Ultrastructural Alterations of the Glomeruli after Prolonged Immunization Correlation with Urinary Excretion of Kidney Specific Antigens in the Rat

Department of Pathology and Laboratory of Immunology (Faculty of Dental Medicine) Hebrew University-Hadassah Medical School Jerusalem, Israel

Ultrastructural Alterations of the Glomeruli after Prolonged Immunization Correlation with Urinary Excretion of Kidney Specific Antigens in the Rat* Ultrastrukturelle Veranderungen der Rattenglomeruli nach langdauernder Immunisation. Korrelation mit der Ausscheidung nierenspezifischer Antigene im Harn E.

ROSENMANN,

T. DISHON, and

With 8 Figures and

I

J. H. Boss

Table· Received February 14, 1973 . Accepted March 9,1973

Summary Kidney specimens of rats injected every 3rd day with 20 mg of BSA for 9 - I 5 months were examined. Immunohistologically, deposition of rat gammaglobulin and avidity for heterologous complement were demonstrated in a mesangial pattern. Mesangial increase was found by light microscopy. Electronmicroscopically, matrix increase and electron dense deposits were noted in the mesangium in a segmental distribution. Subendothelial accumulations of loose coarsely granular material were also observed. Kidney specific antigens were more often detected in the urine during the early phase than in the late phase. Chronic glomerulonephritis did not develop despite prolonged administration of antigen and the renal findings are indicative of sequelae of a glomerulonephritic process. The pathogenesis and significance of these findings are discussed.

A mixed membranous and proliferative glomerulonephritis develops in rats repeatedly injected with a foreign protein for 2 - 8 weeks. Kidney specific antigens are excreted in the urine of these animals (DISHON et aI., 1971).

* This investigation was supported by a grant from David

Nessim Gaon of Geneva.

228 .

E.

ROSENMANN,

T.

DISHON,

and J. H. Boss

The purpose of the present investigation is to correlate the urinary excretion of renal tissue constituents with the structural alterations of the kidneys after exposure to an antigen for periods up to 15 months.

Material and Methods Animals. Albino rats of both sexes of the Hebrew University (Sabra) strain, weighing 100 g at the beginning of the experiment, were used. The animals were given regular chow and water ad libitum. They were housed two to a cage in air-conditioned quarters. Experimental Design. Group I: Fifty normal rats served as controls. Urine specimens were collected every fortnight for a period of 9 -15 months. Every 4 weeks, as of the 9th month, the kidneys of 6 - 8 rats were removed for histological examination. Group 2: Twenty-five rats were injected subcutaneously in the interscapular region with 10 mg of bovine serum albumin* (BSA) in 0.5 ml of saline emulsified in 0.5 mlof Freund's complete adjuvant (Difco). As of the 2nd week, the rats were injected intraperitoneally every 3 rd day with 20 mg of BSA in I ml of saline. The injections were continued till the animals were sacrificed or died, as detailed in Table 1. With few exceptions, urine was collected every fortnight. Preparation and Testing of Kidney Specific Antisera. Immunization of rabbits with rat kidney fractions, absorption procedures and examination of the antisera by immunodiffusion and immunofluorescence microscopy were described in detail previously (RoSENMANN et aI., 1971; DISHON et aI., 1971). The antisera to the renal sediment, particulate and soluble fractions were kidney specific inasmuch as they did not contain precipitating antibodies reacting with components of rat blood, heart, lung, liver and spleen. The antisera to the renal sediment, particulate and soluble fractions contained one, two and three precipitating antibodies, respectively. Absorption of the antisera with each of the three renal fractions disclosed the presence of three kidney specific antibodies. Immunohistologically, the homologous antigens were found to be distributed in the cell membranes, including the brush border, and cytoplasm of the proximal tubular epithelium as well as within the mesangial region of the glomeruli (NAIRN et aI., 1962; ROSENMANN et aI., 1971). Collection and Testing of Urine. Rats were placed in metabolism cages over urine-feces separators and urine was collected for 24 hours; food was withheld but the animals had free access to water. The urine specimens were reacted with each of the three antirenal fraction sera by immunodiffusion in agarose gel. The plates were examined for 3 days, washed in saline, stained with amido black and rechecked. A urine specimen giving one or more precipitation lines with anyone of the antisera was scored positive. Examination of Rat Sera. Blood was drawn from the tail vein at the end of the 4 th month of the experiment. The sera were tested for precipitating anti-BSA antibodies by immunodiffusion. Morphological Studies. Nine of the 25 rats of group 2 died intercurrently, mainly of chronic respiratory disease; their kidneys were not examined histologically since autolysis was evident. The remaining 16 animals were killed at various time intervals (Table I). Samples of the kidneys were fixed in 10% buffered formalin, embedded in paraffin and 6 [L sections were stained with hematoxylin and eosin, periodic acid Schiff and

* Bovine albumin, Cohn fraction V, 96-99% albumin, Sigma Chemical Company.

Glomerular Alterations after Immunization' 229 Masson's trichrome. For electron microscopy, small blocks of kidney tissue were fixed in glutaraldehyde, post-fixed in osmium tetroxide and embedded in epon. Ultrathin sections were stained with uranyl acetate and lead citrate. The sections were examined by a Philips EM 300 electron microscope. Five or more glomeruli were scrutinized in each instance. Cryostat sections of snap frozen specimens of the kidneys from selected cases were prepared for immunofluorescence microscopy. The sections were incubated with rabbit antirat gamma globulin serum in a moist chamber for 30 minutes at room temperature. Following repeated washings, the sections were incubated with goat anti-rabbit gammaglobulin serum conjugated with fluorescein isothiocyanate. Consecutive sections were incubated with fresh human serum, as source of complement, washed, treated with rabbit anti-human complement (beta I C / I A) serum and stained with fluoresceinated horse anti-rabbit gammaglobulin serum. The sections were mounted in buffered glycerol, pH 7.2, and examined with an Ortholux Leitz fluorescence microscope. Appropriate control tests for specificity of the staining were carried out (ULlv!ANSKY et aI., I972).

Results The results of urine examinations, using the three antirenal fraction sera, are presented in Table I. A positive score denotes that at least one urine specimen within the respective month contained kidney specific antigens. All urine samples of the control animals were negative. Renal antigens were found in the urine of 19 of the 25 experimental rats on one or more occasions. It is of note that no antigens were detected in the urine obtained during the I st month of the experiment. Thirteen of the 19 animals excreted the antigens in the course of the 2nd and/or 3 rd month. In the remaining 6 rats a positive urine sample was discovered only after the 3 rd month, in 2 animals as late as the 8 th month. Precipitating anti-BSA antibodies were detected in the sera of all immunized rats. The kidneys of 4 experimental and 4 untreated rats were examined immunohistologically. No specific fluorescce was observed in kidney sections of the control rats. The kidneys of the experimental rats disclosed specific glomerular staining in a focal and segmental distribution, inasmuch as about one third of the glomeruli exhibited specific fluorescence involving portions of the tufts only. The specific fluorescence was coarsely granular and appeared to be located in the centro lobular regions. This pattern of specific staining was identical in sections studied for the deposition of rat gammaglobulin and avidity for heterologous complement. There was no specific staining of the tubules and blood vessels. The appropriate control sections were negative. Light microscopical examination of kidney sections of the control rats revealed no glomerular changes. There were glomerular lesions in the kidneys of 12 of the 16 experimental animals. The majority of the glomeruli were essentially normal. Some glomeruli, however, exhibited diffuse or 16 Beitr. Path. Bd. 149

230 . E. RosENMANN, T. DISHON, and J. H. Boss Table I. Incidence of urinary excretion of kidney-specific antigens in rats subjected to prolonged immunization

2

Month Rat N o.

-\-\-\-

2 }

-\-\-\-

8

12 14 15 16 17

-\-

1I

12

13

14

ND ND ND ND

-\-

D S S S

-\-

-\-

S

D S

-\-

D -\-

-\-

D D -\-

S

S

-\-

ND ND -

S S

-\-

S S -\-

-\-

-\-\-

2}

-\-

15

S

22

25

10

D

n

24

9

D

IS

19 20

8

D -\-

9 [0

I}

-\-\D

7

-\-

4 5 6 7

II

6

4

-\-\-

-\-

S S

-\-

S S

TOTAL 0/21 9/25 8/21 2/ 17 2/ 17 0/ 17 2/17 2/ 17 1/ 15 0/ 13 1/ 12 1/ 10 1/4 0/2 ND = Not done, D = Died, S = Sacrificed

segmental widening of the mesangial regions (Fig. I) with rare capsulotuftal adhesions, while others were segmentally sclerotic. Many hyaline droplets and vacuoles containing a brownish granular pigment were noticed in the proximal tubular epithelium. Slight focal interstitial in@trations of mononuclear cells were found in sections of the control and experimental animals. The blood vessels were normal. The ultrastructural changes of the glomeruli were segmental in distribution. The mesangial areas of the involved lobules were widened due to matrix increase and in occasional instances due to cellular proliferation (Fig.

Glomerular Alterations after Immunization . 23 1

Fig. I. Glomerulus of rat treated with BSA for increase. Periodic acid Schiff technique. X 520.

I I

months. Note slight diffuse mesangial

¥ .

Fig. 2. Electron micrograph of mesangial area showing matrix increase and hypercellularity. MM - mesangial matrix, MC - mesangial cell, En - endothelial cell, Ep - epithelial cell, CL - capillary lumen, BC - Bowman's capsule. X 6,380.

23 2 . E.

ROSENMANN,

T.

DISHON,

and J. H. Boss

Fig. 3. Irregular thickening of the lamina densa of the peripheral basement membrane. LD -lamina densa, CL - capillary lumen. X 41,040.

2). The peripheral basement membrane was irregularly thickened (Fig. 3). Dome-shaped formations of the lamina densa, projecting towards the epithelial celis, were noticed in some capillaries (Fig. 4). A few of these structures were capped by a material of higher electron density. Some visceral epithelial cells were swollen. The foot processes were in general discretely applied to the basement membrane, though occasionally they were flattened, widened or fused. Subendothelial deposits were conspicuous in most glomeruli and found in two distinct locations. Firstly, finely granular electrondense deposits occurred beneath the basement membrane of the mesangial regions as well as among branches of the basement membrane of the me sangium (Fig. 5, 6). Processes of the mesangial cell cytoplasm oftentimes pro-

Glomerular Alterations after Immunization . 233

Fig. 4- Electron micrograph of a subephithelial dome-shaped projection. Note that upper portion of this structure consists of finely granular electron dense material (arrow). X 25,080.

truded into the deposits, extending to the lamina densa (Fig. 6). The depossits varied considerably in size. Some of them were of large dimension, presenting a nodular appearance (Fig. 7). Secondly, coarse granular deposits were noted at the periphery of the capillary loops, where they caused segmental thickening of the wall (Fig. 8). The granules, which were loosely arranged, were either electron-dense or of basement membrane-like density. The deposits were generally bordered on their luminal aspect by a split-off or newly formed delicate layer of lamina densa (Fig. 8). Infrequently, the deposits were directly covered by the lamina fenestrata of the endothelial cytoplasm. Examination of kidney sections of the control rats revealed no pathological alterations.

234' E. ROSENMANN, T. DISHON, and J. H . Boss

Fig. 5. Mesangial area with marked matrix increase and finely granular electron dense deposits (arrows). X 20,520.

Discussion Bovine serum albumin, in comparatively large amounts, was injected intraperitoneally into rats every 3 rd day for periods up to 15 months. Chronic glomerulonephritis failed to develop, confirming the findings of BOYNS and HARDWICKE (1968) in rabbits given BSA in a schedule designed to preclude the presence of free circulating antibody at any time. That the repeated administration of BSA results in the formation of circulating soluble immune complexes (GERMUTH, 1967) and in their trapping in the glomeruli, is attested to, in our animals, by the immunohistological demonstration of rat gamma globulin and avidity for heterologous complement (BURKHOLDER, 1961). The latter is indicative of deposition of antigen-antibody complexes in the glomeruli. Dixon and his associates (1961) have shown that chronic glomerulonephritis develops during prolonged immunization if the relationship of antibody produced to antigen injected is such that

Glomerular Alterations after Immunization . 235

Fig. 6. Electron micrograph of mesangial region with large, confluent, finely granular electron dense deposits (Dep) located between basement membrane and mesangial cell cytoplasm (MC). Note the cytoplasmic processes (arrows) of the mesangial cell extending into the deposits. X Z 5,080.

low levels of free antibody or antigen are constantly present in the circulation. According to GERMUTH et al. (1967), rabbits receiving large daily doses of BSA develop progressive obliterative glomerulonephritis, whereas those given small amounts of antigen present with membranous glomerulonephritis. In our experiments, local glomerular lesions, hitherto not reported under similar experimental conditions, have been observed. The morphological alterations were easily recognizable by immunofluorescence and electron microscopy, but were rather inconspicuous by light microscopy. Specific fluorescence in sections stained for gammalobulin and studied for comlemcnt avidity was localized to mesangial regions.

236 . E. ROSENMANN, T. DISHON, and

J. H. Boss

Fig. 7. Large nodular electron dense deposit adjacent to the mesangial area. Note fuonsi of foot processes (arrow heads). BM - basement membrane, BS - Bowman's space, Dep deposit, Mes - mesangium, End - endothelial cell, CL - capillary lumen. X 9,120.

The main ultrastructural alterations were mesangial and subendothelial deposits, mesangial matrix increase and irregular thickening of the peripheral basement membrane. Attention should be drawn to the fact that while most of the glomerular structure was intact, the lesions, though observed in practically every tuft, were scattered and involved small portions of the capillary walls and stalks only. It is of note that there were no subepithelial humps. We believe that the described findings represent, for the most part, sequelae ofa previously active inflammatory process, namely glomerulonephritis of chronic serum sickness, which mostly resolved in spite of continued administration of the foreign protein. Moreover, it is thought that immune complexes deposited in the mesangium do not damage the glomeruli (KOFFLER et aI., 1971). Subendothelial expansions and scallopings consisting of loosely arranged granular osmiophilic material, similar to those observed in our animals, have been described in rats injected with turpentine, HCl, carrageenan or lysates of rabbit granulocytes (MANALIGOD et aI., 1969). They are ascribed

Glomerular Alterations after Immunization . 237

Fig. 8. Electron micrograph of portion of capillary showing a subendothelial, loosely arranged, coarsely granular, electron dense material (Dep) bordered on its endothelial aspect by a thin layer of lamina dense-like material (arrow). LD -lamina densa, CL - capillary lumen. X 31,920.

to the injurious effects of granuloytic lysosomal enzymes on the glomerular basement membrane. Small, granular, poorly delineated, electron-dense subendothelial deposits are observed 6 - 12 hours after injection of nephrotoxic serum and are accepted to be characteristic of the heterologous phase of nephrotoxic nephritis (UNANUE and DIXON, 1967; BATTIFORA and MARKOWITZ, 1969). Newly deposited dense material appears in abundance in subendothelial location during the autologous phase and progressively increases in amount up to 4 weeks after the injection. The nature and significance of the subendothelial lesion are controversial; it occurs in many glomerular diseases, not necessarily related to immune mechanisms (LEWY et aI., 1971).

23 8 . E. ROSENMANN, T. DISHON, and J. H. Boss Subendothelial deposits are also observed in rats with glomerulonephritis following sensitization with homologous glomerular basement membrane (SHIBATA et ai., 1971). Whereas the subendothelial lesions are of common occurrence, the electron-dense deposits in the mesangial regions, as described herein, are only rarely observed. They have been described in glomerulonephritis induced in rats by sensitization with homologous glomerular basement membrane (SHIBATA et aI., 1971) and in certain human diseases, namely, Berger's disease, anaphylactoid purpura and lupus nephritis (MOREL-MAROGER ct ai., 1968; URIZAR et ai., 1968; CHURG and GRISHMAN, 1972). In view of the findings in the experimental model described herein, would it be possible to attribute the mesangial deposits in the glomeruli in these conditions to prolonged and continuous immunological insult of a sensitized host? The dome shaped subepithelial projections and the irregular thickening of the basement membrane appear to be due to incorporation of humps into the basement membrane and replacement by basement membrane-like materiai. Subepithelial electron-dense deposits were not observed in the glomeruli of our rats, which were examined between the 9th and 15th month of the experiment. It is well known that subepithelial deposits occur in acute serum sickness nephritis (FISH et ai., 1966). In the chronic form of the disease, the deposits are small and more confined to the substance of the basement membrane (DIXON et ai., 1961). Electron-dense mesangial deposits, similar to those demonstrated herein, are ascribed to phagocytic activity of the mesangial cells and accompany subepithelial humps in immune complex glomerulonephritis (LEWY et aI., 1971). Mesangial matrix increase is a non-specific lesion reflecting a reaction to a variety of injuries. In our rats, it is probably the result of deposition of immune complexes in these areas, corresponding to the specifically fluorescing sites in sections stained for rat gammaglobulin and reacted for complement avidity. Urinary excretion of kidney specific antigens emanating from the proximal tubular epithelium, is a reliable indicator of renal damage (ROSENMANN et ai., 1971; Boss et aI., 1973). In previous experiments, in which urine specimens were tested at close intervals during the early phase of chronic serum sickness nephritis, kidney antigens were detected in most samples obtained after a few weeks of beginning the immunization procedure (DISHON et ai., 1971), an observation confirmed in the present investigation. A significant decline in the incidence of positive urinary findings is noticed as of the 4 th month (Table I), compatible with the fact that chronic glomerulonephritis did not develop. The occasional demonstration of antigens in the urine may indicate isolated episodes of immune insult to the kidney. It seems that glomerular nephritic lesions resolved leaving behind histological alterations

Glomerular Alterations after Immunization . 239

believed to represent sequelae. Our findings can best be explained in the light of previous studies showing that rabbits lose the ability to synthesize specific antibodies when repeated injections of antigen result in continuous circulation of soluble complexes; however, the ensuing tolerant state is not complete as small amounts of antibody are still made (BOYNS and HARDWICKE, 1968b). We suggest, therefore, that a similar state of tolerance is responsible for the lack of progression towards chronic glomerulonephritis in our model. Apparently, it is this incompleteness of tolerance which permits occult injury to the kidneys.

Zusammenfassung Es wurden Nierenschnitte von Ratten untersucht, welche wahrend 9 - I 5 Monaten jeden 3. Tag 20 mg BSA erhalten hatten. Immunhistologisch konnten Ablagerungen von Ratten-Gammaglobulin und Aviditat fUr heterologes Komplement mesangial demonstriert werden. Eine mesangiale Verbreiterung wurde lichtmikroskopisch gefunden. Elektronenmikroskopisch konnten eine Vermehrung der Matrix sowie elektronendichte Ablagerungen im Mesangium in segmentaler Verteilung beobachtet werden. Daneben bestanden subendotheliale Ansammlungen von lockerem grobgranularem Material. Nierenspezifische Antigene konnten im Urin nachgewiesen werden, haufiger in der FrUhphase als in den Spatphasen. Eine chronische Glomerulonephritis hat sich nicht entwickelt trotz prolongierter Verabreichung des Antigens und die morphologischen Veranderungen deuten auf Seguolen eines abgelaufenen glomerulonephritis chen ProzeB. Die Pathogenese und die Bedeutung dieser Befunde werden diskutiert.

References BATTIFORA, H. A. and MARKOWITZ, A. S.: Amer. J. Path. f f, 267 (1969) Boss, J. H., DISHON, T., DURST, A., and ROSENMANN, E.: Israel ]. med. Sci. 9, 490 (1973) BOYNS, A. R. and HARDWICKE, J.: Immunology I4, 367 (1968 a) BOYNS,A. R. and HARDWICKE, J.: Immunology If, 263 (1968 b) BURKHOLDER, P. M.: J. expo Med. II 4,605 (1961) CHURG,]. and GRISHMAN, E. Ann. intern. Med. 16,479 (1972) DISHON, T., ROSENMANN, E., DURST, A., and Boss,]. H.: Exp. molec. Path. If, 126 (1971) DIXON, F.]., FELDMAN, J.D., and VASQUEZ, J.J.: J. expo Med. II}, 899 (1961) FISH, A. J., MICHAEL, A. F., VERNIER, R. J., and GOOD, R. A.: Amer. J. Path. 46, 997 (1966) GERMUTH jr., F. G., SENTERFIT, L. B., and POLLACK, A. D.: Johns Hopk. Med. J. I20, 225 (1967) KOFFLER, D., AGNELLO, V., THOBURN, R., and KUNKEL, H. G.: J. expo Med. I}4, 169S (197 1) LEWY, J. E., SALINAS-MADRIGAL, L., HERDSON, P. B., PI RANI, C. L., and METcoFF, J.: Medicine f O, 453 (197 1)

240 . E. RosENMANN, T. DISHON, and J. H. Boss

MANALIGOD, ]. R., KRAKOWER, C A., and GREENSPON, S. A.: Amer. ]. Path. 1 6, 533 (19 69) MOREL-MAROGER, L., KRAMP, R., LERoux-RoBERT, C, VERGER, D., and RICHET, G.: Presse Med. 76, 559 (1968) NAIRN, R. C, GHOSE, T., FOTHERGILL,]. E., and McENTEGART, M. G.: Nature 196, 385 ( 19 62 ) ROSENMANN, E., DISHON, T., DURST, A., and Boss, J. H.: Brit.]. expo Path. 12, 388 (1971) SHIBATA, S., NAGASAWA, T., MIYAKAWA, Y., and NARUSE, T.: ]. Immunol. 106, 1284 (197 1) ULMANSKY, M., SELA, J., DISHON, T., ROSENMANN, E., and Boss, ]. H.: Israel J. med. Sci. 8, 1971 (1972) UNANUE, E. R. and DIXON, F.].: Advanc. Immunol. 6, I (1967) URIZAR, R. E., MICHAEL, A. F., SISSON, S. P., and VERNIER, R. L.: Lab. Invest. 19,437 (19 68) Dr. E. ROSENMANN, Department of Pathology, Hebrew University-Hadassah Medical School Dr. T. DISHON, Laboratory of Immunology and Virulogy, Faculty of Dental Medicine, Alpha Omega Research and Postgraduate Center, Hebrew University Professor J. H. Boss, Department of Pathology, Hebrew University-Hadassah Medi, cal School] erusalem, Israel