there is copper overload. Penicillamine may offer an approach to the prevention of liver failure in long-standing biliary obstruction.

require

a

We should like to thank Mr. Dudley Sampson for his invaluable technical assistance. R. A. S. was supported by a Wellcome Trust

fellowship. REFERENCES A. G. (1966) in The Metabolic Basis of Inherited Disease (edited by J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson); p. 761. New York. Kunkel, H. G. (1954) J. clin. Invest. 33, 400. (1955) J. Lab. clin. Med. 45, 623. Butt, E. M., Nusbaum, R. E., Gilmour, T. C., Didio, S. L. (1958) Am. J. clin. Path. 30, 479. Gault, M. H., Stein, J., Aronoff, A. (1966) Gastroenterology, 50, 8. Gubler, C. J., Brown, H., Markowitz, H., Cartwright, G. E., Wintrobe, M. M. (1957) J. clin. Invest. 36, 1208. Hansl, N., Hyman, L., Luparello, T., Zimdahl, W. T. (1956) J. Lab. clin. Med. 48, 108. Hunt, A. H., Parr, R. M., Taylor, D. M., Trott, N. G. (1963) Br. med. J. ii, 1498. Mahoney, J. P., Bush, J. A., Gubler, C. J., Moretz, W. H., Cartwright, G. E., Wintrobe, M. M. (1955) J. Lab. clin. Med. 46, 702. Neumann, P. Z., Carr, R. I., Sass-Kortsak, A. (1962) Can. med. Ass. J. 86, 229. Sass-Kortsak, A. (1965) Adv. clin. Chem. 8, 1. Scheinberg, I. H., Sternlieb, I. (1963) Lancet, i, 1420. (1965) A. Rev. Med. 16, 119. Sternlieb, I. (1967) Gastroenterology, 52, 1038. Scheinberg, I. H. (1968) New Engl. J. Med. 278, 352. Tipton, I. H., Cook, M. J. (1963) Hlth Phys. 9, 103. Todd, A. P., Thorpe, M. E. C., Rosenoer, V. M. (1967) J. clin. Path. 20, 276. Vogel, S. F., Kemper, L. (1963) Lab. Invest. 12, 171. Walshe, J. M. (1963) Practitioner, 191, 789. Briggs, J. (1962) Lancet, ii, 763. Worwood, M., Taylor, D. M., Hunt, A. H. (1968) Br. med. J. ii, 344.

Beam, —

—

—

-

—

—

—

DEPOSITS OF IMMUNOGLOBULIN AND FIBRIN IN HUMAN ALLOGRAFTED KIDNEYS I. F. C. MCKENZIE Melb., M.R.A.C.P.

M.D. THIRD

ASSISTANT, UNIVERSITY

OF MELBOURNE DEPARTMENT OF MEDICINE

SENGA WHITTINGHAM M.B. N.Z., D.C.P. SEROLOGIST, CLINICAL RESEARCH UNIT, WALTER AND ELIZA HALL INSTITUTE OF MEDICAL RESEARCH ROYAL MELBOURNE

HOSPITAL,

VICTORIA

3050,

AUSTRALIA

Eighty-two kidney sections from 28 human recipients of renal allografts were examined, by immunofluorescence, for deposits of immunoglobulin, complement, and fibrin. Deposits of IgM and IgG, but not IgA, were detected in vessels of all sizes, lining the endothelium, extending towards the media, and, in larger vessels, in the thickened intima. Linear and granular deposits were also present in glomeruli. Complement and fibrin were present but not albumin or &agr;2-macroglobulin. Of forty-nine sections examined during rejection, immunoglobulin (especially IgM), complement, and fibrin were demonstrated in a significant number, when compared with the incidence in fourteen non-rejecting kidneys. The demonstration of these deposits suggests that humoral antibodies play an important role in the rejection of human allografted kidneys. Summary

Introduction

THE relative contribution of humoral and cellular immune reactions to the rejection of grafted tissue is controversial. In man the appearance of humoral lymphocytotoxic antibodies has correlated with rejection and failure of the graft (Morris et al. 1968, Terasaki et al. 1968). However, there is considerable evidence favouring cellular immune reactions as the cause of graft rejection (Wilson and Billingham 1967). We have examined frozen sections of thirty-one renal for the deposition of immunoglobulin, complement, and fibrin in the vessels and glomeruli of the graft. Patients and Methods 28 patients had received a renal allograft from a cadaver.

allografts

8 had bilateral nephrectomy before the graft. 3 received a second renal graft after the first was rejected. The patients were treated, after grafting, with azathioprine 3 mg. per kg. daily, prednisolone initially 100 mg., later reduced to 10 mg. daily, and three courses of irradiation, 150 rads per course, to the grafted kidney. Some patients were given actinomycin C (Kincaid-Smith et al. 1967). No patient had a blood-pressure greater than 160/100 mm. Hg after transplantation. Specimens of the grafted kidneys were obtained by biopsy during surgery thirty minutes after the completion of the vascular anastomosis (nineteen biopsies), by percutaneous biopsy after the diagnosis of rejection (20 patients, forty-three biopsies), and when there was no evidence of rejection (8 patients, fourteen biopsies), and from six kidneys removed after severe recurrent episodes of rejection. 2 recipients had follow-up biopsies after successful treatment of a rejection crisis. Percutaneous biopsy was done by the technique described by Mathew et al. (1968). Tissue was taken from eight diseased Control " kidneys removed at nephrectomy before grafting. kidney tissue was obtained, immediately after death, from 10 patients without renal disease. Episodes of rejection were diagnosed by an abrupt rise of serum-creatinine of greater than 0-3 mg. per 100 ml., or blood-urea-nitrogen greater than 20 mg. per ml., and were corroborated by the histological changes "

described by Kincaid-Smith (1967).

1314 TABLE I-DEPOSITS IN KIDNEYS IN ALL BIOPSIES

* No. of

patients

shown in italic type.

t

1

patient had hypertension. t4 patients

Renal tissue was examined by immunofluorescence. 2 mm. cubes of renal tissue were placed on a piece of filter-paper in a sealed glass tube and snap-frozen in a bath of solidified carbon dioxide and alcohol. Frozen sections 2 jjt. in thickness were cut in a cryostat at -20°C. Tissue which was not cut immediately was stored for up to two weeks at -85°C after snap-freezing. The frozen sections were washed three times in phosphatebuffered saline (P.B.s.) pH 7-3 and treated for thirty minutes with fluorescein-conjugated rabbit antisera to human IgG, IgA, IgM, complement, fibrin, and albumin, washed twice for thirty minutes in P.B.s. and mounted in 10% P.B.S. in glycerol. Ten sections from rejecting kidneys were tested with fluoresceinconjugated rabbit antisera to 1X.2-macroglobulin. Sections were examined under a Leitz microscope fitted with a source of ultraviolet light. Antisera to human IgG, IgA, and IgM were made by injecting rabbits with the purified immunoglobulins (Whittingham and Mackay 1968) and were conjugated with fluorescein by the method of Wood et al. (1965). Conjugated and nonconjugated antisera to albumin, complement C’3 (antigenically characterised as f31C and its inactivated form PIA), 1X.2-:-macroglobulin, and fibrin were obtained from Australian Hoechst Limited. All reagents gave a single precipitin line when tested by immunoelectrophoresis against human serum (Scheidegger

had

glomerulonephritis.

§ r < 005.

0-05 < P < 0- 10.

detected in the vessels in 21, IgM in 36, IgA in 1, complein 22, and fibrin in 27; none had deposits of albumin or cx2-macroglobulin. Immunoglobulin, complement, and fibrin were found in vessels of all sizes. In peritubular capillaries, venules, arterioles, and afferent arterioles there were fine patchy deposits on the endothelial surface (figs. 1 and 2). In the larger vessels patchy deposits were present on the intimal surface and in deeper layers on the vessel wall (fig. 1). Interlobular vessels which had a thickened intima and narrowed lumen showed patchy staining on the surface and throughout the thickened intima. Deposits of fibrin tended to be more diffuse than deposits of immunoglobulin or complement. Kidneys removed because severe rejection had occurred showed the heaviest and most ment

1955). The specificity of the immunofluorescence reactions was demonstrated by blocking the reaction of fluorescein-conjugated reagents with non-conjugated reagents. Fluorescein-conjugated anti-human albumin and 1X.2-macroglobulin were used to exclude artefacts resulting from non-specific binding of proteins other than antibodies and leakage of plasma proteins from vessels. Fisher’s exact method for 2 x 2 contingency tables was used in tests for statistical significance (Fisher 1934).

Results

1—A specimen from a rejected kidney showing IgM lining t*e intima and deposited in the wall of the arteriole (x 400).

Fig.

CONTROL KIDNEYS

(29 Specimens) The " control " tissue from patients with normal kidneys contained no deposits in vessels or glomeruli. Two of the nineteen specimens taken thirty minutes after completion of the vascular anastomosis had small aggregates of fibrin in the perivascular capillaries, attributable to vascular stasis; immunoglobulins were not present. PATIENTS’

KIDNEYS

(8 Specimens)

patients with glomerulonephritis showed linear deposits in glomeruli of IgG, IgM, complement, and fibrin. 1 patient with malignant hypertension showed deposits of immunoglobulin, complement, fibrin, and albumin in and around vessel walls (table I). 4

ESTABLISHED GRAFTS

(63 Specimens)

Sixty-three sections were examined from two days to twenty-four months after transplantation (table I). Fortynine sections were from kidneys undergoing rejection and fourteen were from kidneys not undergoing rejection. Deposits in Vessels Of the 49

kidneys undergoing rejection, IgG

was

Fig. 2-A grafted kidney showing complement coating the surface of a venule (x 400).

1315 TABLE II-DEPOSITS IN KIDNEYS IN THE FIRST BIOPSY OF THE ESTABLISHED GRAFT

*005
diffuse deposits of immunoglobulin, complement, and fibrin in the vessels. Of the fourteen kidneys not undergoing rejection none had IgG, IgA, albumin, or cc2-macroglobulin in the vessels; and IgM, complement, and fibrin were present in vessels in a significantly smaller proportion than in

kidneys undergoing rejection (table i). In 2 cases tissue was obtained during and after an acute episode of rejection. Deposits of immunoglobulin, complement, and fibrin detected at the time of rejection were no longer demonstrable after the episode had subsided.

Fig. 3-Linear glomerular deposits (reduced to 3/. of x 250).

of

Fig. 4-Granular deposits of IgM in

a

IgG in

a

rejecting kidney

reiecting kidney (

250).

fp<005.

Deposits in Glomeruli Of the

forty-nine specimens examined during rejection, found in glomeruli in sixteen, IgM in twentyIgG seven, complement in eleven, and fibrin in sixteen. None had deposits of IgA, albumin, or cx2-macroglobulin (table i). The deposits were focal in that some glomeruli were spared, as were some capillaries of affected glomeruli. The deposits of IgG, IgM, and complement in capillary loops were both linear (fig. 3) and granular (fig. 4) in appearance. Fibrin was detected in the mesangium and as linear deposits in the capillary loops, and two specimens obtained during acute rejection showed diffuse linear deposits of fibrin in glomeruli. Of the fourteen kidneys not undergoing rejection IgG was detected in the glomeruli of one and IgM in four, but this difference from biopsies of kidneys undergoing rejection was not significant (0-05
Discussion We have found deposits of IgM, IgG, complement, and fibrin in the vessels and glomeruli of human renal allografts ; and the deposits in vessels were present significantly more often in kidneys undergoing rejection than in established grafts. Furthermore, such deposits in vessels were not found in kidneys from patients with no renal disease, nor in tissue taken during the operation, nor, more particularly, in kidneys of patients with glomerulonephritis. We consider that the immunoglobulins and complement in the grafted kidneys represented a specific immune reaction, rather than non-specific deposition of serum-proteins, because albumin and a2-macroglobulin were not demonstrated, and the reaction with fluoresceinconjugated antiglobulin was blocked with non-conjugated antiserum. Deposits of immunoglobulin have been found in vessels in hypertension, especially malignant hypertension (Burkholder 1965), but our patients were not hypertensive after the renal graft. Our finding of immunoglobulins in vessels and glomeruli of human renal allografts is in accord with observations in animals. Horowitz et al. (1965) found y-globulin in the vessels of renal allografts in dogs four

1316

days after transplantation, and Lindquist et al. (1968) found immunoglobulin in the capillaries of renal allogratfs of rats at an early stage. Williams et al. (1967) noted deposits of immunoglobulin in glomeruli and vessels in 17 patients, but there was no association with rejection. Porter et al. (1968) studied seventy-one specimens of renal tissue obtained at biopsy eighteen days to eight years after transplantation, and found both linear and granular deposits of immunoglobulin, complement, and fibrin in glomeruli; immunoglobulins were found in the glomeruli whether or not rejection was occurring. Our finding, that deposits of immunoglobulin, complement, and fibrin in vessels was significantly associated with rejection, suggests that humoral antibody at least contributed to the process of rejection. Hence we suggest, as does Porter (1967), that the interaction of humoral antibody with an antigen in vessels leads to complement fixation, clumping of platelets, and formation of a thrombus with deposition of fibrin; this eventually would cause occlusion of vessels with secondary damage due to ischsemia. In glomerulonephritis deposits of immunoglobulin found by immunofluorescence can be linear, said to indicate a reaction of antibody with glomerular basement membrane antigens (Lerner et al. 1967), or granular, said

deposition of circulating antibody-antigen complexes (Unanue and Dixon 1967). The deposits in the glomeruli of the grafted kidneys in our patients were either granular or linear, as seen in the different types of glomerulonephritis. We cannot state whether the deposits were due to a continuation of the disease which caused the original damage to the recipient’s kidneys, or whether the deposits were due to an allograft reaction. However, the appearance of deposits in both vessels and glomeruli and the association of these deposits with episodes of rejection leads us to suggest that the immune reaction was directed specifically against the allograft. to

AFTER VITAMIN-D THERAPY IN

HYPERPHOSPHATÆMIC RENAL FAILURE N. P. MALLICK M.B., B.Sc. Manc., M.R.C.P. G. M. BERLYNE M.D. Manc., M.R.C.P.

From the

University Department of Medicine, Royal Infirmary, Manchester 13

Vitamin-D treatment caused widespread arterial metastatic calcification in two patients with advanced hyperphosphatæmic chronic renal failure without producing hypercalcæmia. Arterial damage Summary

prevent regular hæmodialysis treatment, and made subsequent renal transplantation difficult. was so severe as to

Vitamin D should only be given to patients with hyperphosphatæmic renal failure provided that steps are simultaneously taken to lower the elevated seruminorganic-phosphorus levels, so that a serum-calcium x phosphorus product of less than 75 is maintained. This can be achieved by dialysis or by oral phosphate-binding agents such as aluminium hydroxide or calcium carbonate.

indicate

We thank Mrs. C. Muschamp for technical assistance, Dr. B. M. Dr. J. Eremin, Dr. J. K. Dawborn, and Dr. T. H. Mathew who did the biopsies, Prof. R. R. H. Lovell and Dr. Ian Mackay for extensively reviewing the manuscript, Dr. J. D. Mathews for help with statistics, and Dr. P. Kincaid-Smith and Mr. Vernon Marshall who allowed us to study patients under their care. 1. F. C. McK. worked with the aid of a grant from the Felton Bequests’ Committee, and S. W. with the aid of a grant from the National Health and Medical Research Council of Australia. Requests for reprints should be addressed to: 1. F. C. McK., University of Melbourne Department of Medicine, Royal Melbourne Hospital, Victoria 3050, Australia.

Saker,

REFERENCES Burkholder, P. M. (1965) Archs Path. 80, 583. Fisher, R. A. (1934) Statistical Methods for Research Workers. Edinburgh. Horowitz, R. E., Burrows, L., Paronetto, F., Dreiling, D., Kark, A. E. (1965) Transplantation, 3, 318. Kincaid-Smith, P. (1967) Lancet, ii, 849. Marshall, V. C., Mathew, T. H., Eremin, J., Brown, R. B., Johnson, N., Lovell, R. R. H., McLeish, D. G., Fairley, K. F., Allock, F. A., Ewing, M. R. (1967) ibid. p. 59. Lerner, R. A., Glassock, R. J., Dixon, F. J. (1967) J. exp. Med. 126, 989. Lindquist R. R., Guttman, R. D., Merrill, J. P. (1958) in Advance in -

Transplantation; p. 1611. Copenhagen. Mathew, T. H., Kincaid-Smith, P., Eremin, J., Marshall, V. C. (1968) Med. J. Aust. i, 6. Morris, P. J., Williams, G. M., Hume, D. M., Mickey, M. R., Terasaki, P. I. (1968) Transplantation, 6, 392. Porter, K. A. (1967) J. clin. Path. 20, suppl. p. 518. Andres, G. A., Calder, M. W., Dossetor, J. B., Hsu, K. C., Rendall, J. M., Seegal, B. C., Starzl, T. E. (1968) Lab. Invest. 18, 159. Scheidegger, J. J. (1955) Int. Archs Allergy, 7, 103. Terasaki, P. I., Thrasher, D. L., Hauber, T. H. (1968) in Advance in Transplantation; p. 255. Copenhagen. Unanue, E. R., Dixon, F. J. (1967) Adv. Immunol. 6, 1. Whittingham, S., Mackay, I. R. (1968) Unpublished. Williams, G. M., Lee, H. M., Weymouth, R. F., Harlan, W. R., Holden, K. R., Stanley, C. M., Millington, G. A., Hume, D. M. (1967) Surgery, St. Louis, 62, 204. Wilson, D. B., Billingham, R. E. (1967) Adv. Immun. 7, 189. Wood, B. T., Thompson, S. H., Goldstein, G. (1965) J. Immun. 95, 225. —

ARTERIAL CALCIFICATION

Introduction TREATMENT with vitamin D of hypocalcxmia in advanced hyperphosphatasmic renal failure is particularly hazardous. There is always the well-recognised risk of further deterioration in renal function if hypercalcxmia develops (Dent et al. 1961, Stanbury 1962). In addition we have become increasingly aware that widespread metastatic arterial calcification may occur without hypercalcaemia. We describe here two cases of arterial metastatic calcification which was so extensive that regular h2emodialysis treatment was made difficult or impossible, and in one of the patients subsequent renal transplantation was made particularly difficult.

We draw attention to the danger of using vitamin D in hyperphosphatsemic renal failure unless a massive rise in the serum calcium x phosphorus product (to greater than 75) is prevented by simultaneously lowering seruminorganic-phosphorus levels by oral phosphate-binding agents such as aluminium hydroxide or calcium carbonate, or

by dialysis. The patients

Materials and Methods attending the renal clinic of

were

one

of

us

(G. M. B.) in Manchester Royal Infirmary, and were later admitted to the professorial medical unit for dialysis, and before

transplantation. Serum calcium, AutoAnalyzer ’.

phosphate, and

urea were

determined by

Case-records Case 1 (table l) A 35-year-old housewife presented with sterile calculous pyelonephritis in April, 1965. She was found to be in advanced renal failure with a urea clearance of 3 ml. per minute, a blood-urea level of 325 mg. per 100 ml., and a serum-creatinine of 14 mg. per 100 ml. The serum-calcium was 6-1 mg. per 100 ml. and the serum-inorganic-phosphorus was 8-8 mg. per 100 ml. Calciferol wasadministered in a dose of 100,000 units a day, and she was discharged home from hospital on a lowprotein regimen (Shaw et al. 1965) and the same dose of calciferol. At this time she had neither X-ray nor clinical evidence of metastatic calcification. After eight months the level of her serum-calcium was found to be 15-7 mg. per 100 ml., her serum-inorganic-phosphorus was 4-0 mg. per 100 ml., and she was vomiting. She was given prednisone, and