Membranous glomerulonephritis mediated by renal tubular epithelial antigen-antibody complex

Membranous glomerulonephritis mediated by renal tubular epithelial antigenantibody complex Takuji Naruse, M.D., Yuzo Miyakawa, and Seiichi Shibata, M.D. Maebashi md

M.D., I’okyo,

Kiyoshi Japa?L

Kitamura,

M.D.,

Autologous renal tubular epithelial antigen was demonstrated together with immunoglobulins and /3,0 along the glomerular capillary walls ,in 4 out of 9 patients with idiqathio m.emlwa.now glqmerulonephritis, and the existence of the clinical entity of glomerulonephritis mediated by tu$ular epithelial antigen-antibody cornplexes was suggested. Althotigh no differences in laboratory data were noted between tubular antigen-positive and -negative groups, clinical remission ocozlp-red only in the negative group. The fact that no remission was observed in the t&s&r antigenpositive group may be explained by the oontilzuow supply of the antigen from endogerwzls sources that maintained the production of immune complemes and subsequent glomerular injury.

Idiopathic membranous glomerulonephritis has attracted increasing attention for its peculiar pathologic findings and clinical features.lS3 The pathogenic role of immune complexes in the development of this disease is strongly suspected, since typical granular deposition of immunoglobulins is unequivocally detected in the glomerular capillary walls by fluorescent antibody techniques. The antigenic factor(s) that might be included in the immune complexes, however, has not yet been identified. Recently, we have demonstrated,4 in 3 casesof idioiathic membranous glomerulonephritis, autologous renal tubular epithelial antigen together with immunoglobulins and plc in the glomerular capillary walls by the immunofluorescent method. Since then, an additional case was studied and clinical features and laboratory data of these patients have been analyzed in detail. The purpose of the present communication is to extend the studies of this new group of membranous glomerulonephritis casesand to report some of their clinical characteristics that may help distinguish them from the other cases of membranous glomerulonephritis. PATIENTS

AND METHODS

Nine patients mere hospitalized and received percutaneous clinical tests. The diagnosis of membranous glomerulonephritis

renal biopsy as well as general was established according to

From the Third Department of Internal Medicine, Faculty of Medicine, University of cfunm~, Maebashi and the Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Tokyo. Received for publication Jan. 2, 1974. Reprint requests to: Takuji Saruse, N.D., The Third Department of lnternal Medicine, Faculty of Medicine, University of Gunma, Maebashi, 371 Japan. Vol.

54, No.

5,

pp. 311-918

312

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fl0. 1. Normal tubular antigen. tubular epithelial

human kidney section Specific fluorescence cells. (x400.)

stained with FITC-conjugated is observed only in the luminal

CLIN. IMMUNOL. NOVEMBER 1974

antibody layer

these immunopathologic criteria: granular immunoglobulin deposits distributed uniformly in the glomerular capillary walls with little or no associated cellular None of these patients had clinicttl or pathologic evidence of coexistent diseases.

light

to of

human proximal

diffusely and proliferation.

microscopy

Renal tissue was fixed in 10 per cent formalin cut at 2 fi and stained with hematoxylin and eosin, acid-silver methenamine (PAM).

lmmunofk4orercence

and embedded in paraffin. periodic acid-&hit? (PAS),

Sections were and period%

micrerctlpy

Renal tissue was embedded in a piece of rat liver and quick-frozen for 2 minutes in n-hexane, which had been precooled in an acetone-dry ice bath. Cryostat sections cut at 4 c were dried in the air and stained with fluorescein isothiocyanate (FITC)-conjugated antisera against human IgG, IgA, IgM, p,c, fibrinogen, and albumin (Behringwerke, Mahrburg Lahn, West Germany), as well as with FITC-conjugated antibodies against human renal tubular epithelial antigen (Tub-Ag) prepared by the method described below. As a co&roJ study, 6 patients with lupus nephritis, 25 patients with chronic glomerulonaphritis, and 9 patients with other miscellaneous diseases were examined.

Prepordon epithelial

of FITC-con-ted antigen

r&bit

o#&dy

Tub-Ag was prepared from normal kidneys lowing the method described by Edgington and Tub-Ag were raised as previously reported.a.After volume of normal human sera, y-globuIin fraction sulfate precipitation method and conjugated with luminal layer of tubular epithelial cells (Fig. 1).

Partial

elution

Cryostat After three anti-Tub-Ag

of u-gb&w&ns

sections washings reagent.

were with

from.

to hunwn

red

t&u&w

removed at autopsy (blood group 0), folassociates,% 0 and the rabbit a&&era to the absorption of the antiseta with a te&h was obtained by the 33 per cent ammotium FITC. This reagent specSally stained the

immttne

incubated in 0.02 M citrate phosphate-buffered saline,

cumptexes buffer (pH 3.2) they were stained

at 37” C. for 2.5 hours. with ~~~~~~~~~

VOLUME

54

Membranous

NUMBER 5

FIG. 2. Renal sections from capillary walls, A, without tion. (PAM stain; x900.)

the patient in Case cellular proliferation.

1. Moderate (PAS stain;

glomerulonephritis

313

thickening of the glomerular x200.) 8, Typical spike forma-

CASE REPORTS Case 1 H. I., a 17-year-old boy, was in good edema. He consulted his physician, who hospital with the diagnosis of nephrotic steroids without significant change, and hospitalization, the patient was referred to 1971.

health until June, 1971, when he developed ankle noted massive proteinuria and admitted him to a syndrome. The patient was treated with corticothis therapy was discontinued. After 3 months of our hospital for further examinations in November,

On admission, he had severe, generalized anasarea. The blood pressure was 118/74 mm. Hg, urinary protein excretion was 15 to 25 Gm. per 24 hours, and the sediment contained 5 red cells and 10 white cells per high-power field, with some fine granular casts. The hemoglobin level was 15 Gm. per 100 ml., red blood cells 490 x 10’ per cubic millimeter, white blood cells 6,800 per cubic millimeter, with a normal differential count. Total serum protein was 4.1 Gm., serum cholesterol 320 mg., and blood urea nitrogen (BUN) 17.1 mg. per 100 ml. Phenol sulfonphthalein (PSP) excretion during the first 15 minutes was 45 per cent and creatinine clearance was determined to be 107 ml. per minute. Percutaneous renal biopsy was performed and a diagnosis of typical membranous glomerulonephritis was made by histologic studies (Fig. 2). Bed rest, low-sodium diet, and 60 mg. of prednisolone daily were started. The edema decreased but the albuminuria persisted. The patient was discharged in June, 1972, and has been followed in our outpatient clinic, with steroids reduced and then discontinued. To the present time, no remission of his clinical state has occurred (Pig. 3).

Case 4 S. H., a 43-year-old housewife, noticed general lassitude and edema in the lower extremities in June, 1971. She was admitted to a hospital and a diagnosis of nephrotic syndrome was made. She received steroid therapy for 2 weeks without any observed effect. She was discharged from the hospital a year later, since her edema disappeared completely with bed rest, low-sodium diet, and administration of diuretics. However, in November, 1972, massive and generalized edema reappeared and she was hospitalized for the second time. Finally, the patient was transferred to our hospital in March, 1973. On admission, the patient had massive edema of the legs. The blood pressure was 140/88 mm. Hg, hemoglobin level 11.1 Gm. per 100 ml., red blood cells 360 x 10’ per cubic millimeter, white blood cells 6,500 per cubic millimeter. Urinalysis showed protein of 1,300 mg. per 100 ml. with 5 red cells and 5 white cells per high-power Aeld and many granular casts. The 24

314

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J. ALLERGY

et al.

cLk-4. h4MuNol. NCWEMBER 1976

I RENAL BIOPSY 5.6

PRosElNg/lObi

CHCN.ES~Sti

270

4.1 320

37 325

41 334

5.4

5.1

427

336

5.6 305

5.4 293

EDEMA

Oct,72Jao.

Apr.

FIG.

3. Clinical

course

findings

of

Jun.

Octi73Jan.

Apr.

Jun.

'71

T&#&E

I. Clinicopathologic

Comparison

Potbent: Core Qm -x

of tubular

positive

9

patients

(Group

A) and

with

1.

membranous

negative

(Group

glomerulonsphritis: B) cases

No.,

Group A: 1. w. I. 17, hi* 2. T. S. 36, F 34.. Group 5. 6. 7. 8. 9.

antigen

of Case

E&. 1 ;3 , R: H. Y. 29, T. N. 33, H. 0. 33, K. X4. 43, H. S. 44,

FF” M F* M* F F

24 ( 6) 16 ( 6) 102 24 (84) (21) 30 16 41 46 21

(23) (14) ( 6) (24) ( 4)

25 10

3.30 5.23

;; 28 20 17 ‘76

4.97 5.60

726 477 361 385

’ ki 1.2 1.5

3+ 3c 3+ 3+

& 2+ 2c

2+ 2+ 2+ 2+

5.02 5.64 5.96 5.80 6.30

452 277 360 353 304

1.2 1.1

3+ 3+ 3+ 3+ 2+

2+ 2+ 2+ + +

--

*Received corticosteroid therapy before renal tValues at the time of renal biopsy are given.

1.0

0.9 1.2

:z No No

No Yes

Yes Yes No

biopsy.

hour urinary excretion of protein was estimated to be 18 Gm. The serum protein was 4,Si’ Gm., total cholesterol 387 mg., and RUN 16 mg. per 100 ml. Creatinine clearance was 71 ml. per minute. One week after admission, percutaneous renal biopsy was performed. Histologic oxamination revealed characteristic immunopathologie flndiugs of membranous glumerubn@uitia, i.e., beadlike deposition of IgG (Fig. 4) and &c. Prednisolone in a daily dose of 46 mg. was given immediately. During 3 months of hospitaliaation, the patient did not show any sign of remission, and the dose of steroid was gradually decreased. She is now visiting our outpatient clinic. Cases 2 and 3 showed essentially the same clinical course aa the two cases presented.

VOLUME NUMBER

FIG. Uniform

54 5

4.

lmmunofluorescent granular staining

Membranous

staining for IgG is observed along

315

glomerulonephritis

of renal specimen from the glomerular capillary

patient walls.

in Case (x400.1

4.

FIG. 5. Kidney specimens of patient in Case 1, A, and Case 4, B, stained with FITCconjugated antibody to human tubular antigen. Uniform granular fluorescence is seen along the glomerular capillary walls in a fashion similar to that of IgG deposition. Specific fluorescence is also found in the tubular epithelial cells in the left of the fields. (x400.)

RESULTS Clinical features and laboratory data of 9 patients with and without Tub-Ag deposits are summarized in Table I. Nephrotic syndrome was manifest at the time of renal biopsy in all cases and persisted through the observation period in 6 cases. Light microscopy examination failed to show any difference between Tub-Ag-negative and positive groups. Moderate thickening of glomerular capillary walls with PAS stain (Fig. 2, A) and typical spike formation with PAM stain (Fig. 2, B) were observed, while the width of capillary lumina was well maintained. In 4 out of 9 cases, the granular deposition of Tub-Ag was demonstrated along the glomerular capillary walls (Fig. 5). The localization of the deposits of Tub-Ag was identical to those of IgG (Fig. 4) and plc by immunofluorescent studies. In Case 9, diffuse and granular staining of IgM was also observed in

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CLIN. IMMUNOL. NOVEMBER 1974

addition to Igcf and &c depositions. IgA, albumin, and fibrinogen were not found in the glomerular capillary walls of all cases. Immunohistologic examination was also carried out in the patients with other diseases.Six patients with lupus nephritis, who showed diffuse granular immunoglobulin and p,c deposits in the glomcrular capillary walls, were negative for Tub-Ag deposition. scithcr the kidney sections from the patients with chronic proliferative glomerulonephritis nor those with the other miscellaneous renal involvement showed any specific staining of Tub-Ag in glomeruli. Elution of deposited immunoglobulins was performed for these cases, as well as for 5 cases of idiopathic membranous glomerulonephritis without Tub-Ag deposits, in an attempt to expose antigenic sit,es to facilitate the subsequent staining by fluorescent anti-Tub-Ag reagent. hut the results were negative in all cases. At the time of renal biopsy, no difference was observed between Tub-&positive and -negative groups in the degree of edema, daily excretion of protein in the urine, and serum protein and cholesterol levels. The findings of light as well as immunofluorescenct: microscopy also failed to reveal any difference between those two groups except the presence of Tub-Ag in the glomerular capillary walls. Corticosteroids were given to all pat.ients except patient 3 in daily doses ranging from 30 to 60 mg. of prednisolone for 1 to 2 months. Remissions were induced in 3 patients in the Tub-Ag-negative group, patients 6 and 8 following st.eroid therapy, and patient 7 spontaneously, and persisted for 1, In, and 13 months, respectively, up to the time of writing. No remission occurred in the Tub-Ag-positive group during the observation period up to 102 months. DISCUSStON Tubular epithelial antigen (Tub-Ag) was prepared from the normal kidneys removed at autopsy, and the antibody was raised in rabbits. Fluoresccinatcd antiTub-Ag thus prepared stained specifically and exclusively the luminal layer of proximal tubular cpithelial cells in normal kidneys. With the use of this reagent, the Tub-Ag was dcmonstratetl together with immunoglobulins and pl(‘, in 4 out of I) patients with idiopathic membranous glomerulonephritis, in granular fashion along the glomerular capillary walls. Immune complexes have been suggested as the causative agents in glomerulonephritis. Some identified antigens, such as deoxyribonucleic acid (DNA) ,? st repto(occus,X malaria,!’ syphilis”’ and Australia” antigens have already been reported as deposits in the glomerular capil1ar.y walls associated with immunoglobulins, resulting in glomerular diseases.l:p to the present time, however, thcrc have been no reports linking autologous tissue antigens with idiopathic membranous glomerulonephritis except for our cases. Heymann,‘? Edgington,” and Classock’” and their co-workers have induced experimental immune complex nephritis in rats by in,jec+on of homologous tubular cpithtlial antigen t>mulsified with Freund’s adjuvant,. Our easesof Tub-&--mediated membranous glomerulonephritis may be regarded as the clinical counterpart of their experimental model. Such pairs of clinical and experimental kidney diseases hare been well

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glomerulonephritis

317

establivhed in (~oodpasture’s syndrome and Masugi nephritis (antiglomerular basement membrane antibody mediated), as well as in lupus nephritis and NZB/NZW mice nephritis (DNA-anti-DNA complex mediated) .I4 Studies of clinical and experimental renal disease are helpful in evaluating the pathogenesis of glomerulonephritis, since the disease process based on the same presumptive mechanism can be investigated simultaneously. Attempts to classify idiopathic membranous glomerulonephritis according to the causative antigen a8 described in this paper would have more nosologic value than depending solely on the histologic observations, and could be useful for better understanding of certain renal diseases. No difference has been noted in laboratory data between Tub-Ag-positive and -negative groups. In their clinical course, however, a definite difference was demonstrated. On the same therapeutic regimen, none of the Tub-Ag-positive patients went into remission within the observation period of 16 to 108 months, while 3 out of 5 Tub-Ag-negative patients did. A high rate of remission has been universally noted in idiopathic membranous glomerulonephritis by many workers.‘“’ lfi The reasons for the different prognosis have not yet been clarified. A continuous supply of antigens seems to be required for the maintenance of immune complex-mediated glomerular injury. Immune complex-mediated nephritis, accompanied by infectious diseases such as streptococcosis, malaria, and syphilis, has been cured when the pathogenic organism disappeared after specific therapy when the source of the antigen for the immune complex disappeared.sy lo In experimental immune complex-mediated nephritis resulting from heterologous protein, repeated injection of antigen is necessary for the chronic development of nephritis. I7 Endogenous antigens such as autologous Tub-Ag would serve as a continuous source of antigens. This may explain our observation that no remissions have occurred in the Tub-Ag-positive group. The findings by Edgington, Lee, and Dixon lx that the autologous tubular antigen-antibody complex nephritis in rats persisted without remission support this view. REFERENCES 1 Bell, E. T.: A clinical and pathological study of subacute and chronic glomerulonephritis, including lipoid nephrosis, Am. J. Pathol. 14: 691, 1938. 2 Jones, D. B. : Nephrotic glomerulonephritis, Am. J. Pathol. 33: 313, 1957. 3 Mellors, R. C., Ortega, L. G., and Holman, H. R.: Role of gamma globulins in pathogenesis of renal lesions in systemic lupus erythematosus and chronic membranous glomerulonephritis, with an observation on the lupus erythematosus cell reaction, J. Exp. Med. 106: 191, 1957. 4 Saruse, ‘I’., Kitamura, K., Miyaknwa, Y., and Shibata, S.: Deposition of renal tubular epithelial antigen along the glomerular oapillary walls of patients with membranous glomerulonephritis, J. Immunol. 110: 1163, 1973. 5 Edgington, T. S., G&sock, R. J., Watson, a. I., and Dixon, F. J.: Characterization and isolation of specific renal tubular epithelial antigens, J. Immunol. 99: 1199, 1967. 6 Edgington, T. S., Glassock, R. J., and Dixon, F. J.: Autologous immune complex nephritis induced with renal tubular antigen. 1. Identification and isolation of the pathogenetic antigen, J. Exp. Med. 127: 555, 1968. 7 Koffler, I)., Agnello, V., Thoburn, R., and Kunkel, H. G.: Systemic lupus erythematosus: Prototype of immune complex nephritis in man, J. Exp. Med. 134: 169s, 1971. 8 Seegal, B. C., Andres, G. A., Hsu, K. C., and Zabrisk, J. R.: Studies on the pathogenesis

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11

12

13

14 15 16 17

18

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CLIN. IMMUNOL. NOVEMBER 1974

of acute and progressive glamerulonephritis in man by immunofluorescein and immunoferritin techniques, Fed. Proe. 24: 100, 1965. for soluble immune complexes in the Ward, P. A., and Kibukamusoke, J. W.: Evidence pathogenesis of the glomerulonephritis of quartan malaria, Lancet 1: 283, 1969. Falls, W. F., Jr., Ford, K. l,., Ashworth, C. T., and Carter, N. W. : Thr ncphrotic: xyndromo in secondary syphilis. Report of a case with renal biopxy findings, Ann. Intern. Med. 63: 1047, 1965. Combes, R., Stastny, I’., Shorey, J., Eigenbrodt, X. H., Harrera, A., Hull, A. X., and Carter, N. W. : Glomerulonephritis with deposition of Australia antigen-antibody complexes in glomerular basement membrane, Lancet 2: 234, 197J. Heymann, W., Hackel, I). H., Earwoods, J., Wilson, S. G. F., and Hunter, J. L. I’. : Production of the nephrotic syndrome in rats by Freund’s adjuvants and rat kidney suspension, Proc. Sot. Exp. Viol. Med. 109: 660, 1959. Glassock, R. J., Edgington, T. S., Watson, J. l., artd Dixon, F. J.: Autologous immune complex nephritis induced with renal tubular antigen. II. The pathogen&e mechanism, J. Exp. Med. 127: 573, 1968. Dixon, F. J. : The pathogenesis of glomorulonephritis, Am. J. Med. 44: 493; 1968. Rastogi, S. I’., Hart-Mercer, J., and Kerr, L). N. S.: Idiopathic membranous glomerdlonephritis in adults: Remission following steroid therapy, Q. J. Med. 39: 335, 1969. Gluek, 51. C., Gallo, G., Lowenstein, J., and Raldwin. 1). S.: Membranous glomerulonephritis. Evaluation of clinical and pathologic features, Ann. Intern. Med. 78: 1, 1973, Dixon, F. J., Feldman, J. I)., and Vazquez, J. J. : Experimental glomerulonephritis. The pathogenesis of a laboratory model resembling the spectrum of human glomerulonephritis, J. Exp. Med. 113: 899, 1961. Edgington, T. S., Lee, S., and Dixon, F. J.: Persistence of the autoimmune pathogenetie process in experimental autologous immune complex nephritis, J. lmmunol. 193: 528, 1969.

The

correct

answers

to questions

in this

Jaurnal

issue

are

as follows:

Qwstion

1 (p. 262):

D,

see freedman, S. 0.: Ciinical imnwno@y, York, 197 1, Harper & Row, Pvb&ers, p. 231.

C&&h

1 (p.

273):

A,

Rae Writer, 2, Boston,

M,, edit% 1971, Little,

Qu&h

3 (p. 287):

C,

see Comroe, go, 1969, 125.

J. H.: Physblogy Year Book Medical

New

Imryn&@cal disawges, d. Brown & comf;rzuvy, p. 887. of respiration, l%bWers,

ChicaInc., p.