Clin. exp. Immunol. (1978) 33,463-473.
Autoimmune glomerulotubular nephropathy in mice W. K. BOLTON, F. R. BENTON & B. C. STURGILL The Departments of Internal Medicine and Pathology, University of Virginia School ofMedicine, Charlottesville, Virginia, USA
(Received 7 March 1978)
SUMMARY
We produced an autoimmune glomerulotubular nephropathy in Swiss-Webster mice using human glomerular antigen in Freund's complete adjuvant. The disease is associated with circulating antibody to both mouse and human glomerular basement membranes (GBM) and tubular basement membranes (TBM). All mouse IgG subgroups are deposited initially in a linear pattern along the GBM and TBM. IgG deposition remains linear, while that of the other subgroups assumes a granular GBM pattern with continued linear TBM deposits. Despite tissue deposition of antibody capable of C-3 fixation, no C-3 is found in vivo along the GBM or TBM, nor is there C-3 fixation in vitro. This appears to be related to spatial limitations of IgG molecule attachment to basement membranes. A unique ultrastructural lesion of the GBM developed, characterized by periodic expansions of the lamina rara externa to form a beaded pattern. Eluate of nephritic kidneys contained all subgroups of IgG, but mainly IgG1 fixed in vitro to mouse kidney and in vivo when injected intravenously into normal mice. Fixation of other IgG subgroups in vivo may have resulted from antibody formation to abnormally formed GBM, thereby accounting for the peculiar ultrastructural findings and tissue fixation characteristics of the eluted immunoglobulin. Abnormal proteinuria without glycosuria or lysozymuria developed in test animals as compared to controls. Our model is similar in certain aspects to previously described models of Steblay nephritis, but differs because of the total involvement of TBMs, unique ultrastructural lesions, and dissimilarity to other reports ofthis model in mice.
INTRODUCTION Experimental autoimmune antiglomerular basement membrane (GBM) glomerulonephritis (Steblay nephritis) induced by immunization with heterologous GBM antigen has been described in sheep, rats, monkeys, goats, rabbits, guinea-pigs and mice (Steblay, 1962; Steblay, 1963; Williams & Steblay, 1965; Steblay & Rudofsky, 1966; Couser, Stilmant & Lewis, 1973; Avasthi et al., 1971; Unanue & Dixon 1967a; Lerner & Dixon, 1968). The nephritis may be severe, with marked golmerular proliferation and rapid death induced by renal failure (Steblay, 1962; Williams & Steblay, 1965), or mild, without significant proliferation or renal dysfunction (Steblay, 1963; Couser et a., 1973). Host antibody is invariably deposited in a linear pattern along the GBM, and some investigators ha-ve noted occasional linear tubular basement membrane (TBM) deposits (Couser et al., 1973; Avasthi et al., 1971). Complement may play a major role in some of these types of autoimmune nephritis (Unanue & Dixon, 1967b), but other types appear to be complement-independent (Couser et al., 1973). Mice immunized with canine GBM develop a severe necrotizing nephritis with perivascular and interstitial infiltrates resembling those Correspondence: Dr W. K. Bolton, Box 133, Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22901, USA. 0099-9104178/0900-463$02.00 (© 1978 Blackwell Scientific Publications
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accompanying transplantation rejection (Avasthi et al., 1971). No data are available about complement fixation in the mouse. We immunized Swiss-Webster mice with human GBM, and produced an autoimmune glomerulotubular nephropathy with features different from those noted in the other models cited above. The disease involved non-complement-fixing anti-GBM deposits and diffuse linear antibody deposition on all TBMs. In contrast to the mouse model previously described (Avasthi et al., 1971), our model was unremarkable for proliferative or necrotizing nephritis, or for any type of cellular infiltrate. In addition, the mice in our study developed a unique ultrastructural glomerular lesion. MATERIALS AND METHOD Animals. Male and female Swiss-Webster mice weighing 25-30 g each were used in our study and were housed under standard conditions in the vivarium. Immunization. Human kidneys were kept frozen at -70'C until they were to be used; they were then thawed at 4VC. The cortices were separated from the medullae, minced, and used to isolate glomeruli by the method of Krakower & Greenspon (1951). We washed the sediment repeatedly in phosphate buffered saline (PBS), pH 7-3, until it contained less than 5% tubules. We then washed the glomeruli in deionized water, lyophilized them and stored them in dessicated form. Preparatory to use in immunization, the glomeruli were suspended in PBS and mixed with an equal amount of Freund's complete adjuvant to deliver 0-75 mg glomeruli dry weight per mouse in 0-15 ml volume. This inoculum was given to mice in the foot pads and subcutaneously as the initial injection; animals were injected subcutaneously thereafter every 2 weeks until the experiment's termination. Control animals received only PBS in Freund's complete adjuvant at 2 week intervals. Protein excretion, blood urea nitrogen (BUN) and tubular function evaluation. Animals were periodically placed in metabolic cages for 24 hr urine collections. Urine was tested for blood, ketones, and glucose with Ames Multistix and the protein excretion was determined by means of 3% sulfosalicylic acid with a bovine serum albumin standard (Davidson & Henry, 1969). Lysozymuria was quantified by radial diffusion with lysozyme Quantiplates (Kallestad Laboratories). We killed the animals serially to obtain blood samples for routine biochemical analysis of BUN and tissue specimens for examination. Immunofluorescence, light and electron microscopy. Renal tissue taken from mice at autopsy or open biopsy was divided into three portions. One specimen was snap-frozen in dry ice-isopentane, another fixed in buffered neutral formalin, and the third fixed in 2% gluteraldehyde, post-fixed in 2% osmium tetroxide, and serially dehydrated in ethanol. Thin sections of specimens embedded in Araldite (Ladd Co.) were stained with lead citrate and examined with a Zeiss electron microscope. We used the direct technique to examine frozen tissue with monospecific rhodamine-labelled rabbit antimouse IgG and fluorescein-labelled antisera (Cappel Laboratory) to mouse C-3, IgM, fibrinogen, and albumin (Bolton et al., 1976). Staining intensity was graded 0 to 4+ and the staining patterns classified as mesangial, GBM, TBM, granular or linear. Selected kidneys from mice with linear GBM or GBM plus TBM deposits of IgG were examined by indirect fluorescence techniques to determine the subtype of IgG deposited. Monospecific rabbit antimouse IgG1, IgG2A, IgG2B, and IgG3 (Miles Laboratory) were layered onto test kidney sections. After washing in PBS, fluorescein-labelled anti-rabbit IgG, previously absorbed with mouse IgG linked to Sepharose, was applied. We recorded the pattern, intensity and distribution of the IgG subgroups for each animal, doing so without knowledge of the clinical or immunization state of the animal. Kidney tissue fixed in buffered formalin was embedded in paraffin and cut serially at 3 gm and stained with haematoxylin and eosin (H&E), periodic acidSchiff reagent (PAS), and colloidal iron. The sections were examined for incidence of proliferation, crescents, interstitial infiltrates, tubular atrophy, arteritis, or casts, without knowledge of the study group from which the tissue samples were taken. Circulating anti-GBM antibodies. Sera selected at random from test and control mice were serially diluted with PBS and layered onto normal mouse and human kidney sections. We applied unlabelled rabbit antimouse IgG1 antiserum and followed this with fluorescein-labelled goat anti-rabbit IgG previously absorbed by mouse IgG linked to Sepharose. Titres were calculated as the reciprocals of the dilutions. Certain sera containing anti-GBM antibody were tested for complement fixation using normal mouse and human kidney. Elution studies. Kidneys of mice with tissue-proven IgG deposits were minced, washed in PBS, and eluted with citric acid-citrate buffer, 0-02 M, pH 3-2 (Bolton et al., 1976; McPhaul & Dixon, 1970). The eluate was neutralized and tested by indirect fluorescence against normal mouse kidney, muscle, liver, lung and heart. We gave an intravenous injection of 0- 1 ml ofeluate to normal mice; these mice were killed 24 hr later. Complement fixation studies. We assessed in vivo fixation of complement (C-3) by examining kidney tissue with fluoresceinated antimouse C-3. In vitro tests of complement fixation were performed as described by Burkholder (1961), using normal human serum and fluorescein-labelled goat antihuman C-3. We used positive controls with antinuclear antibody containing serum and negative controls with heat-inactivated serum. Colloidal carbon studies. Selected test and control animals were given 70 mg/100 g body weight of colloidal carbon (C 11/ 1431 A, Gunther-Wagner, W. Germany) with heparin 50 iu/ml intravenously 24 hr prior to being killed; any alteration of mesangial function induced by the anti-GBM antibodies was then noted. Animals examined in this way had received alternateweek immunizations over a period of 25 weeks. Tissue sections were examined for the amount and distribution of carbon as described by Hoyer, Elema & Vernier (1976).
Autoimmune nephropathy in mice
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RESULTS Animals Thirty-four male and thirty-two female mice were immunized in three different experiments. Fourteen mice of each sex served as controls and received Freund's adjuvant alone. The mice had received a total amount of up to 9 mg of antigen by the end of the study. All mice tolerated the injections well; only a few developed abscesses and none failed to thrive or died as a result of immunizations. Animals in the first two series were killed serially every 2 to 4 weeks. Subsets of mice in the third series were biopsied every week for the first 8 weeks. Protein excretion, BUN and tubular function Baseline protein excretion rates of male mice, antigen recipients and controls, were similar (Fig. 1), as were the baselines of the female mice. Proteinuria in males was much greater than in females and was more variable. Proteinuria increased over time, both in test and control male mice but, by the fifth week, the protein excretion rates of test animals were significantly greater than those of the age-matched controls (P< 0.05). Abnormal proteinuria was not detected in female mice by the fifteenth week. BUN values revealed little difference between test and control mice, male or female. Multistix evaluations of urines were normal other than those indicating proteinuria, and no differences in lysozymuria were detected.
Immunofluorescence microscopy Direct. In the first 4 weeks after the initial immunization, normal mice, control animals and test animals demonstrated only granular mesangial deposits of IgM, IgG and, occasionally, C-3. By the fourth week, the antigen recipients had developed fine trace linear deposits of IgG alone the GBM (Fig. 2a). With time these deposits showed an increase in staining intensity and they gradually assumed a linear-granular appearance. Later, as the nephropathy progressed in these test animals, GBM deposits were shown by staining to be primarily granular and only slightly linear. As staining intensity increased with time, thickening of the GBM was also noted. In older animals, the GBM seemed stiff, with open lumina and a wire loop pattern (Fig. 2b). In the first weeks after GBM deposits were detectable, no IgG was present on the TBMs; however, distinct linear IgG deposition subsequently occurred, increasing in intensity with time (Fig. 2c). In contrast to the GBM deposits, IgG deposited on the TBM never
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FIG. 1. Graph of 24 hr urinary protein excretion in male mice. Test animals (-) received GBM in Freund's complete adjuvant, while controls (Z) received only Freund's complete adjuvant. Test animals excreted significantly more protein than controls after the fourth week. (*) P< 0 05.
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&
B. C. Sturgill
FIG. 2. Immunofluorescent findings. (a) Photomicrograph demonstrating fine linear deposits of mouse IgG1 along the GBM, from an animal 7 weeks after immunization with GBM. Rabbit antimouse IgG1/fluoresceinlabelled goat anti-rabbit IgG (magnification x 1660). (b) Kidney section from a test mouse showing intense staining for IgG along the GBM and TBM, thickening of the basement membranes, and granularity of the GBM with convolution of the TBM. From a mouse after 15 weeks of immunization. Rhodamine-labelled rabbit anti-mouse IgG (magnification x 620). (c) Section of test mouse kidney showing linear deposits of mouse IgG along the TBM (magnification x 640). (d) Section of kidney from a control mouse injected every 2 weeks with Freund's complete adjuvant for 25 weeks stained with anti-mouse IgG. Heavy granular mesangial deposits and trace segmental granular GBM deposits are present, but no anti-GBM antibody is detectable (magnification x 700). (e) Photomicrograph illustrating discrete beads (right-hand arrow) continuous with the GBM (left-hand arrow). From an animal after 21 weeks of immunization. Rabbit antimouse IgG2A/fluoresceinated goat anti-rabbit IgG (magnification x 1620). (f) Mouse kidney from the test animal in (b) demonstrating fixation of human C-3 by mesangial deposits only. Stained with fluoresceinated rabbit anti-human C-3
(magnification
x
800).
Autoimmune nephropathy in mice
467
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FIG. 3. Distribution of IgG subgroups in GBM deposits as a function of length of immunization. Only animals showing distinctly linear and/or granular deposits are noted. Other mice with a mixture of these two patterns or a fragmented linear GBM pattern are not presented in the graph. ( 0) Negative; (0) linear and (A) granular.
modified from a linear to a primarily granular pattern, but the TBM did thicken and become convoluted as the nephropathy progressed (Fig. 2b). C-3 deposition was usually confined to the mesangium, with only an occasional animal showing trace diffuse linear or focal segmental linear GBM deposits. Complement was not detected on the TBM. IgM was not found along either the GBM or TBM, and fibrin appeared only in rare segmental deposits in areas of sclerosis. Older control mice had heavy deposits of lgG, IgM, and C-3 in the mesangium and, occasionally, slight granular GBM deposits; this pattern of deposition (Fig. 2d) did not differ from that of age-matched normal animals. Indirect. The kidneys of forty test mice were studied for the presence and distribution of IgG subgroups from 1 to 25 weeks after the beginning of immunizations (Fig. 3). Sections of kidneys from mice immunized for 6 or more weeks were read without knowledge of the duration of immunizations. IgG was not detected in mice during the first 3 weeks after immunization. By the fourth week, IgG1, deposited in a lacy, linear pattern along the GBM, was detectable in 75% of the mice; no other subgroup was present. By the fifth week, the other subgroups were detectable in half of the biopsied mice. Thereafter, all IgG subgroups were found in animals with deposits. The pattern of staining was always distinctly linear in the first 4 weeks, but thereafter it began to assume a granular pattern that roughly correlated with the specific subgroup (Fig. 2e). IgG3 showed the greatest propensity to assume a granular pattern, with two thirds of the animals immunized for 10 weeks or more having granular IgG3. IgG2B showed an essentially similar pattern, while IgG2A remained in a linear pattern until 18 weeks. In contrast to the other subgroups, IgG1 maintained the linear pattern of deposition in most animals, even when the other subgroups were found in distinctly granular patterns. Fig. 3 depicts only those animals with wholly linear or distinctly granular deposits. Approximately half of the kidneys examined had a fragmented linear GBM pattern with characteristics of both linear and granular immunoglobulin. All of the IgG subgroups were present on all TBMs in a wholly linear pattern of slightly less staining intensity than GBM deposits; they never became granular, regardless of the corresponding GBM patterns.
Histopathology Examination of serial sections by H&E, PAS and colloidal iron revealed only minimal histological alterations during the first 6 to 10 weeks after immunization. Thereafter, a spectrum of changes was noted in immunized animals. Approximately a third of the test mice showed some degree of thickening of capillary walls by 20 weeks and half by 25 weeks. Sometimes this was striking and characterized
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W. K. Bolton, F. R. Benton & B. C. Sturgill
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FIG. 4. (a) Glomerulus from an animal 25 weeks after immunization illustrating thick, beaded capillary walls and absence of proliferation. PAS (Magnification x 250). (b) Glomerulus from an animal 16 weeks after immunization showing proliferation of Bowman's capsular epithelium. H&E (Magnification x 250).
by a beaded appearance suggestive of the lesion seen in idiopathic membranous glomerulopathy in humans (Fig. 4a). These changes were generally associated with a comparable degree of tubular atrophy and hyaline tubular casts. No significant interstitial or perivascular infiltrates were seen. Two mice that received GBM antigen had a proliferative nephritis (Fig. 4b). Colloidal iron staining was not diminished until late in the immunization period and was not consistently related to histological abnormalities, being slightly diminished in some histologically normal glomeruli, and normal in some histologically abnormal glomeruli. There was also no apparent correlation between colloidal iron staining and glomerular ultrastructure. Colloidal carbon studies Six test animals and six controls received colloidal carbon. The test animals had heavy deposits of IgG along the GBM, but there were no detectable differences between test and control animals with regard to mesangial carbon uptake as judged by double-blind analysis. Electron microscopy The glomeruli of antigen-recipient animals were ultrastructurally normal for the first 6 to 10 weeks after immunization with GBM began. Thereafter, the GBM gradually thickened, although all layers were quite distinct and of normal density. Within 12 to 15 weeks, the GBM had definitely thickened and developed structural irregularities. These changes appeared to result from progressive alterations in the lamina rara externa, which developed a periodic beading contiguous to normal areas. Regions of increased lamina rara were indistinguishable from normal basement membrane and did not involve the lamina densa. Foot process obliteration and condensation of epithelial cell cytoplasm occurred over the extrusions. As the lesion developed, it assumed a characteristic string-bead appearance which affected the length of any GBM region (Figs 5 and 6). The masses, separated by fingers of epithelial cell cytoplasm, continually enlarged. At all times the subepithelial masses appeared to have the same density and homogeneity as the lamina rara externa. The lamina rara interna and lamina densa remained normal and there were no alterations of endothelial cells. Only occasional electron-dense deposits were seen in the mesangium of some animals. The TBMs also showed a similar, albeit less striking, beading on the luminal side. We found no fibrin, collagen or inflammatory cells.
Circulating anti-GBM-TBM antibody Sera taken from animals at various times after immunizations began were tested as described above.
Autoimmune nephropathy in mice
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FIG. 5. Electron micrograph of glomerulus from an animal 25 weeks post-immunization demonstrating extensive beading of the GBM. CL = capillary lumen; US = urinary space. (Magnification x 2550).
Two thirds of the animals had circulating antibody to GBM and TBM, with titres ranging from 4 to 32 Higher titres of anti-GBM-TBM were recorded when normal human kidney was used as the substrate. Antinuclear factor was detected in one third of the animals, and was associated with a low titre or undetectable antibody to GBM and TBM.
Elution studies The eluate concentrated to 1*0 mg/ml protein contained IgG which bound selectively to normal mouse GBM and TBM, but not to heart, muscle, liver or lung. Kidneys of normal mice given 0 1 ml of the eluate intravenously showed linear staining along the GBM and TBM. IgG1 was found in the greatest quantities, with IgG2A, IgG2B and IgG3 detected in trace amounts only along the GBM. Immunoglobulin eluted from mouse kidneys fixed slightly to human GBM and TBM
Cornplementfixation studies Mouse C-3 was detectable in vivo in a mesangial pattern, but only rarely in a linear GBM distribution. Two to 25 weeks after immunizations began, kidneys were examined using human serum as a source of complement, and our studies revealed intense C-3 fixation by granular mesangial deposits, but not by linear deposits (Fig. 2f). Granular GBM complement fixation was seen in one mouse with a distinctly granular GBM pattern of IgG. The in vivo fixation was abolished by heating the human serum at 560C for 30 min. The amount of C-3 fixed in the mesangium was greater in older test animals, but similar to that of age-matched controls. Sera from mice with circulating anti-mouse/human GBM-TBM activity avidly fixed human complement when human kidney was the substrate, but failed to do so with mouse kidney substrate. Eluate weakly reactive with human GBM-TBM did not fix complement. Non-complement fixing linear GBM-TBM deposits of IgG in mouse kidneys did show fixation of human C-3 if rabbit anti-mouse IgG, was applied prior to the fresh human serum. This fixation was abolished by heating the serum.
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W. K. Bolton, F. R. Benton C B. C. Sturgill
FIG. 6. Electron micrograph of a glomerulus from an animal 25 weeks after immunization showing pronounced beading (B) of the GBM which appears to be largely the result of periodic expansion of the lamina externa. Note the well defined lamina densa (arrows). Carbon particles are evident in the capillary lumen. CL = capillary lumen, US = urinary space, EP = epithelial cell. (Magnification x 6400).
DISCUSSION Our studies describe production of autoimmune glomerulotubular nephropathy in mice using human GBM antigen. The anti-GBM pattern of deposition is initially similar to that reported in this disease model in other species (Steblay, 1962; Steblay, 1963; Williams & Steblay, 1965; Steblay & Rudofsky, 1966; Couser et at., 1973; Avasthi et at., 1971); Unanue & Dixon, 1967a; Lerner & Dixon, 1968). Our studies demonstrate specificity of the mouse IgG for GBM and TBM by our detection in test animals of circulating antibodies against normal kidney, by the in vitro fixation of the eluate from test animals to normal mouse and human GBM and TBM, and by the in vivo fixation of the eluate to mouse kidney GBM-TBM. The lesion evolves over time into a unique linear-granular GBM pattern. Granular GBM deposits with anti-GBM disease have been described before, but have resulted either from GBM fragmentation or deposition of electron-dense material (Lerner & Dixon, 1968; Agodoa et at., 1976; Steinmuller et at., 1976). In our studies, GBM granularity was not secondary to GBM fragmentation. Autoimmune nephropathy in guinea-pigs is associated with somewhat similar ultrastructural lesions (Couser et at., 1975). In contrast to mice, the IgG deposition by immunofluorescence in guinea-pigs is linear-GBM, even with extensive ultrastructural lesions (Couser et at., 1975). Our model of autoimmune nephropathy differs from others in terms of the extensive IgG deposits on the TBM. We found antibody on the TBM several weeks after it first appeared on the GBM, and noted uniform involvement of all tubular segments. While the TBMs became convoluted over time, no pattern of granularity appeared. Other investigators have described linear TBM deposits (Couser et at., 1973; Avasthi et at., 1971), but these have been of mild intensity, isolated to portions of the tubular network, and affected only some of the experimental animals.
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Examination of normal and control mice with antisera to mouse IgG subgroups demonstrated all subgroups in mesangial deposits. These deposits were associated with C-3 and IgM deposits and fixation of complement in vitro. Prior to detection of GBM IgG, the mesangial deposits in test mice were similar to those in controls. Within 4 to 7 weeks of the initial immunization, test mice had IgG1 deposited in a linear pattern along the GBM only. Two to 3 weeks later, the other IgG subgroups were detected along the GBM, and all subgroups were found on the TBM. IgG1 deposits on the GBM were generally linear from the time of their detection until the time when the animal was killed (as late as 25 weeks). In contrast, the other subgroups gradually assumed a granular pattern of GBM distribution, often superimposed on a linear background. The transition to granularity occurred first with IgG2B and IgG3, but it was also noted later with IgG2A. Since the eluate of nephropathic kidneys contained antibody activity to normal GBM and TBM primarily of the IgG1 subgroup, the other subgroups may have been directed to an abnormal type of GBM. Alternatively, the other subgroups may have combined with an unidentified antigen to produce soluble immune complex disease with deposits in the lamina rara externa; however, the absence of ultrastructural electron-dense deposits and the periodicity of the deposition both argue against these hypotheses. It is also possible that the other subgroups were directed against fixed GBM antigens located in the lamina rara externa or at the epithelial slit pores. This has recently been postulated to be the mechanism of GBM deposits in autologous immune complex disease in rats (Van Damme et al., 1976). This appears unlikely here, since no antibody activity to proximal tubule brush border was present in test sera or eluate. The most plausible explanation for the GBM beading in our test mice would be an alteration in the metabolism of the epithelial cells with an elaboration of excess amounts of lamina rara externa or a decrease in the turnover of this layer. The morphologically abnormal lamina rara externa may have been antigenically abnormal, leading to the formation of antibodies with specificity for this substance, separate from antibody to normal GBM. This would explain the relative decrease in IgG2A, IgG2B and IgG3 binding to normal GBM in vivo in normal mice given eluate intravenously, the presence of relatively large amounts of IgG subgroups in test animals, and the coincidental presence of beading in these subgroups. Differences in anti-GBM antibody specificity has been demonstrated in man (McCoy et al., 1976).
Our ultrastructural studies support the idea that abnormal GBM metabolism was responsible for the
granular appearance seen by immunofluorescence. Early lesions consisted of segmental thickening of the GBM, followed later by extremely regular, periodic out-pouching of the lamina rara externa involving all loops. Since the three layers comprising the GBM in mice are so distinct, it was easy to demonstrate that the beads
were
limited
to
the lamina rara externa with a normal, intact lamina densa and lamina
rara interna. The extensions of lamina rara externa were homogeneous and of the same density as normal externa. we found slight abnormalities suggesting non-homogeneity in some beads, but this was not usual. A somewhat similar lesion has been reported in guinea-pigs (Couser et al., 1975) but was less was associated with some abnormalities of the lamina densa and involved sub-
Occasionally,
diffuse, epithelial GBM expansions of a characteristically 'moth-eaten' appearance. Further, the ultrastructural abnormality in guinea-pigs was not associated with any granularity by immunofluorescence. Lack of complement fixation by immunoglobulin deposits in autoimmune nephropathy has been reported in guinea-pigs (Couser et al., 1973), but not in mice. Our mice had large quantities of all IgG subgroups along the GBM and TBM. Even though IgG2A and IgG2B have complement-fixing ability (Herzenberg & Herzenberg, 1973), they did not fix complement in this model. There was in vitro fixation of human C-3 by mesangial deposits in all animals. Fixation of the immunoglobulin to antigens in the
GBM and TBM may have altered the immunoglobulins rendering them incapable of C-3 fixation. A likely explanation would be spatial limitation with an inadequate density of individual IgG molecules. Fixation of human complement along the GBM and TBM of normal human kidney after layering with test sera containing circulating antibodies strongly supports this hypothesis, as does fixation of C-3 by the rabbit anti-mouse IgG1 after its attachment to the linear GBM-TBM deposits. A similar explanation could account for discrepancies in studies of C-3 fixation in the human kidney by IgG subgroups (Lewis, Burch & Schur, 1970; McPhaul & Dixon, 1971). Measurement of albumin excretion has been considered more sensitive than measurement of total more
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W. K. Bolton, F. R. Benton & B. C. Sturgill
protein excretion in mice, but is allegedly a poor indicator of glomerular damage (Hoffsten, Hill & Klahr, 1975). The protein excretion rates of our mice was quite variable but, after the fifth week, test mice excreted more protein per 24 hr than controls (P< 0.05), while no abnormal proteinuria was noted in test females. This may be related to hormonal factors known to affect protein excretion in rodents (Bolton et al., 1976). There were no significant differences between test and control animals' BUNs. This probably resulted from the lack of glomerular proliferation and damage. Abnormal tubular function manifested as glycosuria or lysozymuria was not detected. Although test animals had greater proteinuria than controls, we were not able to demonstrate differences in mesangial uptake of colloidal carbon, in contrast to what has been shown in proteinuric rats (Hoyer et al., 1976). The mechanism of proteinuria in this model is not entirely clear. Complement has been shown to enhance protein excretion in other species with anti-GBM antibody disease (Unanue & Dixon, 1974), but is not involved in the proteinuria of guinea-pigs (Couser et al., 1973) or our mice. The onset of abnormal proteinuria in the guinea-pig is associated with the appearance of 'moth-eaten' expansions of the GBM ultrastructurally and loss of staining for sialoprotein (Couser et al., 1975). Despite proteinuria and ultrastructural changes of the GBM lamina rara externa in our mice, only minimal decreases in sialoprotein were detectable in a few animals. Alteration of the polyanionic sialoprotein coat covering the epithelial side of the GBM results in foot process obliteration, while reconstitution of the negative charge restores normal foot process morphology (Seiler, Venkatachalam & Cotran, 1975). Since the sialoprotein polyanionic coat appears intact in the present model, despite GBM changes, foot process obliteration and proteinuria, it is possible that mechanisms other than alteration of the sialoprotein charge are responsible for proteinuria. Circulating lymphokines have been incriminated in the production of proteinuria in certain cases of nephrotic syndrome in man (Lagrue et al., 1975; Shalhoub, 1974). The mechanism of GBM permeability alteration and proteinuria in autoimmune glomerulotubular nephropathy in mice has yet to be clarified. We would like to acknowledge the additional technical assistance of Mr M. Nickerson, Ms D. Shifflett and Mr R. Kirkpatrick; the secretarial assistance of Ms A. Brent and the editorial assistance ofMs M. Day.
REFERENCES AGODOA, L.C.Y., STRIKER, G.E., GEORGE, C.R.P., GLASSOCK, R. & QUADRACCI, L.J. (1976) The appearance of non-linear deposits of immunoglobulins in Goodpasture's syndrome. Am. 7. Med. 61, 407. AVASTHI, P.S., AvATsii, P., TOKUDAS, S., ANDERSON, R.E. & WILLIAMS, R.C. JR. (1971) Experimental glomerulonephritis in the mouse. I. The model. Clin. exp. Immunol. 9,667. BOLTON, W.K., BENTON, F.R., MACLAY, J.G. & STURGILL, B.C. (1976) Spontaneous glomerular sclerosis in ageing Sprague-Dawley rats. Am..j. Path. 85, 277. BURKHOLDER, P.M. (1961) Complement fixation in diseased tissues. I. Fixation of guinea-pig complement in sections of kidney from humans with membranous glomerulonephritis and rats injected with anti-rat kidney serum. J. exp. Med. 114,605. COUSER, W.G., SPARGo, B.H., STILMANT, M. & LEWIS, E.J. (1975) Experimental glomerulonephritis in the guinea pig. II. Ultrastructural lesions of the basement membrane associated with proteinuria. Lab. Invest. 32, 46. COUSER, W.G., STILMANT, M.M. & LEWIS, E.J. (1973) Experimental glomerulonephritis in the guinea pig. I. Glomerular lesions associated with anti-glomerular basement membrane antibody deposits. Lab. Invest. 29, 236. DAVIDSON, J. & HENRY, J.B. (1969) Clinical Diagnosis by Laboratory Methods (14th edn), p. 48. W. B. Saunders Co., Philadelphia.
HERzENBERG, L.A. & HERZENBERG, L.A. (1973) Mouse immunoglobulin allotypes: Description and special methodology. Handbook of Experimental Immunology 2nd edn (ed. by D. M. Weir), Ch. 13. Blackwell Scientific Publications Ltd., Oxford. HOFFSTEN, P.E., HILL, C.L. & KLAH, S. (1975) Studies of albuminuria and proteinuria in normal mice and mice with immune complex glomerulonephritis. 5. Lab. Clin. Med. 86, 920. HoYER, J.R., ELEMA, J.D. & VERNIER, R.L. (1976) Unilateral renal disease in the rat. II. Glomerular mesangial uptake of colloidal carbon in unilateral aminonucleoside nephrosis and nephrotoxic serum nephritis. Lab. Invest. 34, 250. KRAKowER, C.A. & GREENSPON, S.A. (1951) Localization of the nephrotoxic antigen within the isolated renal glomerulus. A.M.A. Arch. Pathol. 51, 629. LAGRUE, G., XHENEUMONT, S., BRANELLEC, A. & WEIL, B. (1975) Lymphokines and nephrotic syndrome. Lancet, i,
271. LxERNm, R.A. & DixoN, F.J. (1968) The induction of acute glomerulonephritis in rabbits with soluble antigens isolated from normal homologous and autologous urine. J. Immunol. 100, 1277. LEWIS, E.J., BuRcH, G.H. & ScHuR, P.H. (1970) Gamma G globulin subgroup composition of the glomerular deposits in human renal diseases. J. Clin. Invest. 49, 1103. McCoy, R.C., JoHNSoN, H.K., STONE, W.J. & WILSON, C.D. (1976) Variation in glomerular basement membrane
Autoimmune nephropathy in mice antigens in hereditary nephritis. Lab. Invest. 34, 325, abstr. MCPHAUL, J.J., JR. & DIXON, F.J. (1970) Characterization of human anti-glomerular basement membrane antibodies eluted from glomerulonephritic kidneys. A. Clin. Invest. 49,308. MCPHAUL, J.J., JR. & DIXON, F.J. (1971) Characterization of immunoglobulin G anti-glomerular basement memmembrane antibodies eluted from kidneys of patients with glomerulonephritis. II. IgG subtypes and in vitro complement fixation. J. Immunol. 107, 678. SEILER, M.W., VENKATACHALAM, M.A. & ComuN, R.S. (1975) Glomerular epithelium: Structural alterations induced by polycations. Science, 189,390. SHALHOUB, R.J. (1974) Hypothesis. Pathogenesis of lipoid nephrosis: A disorder of T-cell function. Lancet, ii, 556. STEBLAY, R.W. (1962) Glomerulonephritis induced in sheep by injections of heterologous glomerular basement membrane and Freund's complete adjuvant. 3. exp. Med. 116,253. STEBLAY, R.W. (1963) Glomerulonephritis induced in monkeys by injection of heterologous glomerular basement membrane and Freund's adjuvant. Nature (Lond.), 197, 1173. STEBLAY, R.W. & RUDOFSKY, U. (1966) Autoimmune nephritis in rats. I. Nephritis induced in rats by injection
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of human glomerular basement membrane (HGBM) and Freund's adjuvant (FA). Clin. Res. 14, 449, abstract. STEINMULLER, D.A., COUSER, W.G., STILMANT, M.D., IDELSON, B.A., LEWIS, E.J., MONACO, A.P. & DAvIS, R.C. (1976) DeNovo development of membranous nephropathy (MN) in cadavar renal transplants (TP). 9th Annual Meeting, American Society of Nehprology, Washington, D.C. Kidney Int. 10, 610, abstr. UNANUE, E.R. & DIXON F.J. (1967a) Experimental allergic glomerulonephritis induced in the rabbit with heterologous renal antigens. J. exp. Med. 125, 149. UNANUE, E.R. & DIxoN, F.J. (1967b) Experimental glomerulonephritis: Immunological events and pathogenetic mechanisms. Advances in Immunology Vol. 6 (ed. by F. J. Dixon & J. H. Humphrey), p. 1. Academic Press, New York & London. UNANLIE, E.R. & DIXON, F.J. (1974) Experimental glomerulonephritis: IV. Participation of complement in nephrotoxic nephritis. J. exp. Med. 119, 965. VAN DAMME, B., FLEUREN, G.J., BAKKER, W.W., HOEDEMAEKER, P.J. & VERNIER, R.L. (1976) Fixed glomerular antigens in the pathogenesis of heterologous immune complex glomerulonephritis. Kidney Int. 10, 551 abstr. WILLIAMS, R. & SEBLAY, R.W. (1965) Glomerulonephritis induced in goats by injections of human glomerular basement membrane (HGBM) and Freund's adjuvant (FA). Fed. Proc. 24, 243 abstr.
Autoimmune glomerulotubular nephropathy in mice W. K. BOLTON, F. R. BENTON & B. C. STURGILL The Departments of Internal Medicine and Pathology, University of Virginia School ofMedicine, Charlottesville, Virginia, USA
(Received 7 March 1978)
SUMMARY
We produced an autoimmune glomerulotubular nephropathy in Swiss-Webster mice using human glomerular antigen in Freund's complete adjuvant. The disease is associated with circulating antibody to both mouse and human glomerular basement membranes (GBM) and tubular basement membranes (TBM). All mouse IgG subgroups are deposited initially in a linear pattern along the GBM and TBM. IgG deposition remains linear, while that of the other subgroups assumes a granular GBM pattern with continued linear TBM deposits. Despite tissue deposition of antibody capable of C-3 fixation, no C-3 is found in vivo along the GBM or TBM, nor is there C-3 fixation in vitro. This appears to be related to spatial limitations of IgG molecule attachment to basement membranes. A unique ultrastructural lesion of the GBM developed, characterized by periodic expansions of the lamina rara externa to form a beaded pattern. Eluate of nephritic kidneys contained all subgroups of IgG, but mainly IgG1 fixed in vitro to mouse kidney and in vivo when injected intravenously into normal mice. Fixation of other IgG subgroups in vivo may have resulted from antibody formation to abnormally formed GBM, thereby accounting for the peculiar ultrastructural findings and tissue fixation characteristics of the eluted immunoglobulin. Abnormal proteinuria without glycosuria or lysozymuria developed in test animals as compared to controls. Our model is similar in certain aspects to previously described models of Steblay nephritis, but differs because of the total involvement of TBMs, unique ultrastructural lesions, and dissimilarity to other reports ofthis model in mice.
INTRODUCTION Experimental autoimmune antiglomerular basement membrane (GBM) glomerulonephritis (Steblay nephritis) induced by immunization with heterologous GBM antigen has been described in sheep, rats, monkeys, goats, rabbits, guinea-pigs and mice (Steblay, 1962; Steblay, 1963; Williams & Steblay, 1965; Steblay & Rudofsky, 1966; Couser, Stilmant & Lewis, 1973; Avasthi et al., 1971; Unanue & Dixon 1967a; Lerner & Dixon, 1968). The nephritis may be severe, with marked golmerular proliferation and rapid death induced by renal failure (Steblay, 1962; Williams & Steblay, 1965), or mild, without significant proliferation or renal dysfunction (Steblay, 1963; Couser et a., 1973). Host antibody is invariably deposited in a linear pattern along the GBM, and some investigators ha-ve noted occasional linear tubular basement membrane (TBM) deposits (Couser et al., 1973; Avasthi et al., 1971). Complement may play a major role in some of these types of autoimmune nephritis (Unanue & Dixon, 1967b), but other types appear to be complement-independent (Couser et al., 1973). Mice immunized with canine GBM develop a severe necrotizing nephritis with perivascular and interstitial infiltrates resembling those Correspondence: Dr W. K. Bolton, Box 133, Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22901, USA. 0099-9104178/0900-463$02.00 (© 1978 Blackwell Scientific Publications
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accompanying transplantation rejection (Avasthi et al., 1971). No data are available about complement fixation in the mouse. We immunized Swiss-Webster mice with human GBM, and produced an autoimmune glomerulotubular nephropathy with features different from those noted in the other models cited above. The disease involved non-complement-fixing anti-GBM deposits and diffuse linear antibody deposition on all TBMs. In contrast to the mouse model previously described (Avasthi et al., 1971), our model was unremarkable for proliferative or necrotizing nephritis, or for any type of cellular infiltrate. In addition, the mice in our study developed a unique ultrastructural glomerular lesion. MATERIALS AND METHOD Animals. Male and female Swiss-Webster mice weighing 25-30 g each were used in our study and were housed under standard conditions in the vivarium. Immunization. Human kidneys were kept frozen at -70'C until they were to be used; they were then thawed at 4VC. The cortices were separated from the medullae, minced, and used to isolate glomeruli by the method of Krakower & Greenspon (1951). We washed the sediment repeatedly in phosphate buffered saline (PBS), pH 7-3, until it contained less than 5% tubules. We then washed the glomeruli in deionized water, lyophilized them and stored them in dessicated form. Preparatory to use in immunization, the glomeruli were suspended in PBS and mixed with an equal amount of Freund's complete adjuvant to deliver 0-75 mg glomeruli dry weight per mouse in 0-15 ml volume. This inoculum was given to mice in the foot pads and subcutaneously as the initial injection; animals were injected subcutaneously thereafter every 2 weeks until the experiment's termination. Control animals received only PBS in Freund's complete adjuvant at 2 week intervals. Protein excretion, blood urea nitrogen (BUN) and tubular function evaluation. Animals were periodically placed in metabolic cages for 24 hr urine collections. Urine was tested for blood, ketones, and glucose with Ames Multistix and the protein excretion was determined by means of 3% sulfosalicylic acid with a bovine serum albumin standard (Davidson & Henry, 1969). Lysozymuria was quantified by radial diffusion with lysozyme Quantiplates (Kallestad Laboratories). We killed the animals serially to obtain blood samples for routine biochemical analysis of BUN and tissue specimens for examination. Immunofluorescence, light and electron microscopy. Renal tissue taken from mice at autopsy or open biopsy was divided into three portions. One specimen was snap-frozen in dry ice-isopentane, another fixed in buffered neutral formalin, and the third fixed in 2% gluteraldehyde, post-fixed in 2% osmium tetroxide, and serially dehydrated in ethanol. Thin sections of specimens embedded in Araldite (Ladd Co.) were stained with lead citrate and examined with a Zeiss electron microscope. We used the direct technique to examine frozen tissue with monospecific rhodamine-labelled rabbit antimouse IgG and fluorescein-labelled antisera (Cappel Laboratory) to mouse C-3, IgM, fibrinogen, and albumin (Bolton et al., 1976). Staining intensity was graded 0 to 4+ and the staining patterns classified as mesangial, GBM, TBM, granular or linear. Selected kidneys from mice with linear GBM or GBM plus TBM deposits of IgG were examined by indirect fluorescence techniques to determine the subtype of IgG deposited. Monospecific rabbit antimouse IgG1, IgG2A, IgG2B, and IgG3 (Miles Laboratory) were layered onto test kidney sections. After washing in PBS, fluorescein-labelled anti-rabbit IgG, previously absorbed with mouse IgG linked to Sepharose, was applied. We recorded the pattern, intensity and distribution of the IgG subgroups for each animal, doing so without knowledge of the clinical or immunization state of the animal. Kidney tissue fixed in buffered formalin was embedded in paraffin and cut serially at 3 gm and stained with haematoxylin and eosin (H&E), periodic acidSchiff reagent (PAS), and colloidal iron. The sections were examined for incidence of proliferation, crescents, interstitial infiltrates, tubular atrophy, arteritis, or casts, without knowledge of the study group from which the tissue samples were taken. Circulating anti-GBM antibodies. Sera selected at random from test and control mice were serially diluted with PBS and layered onto normal mouse and human kidney sections. We applied unlabelled rabbit antimouse IgG1 antiserum and followed this with fluorescein-labelled goat anti-rabbit IgG previously absorbed by mouse IgG linked to Sepharose. Titres were calculated as the reciprocals of the dilutions. Certain sera containing anti-GBM antibody were tested for complement fixation using normal mouse and human kidney. Elution studies. Kidneys of mice with tissue-proven IgG deposits were minced, washed in PBS, and eluted with citric acid-citrate buffer, 0-02 M, pH 3-2 (Bolton et al., 1976; McPhaul & Dixon, 1970). The eluate was neutralized and tested by indirect fluorescence against normal mouse kidney, muscle, liver, lung and heart. We gave an intravenous injection of 0- 1 ml ofeluate to normal mice; these mice were killed 24 hr later. Complement fixation studies. We assessed in vivo fixation of complement (C-3) by examining kidney tissue with fluoresceinated antimouse C-3. In vitro tests of complement fixation were performed as described by Burkholder (1961), using normal human serum and fluorescein-labelled goat antihuman C-3. We used positive controls with antinuclear antibody containing serum and negative controls with heat-inactivated serum. Colloidal carbon studies. Selected test and control animals were given 70 mg/100 g body weight of colloidal carbon (C 11/ 1431 A, Gunther-Wagner, W. Germany) with heparin 50 iu/ml intravenously 24 hr prior to being killed; any alteration of mesangial function induced by the anti-GBM antibodies was then noted. Animals examined in this way had received alternateweek immunizations over a period of 25 weeks. Tissue sections were examined for the amount and distribution of carbon as described by Hoyer, Elema & Vernier (1976).
Autoimmune nephropathy in mice
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RESULTS Animals Thirty-four male and thirty-two female mice were immunized in three different experiments. Fourteen mice of each sex served as controls and received Freund's adjuvant alone. The mice had received a total amount of up to 9 mg of antigen by the end of the study. All mice tolerated the injections well; only a few developed abscesses and none failed to thrive or died as a result of immunizations. Animals in the first two series were killed serially every 2 to 4 weeks. Subsets of mice in the third series were biopsied every week for the first 8 weeks. Protein excretion, BUN and tubular function Baseline protein excretion rates of male mice, antigen recipients and controls, were similar (Fig. 1), as were the baselines of the female mice. Proteinuria in males was much greater than in females and was more variable. Proteinuria increased over time, both in test and control male mice but, by the fifth week, the protein excretion rates of test animals were significantly greater than those of the age-matched controls (P< 0.05). Abnormal proteinuria was not detected in female mice by the fifteenth week. BUN values revealed little difference between test and control mice, male or female. Multistix evaluations of urines were normal other than those indicating proteinuria, and no differences in lysozymuria were detected.
Immunofluorescence microscopy Direct. In the first 4 weeks after the initial immunization, normal mice, control animals and test animals demonstrated only granular mesangial deposits of IgM, IgG and, occasionally, C-3. By the fourth week, the antigen recipients had developed fine trace linear deposits of IgG alone the GBM (Fig. 2a). With time these deposits showed an increase in staining intensity and they gradually assumed a linear-granular appearance. Later, as the nephropathy progressed in these test animals, GBM deposits were shown by staining to be primarily granular and only slightly linear. As staining intensity increased with time, thickening of the GBM was also noted. In older animals, the GBM seemed stiff, with open lumina and a wire loop pattern (Fig. 2b). In the first weeks after GBM deposits were detectable, no IgG was present on the TBMs; however, distinct linear IgG deposition subsequently occurred, increasing in intensity with time (Fig. 2c). In contrast to the GBM deposits, IgG deposited on the TBM never
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FIG. 1. Graph of 24 hr urinary protein excretion in male mice. Test animals (-) received GBM in Freund's complete adjuvant, while controls (Z) received only Freund's complete adjuvant. Test animals excreted significantly more protein than controls after the fourth week. (*) P< 0 05.
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W. K. Bolton, F. R. Benton
&
B. C. Sturgill
FIG. 2. Immunofluorescent findings. (a) Photomicrograph demonstrating fine linear deposits of mouse IgG1 along the GBM, from an animal 7 weeks after immunization with GBM. Rabbit antimouse IgG1/fluoresceinlabelled goat anti-rabbit IgG (magnification x 1660). (b) Kidney section from a test mouse showing intense staining for IgG along the GBM and TBM, thickening of the basement membranes, and granularity of the GBM with convolution of the TBM. From a mouse after 15 weeks of immunization. Rhodamine-labelled rabbit anti-mouse IgG (magnification x 620). (c) Section of test mouse kidney showing linear deposits of mouse IgG along the TBM (magnification x 640). (d) Section of kidney from a control mouse injected every 2 weeks with Freund's complete adjuvant for 25 weeks stained with anti-mouse IgG. Heavy granular mesangial deposits and trace segmental granular GBM deposits are present, but no anti-GBM antibody is detectable (magnification x 700). (e) Photomicrograph illustrating discrete beads (right-hand arrow) continuous with the GBM (left-hand arrow). From an animal after 21 weeks of immunization. Rabbit antimouse IgG2A/fluoresceinated goat anti-rabbit IgG (magnification x 1620). (f) Mouse kidney from the test animal in (b) demonstrating fixation of human C-3 by mesangial deposits only. Stained with fluoresceinated rabbit anti-human C-3
(magnification
x
800).
Autoimmune nephropathy in mice
467
Mouse IgG subgroups
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modified from a linear to a primarily granular pattern, but the TBM did thicken and become convoluted as the nephropathy progressed (Fig. 2b). C-3 deposition was usually confined to the mesangium, with only an occasional animal showing trace diffuse linear or focal segmental linear GBM deposits. Complement was not detected on the TBM. IgM was not found along either the GBM or TBM, and fibrin appeared only in rare segmental deposits in areas of sclerosis. Older control mice had heavy deposits of lgG, IgM, and C-3 in the mesangium and, occasionally, slight granular GBM deposits; this pattern of deposition (Fig. 2d) did not differ from that of age-matched normal animals. Indirect. The kidneys of forty test mice were studied for the presence and distribution of IgG subgroups from 1 to 25 weeks after the beginning of immunizations (Fig. 3). Sections of kidneys from mice immunized for 6 or more weeks were read without knowledge of the duration of immunizations. IgG was not detected in mice during the first 3 weeks after immunization. By the fourth week, IgG1, deposited in a lacy, linear pattern along the GBM, was detectable in 75% of the mice; no other subgroup was present. By the fifth week, the other subgroups were detectable in half of the biopsied mice. Thereafter, all IgG subgroups were found in animals with deposits. The pattern of staining was always distinctly linear in the first 4 weeks, but thereafter it began to assume a granular pattern that roughly correlated with the specific subgroup (Fig. 2e). IgG3 showed the greatest propensity to assume a granular pattern, with two thirds of the animals immunized for 10 weeks or more having granular IgG3. IgG2B showed an essentially similar pattern, while IgG2A remained in a linear pattern until 18 weeks. In contrast to the other subgroups, IgG1 maintained the linear pattern of deposition in most animals, even when the other subgroups were found in distinctly granular patterns. Fig. 3 depicts only those animals with wholly linear or distinctly granular deposits. Approximately half of the kidneys examined had a fragmented linear GBM pattern with characteristics of both linear and granular immunoglobulin. All of the IgG subgroups were present on all TBMs in a wholly linear pattern of slightly less staining intensity than GBM deposits; they never became granular, regardless of the corresponding GBM patterns.
Histopathology Examination of serial sections by H&E, PAS and colloidal iron revealed only minimal histological alterations during the first 6 to 10 weeks after immunization. Thereafter, a spectrum of changes was noted in immunized animals. Approximately a third of the test mice showed some degree of thickening of capillary walls by 20 weeks and half by 25 weeks. Sometimes this was striking and characterized
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FIG. 4. (a) Glomerulus from an animal 25 weeks after immunization illustrating thick, beaded capillary walls and absence of proliferation. PAS (Magnification x 250). (b) Glomerulus from an animal 16 weeks after immunization showing proliferation of Bowman's capsular epithelium. H&E (Magnification x 250).
by a beaded appearance suggestive of the lesion seen in idiopathic membranous glomerulopathy in humans (Fig. 4a). These changes were generally associated with a comparable degree of tubular atrophy and hyaline tubular casts. No significant interstitial or perivascular infiltrates were seen. Two mice that received GBM antigen had a proliferative nephritis (Fig. 4b). Colloidal iron staining was not diminished until late in the immunization period and was not consistently related to histological abnormalities, being slightly diminished in some histologically normal glomeruli, and normal in some histologically abnormal glomeruli. There was also no apparent correlation between colloidal iron staining and glomerular ultrastructure. Colloidal carbon studies Six test animals and six controls received colloidal carbon. The test animals had heavy deposits of IgG along the GBM, but there were no detectable differences between test and control animals with regard to mesangial carbon uptake as judged by double-blind analysis. Electron microscopy The glomeruli of antigen-recipient animals were ultrastructurally normal for the first 6 to 10 weeks after immunization with GBM began. Thereafter, the GBM gradually thickened, although all layers were quite distinct and of normal density. Within 12 to 15 weeks, the GBM had definitely thickened and developed structural irregularities. These changes appeared to result from progressive alterations in the lamina rara externa, which developed a periodic beading contiguous to normal areas. Regions of increased lamina rara were indistinguishable from normal basement membrane and did not involve the lamina densa. Foot process obliteration and condensation of epithelial cell cytoplasm occurred over the extrusions. As the lesion developed, it assumed a characteristic string-bead appearance which affected the length of any GBM region (Figs 5 and 6). The masses, separated by fingers of epithelial cell cytoplasm, continually enlarged. At all times the subepithelial masses appeared to have the same density and homogeneity as the lamina rara externa. The lamina rara interna and lamina densa remained normal and there were no alterations of endothelial cells. Only occasional electron-dense deposits were seen in the mesangium of some animals. The TBMs also showed a similar, albeit less striking, beading on the luminal side. We found no fibrin, collagen or inflammatory cells.
Circulating anti-GBM-TBM antibody Sera taken from animals at various times after immunizations began were tested as described above.
Autoimmune nephropathy in mice
469 *us
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FIG. 5. Electron micrograph of glomerulus from an animal 25 weeks post-immunization demonstrating extensive beading of the GBM. CL = capillary lumen; US = urinary space. (Magnification x 2550).
Two thirds of the animals had circulating antibody to GBM and TBM, with titres ranging from 4 to 32 Higher titres of anti-GBM-TBM were recorded when normal human kidney was used as the substrate. Antinuclear factor was detected in one third of the animals, and was associated with a low titre or undetectable antibody to GBM and TBM.
Elution studies The eluate concentrated to 1*0 mg/ml protein contained IgG which bound selectively to normal mouse GBM and TBM, but not to heart, muscle, liver or lung. Kidneys of normal mice given 0 1 ml of the eluate intravenously showed linear staining along the GBM and TBM. IgG1 was found in the greatest quantities, with IgG2A, IgG2B and IgG3 detected in trace amounts only along the GBM. Immunoglobulin eluted from mouse kidneys fixed slightly to human GBM and TBM
Cornplementfixation studies Mouse C-3 was detectable in vivo in a mesangial pattern, but only rarely in a linear GBM distribution. Two to 25 weeks after immunizations began, kidneys were examined using human serum as a source of complement, and our studies revealed intense C-3 fixation by granular mesangial deposits, but not by linear deposits (Fig. 2f). Granular GBM complement fixation was seen in one mouse with a distinctly granular GBM pattern of IgG. The in vivo fixation was abolished by heating the human serum at 560C for 30 min. The amount of C-3 fixed in the mesangium was greater in older test animals, but similar to that of age-matched controls. Sera from mice with circulating anti-mouse/human GBM-TBM activity avidly fixed human complement when human kidney was the substrate, but failed to do so with mouse kidney substrate. Eluate weakly reactive with human GBM-TBM did not fix complement. Non-complement fixing linear GBM-TBM deposits of IgG in mouse kidneys did show fixation of human C-3 if rabbit anti-mouse IgG, was applied prior to the fresh human serum. This fixation was abolished by heating the serum.
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W. K. Bolton, F. R. Benton C B. C. Sturgill
FIG. 6. Electron micrograph of a glomerulus from an animal 25 weeks after immunization showing pronounced beading (B) of the GBM which appears to be largely the result of periodic expansion of the lamina externa. Note the well defined lamina densa (arrows). Carbon particles are evident in the capillary lumen. CL = capillary lumen, US = urinary space, EP = epithelial cell. (Magnification x 6400).
DISCUSSION Our studies describe production of autoimmune glomerulotubular nephropathy in mice using human GBM antigen. The anti-GBM pattern of deposition is initially similar to that reported in this disease model in other species (Steblay, 1962; Steblay, 1963; Williams & Steblay, 1965; Steblay & Rudofsky, 1966; Couser et at., 1973; Avasthi et at., 1971); Unanue & Dixon, 1967a; Lerner & Dixon, 1968). Our studies demonstrate specificity of the mouse IgG for GBM and TBM by our detection in test animals of circulating antibodies against normal kidney, by the in vitro fixation of the eluate from test animals to normal mouse and human GBM and TBM, and by the in vivo fixation of the eluate to mouse kidney GBM-TBM. The lesion evolves over time into a unique linear-granular GBM pattern. Granular GBM deposits with anti-GBM disease have been described before, but have resulted either from GBM fragmentation or deposition of electron-dense material (Lerner & Dixon, 1968; Agodoa et at., 1976; Steinmuller et at., 1976). In our studies, GBM granularity was not secondary to GBM fragmentation. Autoimmune nephropathy in guinea-pigs is associated with somewhat similar ultrastructural lesions (Couser et at., 1975). In contrast to mice, the IgG deposition by immunofluorescence in guinea-pigs is linear-GBM, even with extensive ultrastructural lesions (Couser et at., 1975). Our model of autoimmune nephropathy differs from others in terms of the extensive IgG deposits on the TBM. We found antibody on the TBM several weeks after it first appeared on the GBM, and noted uniform involvement of all tubular segments. While the TBMs became convoluted over time, no pattern of granularity appeared. Other investigators have described linear TBM deposits (Couser et at., 1973; Avasthi et at., 1971), but these have been of mild intensity, isolated to portions of the tubular network, and affected only some of the experimental animals.
Autoimmune nephropathy in mice
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Examination of normal and control mice with antisera to mouse IgG subgroups demonstrated all subgroups in mesangial deposits. These deposits were associated with C-3 and IgM deposits and fixation of complement in vitro. Prior to detection of GBM IgG, the mesangial deposits in test mice were similar to those in controls. Within 4 to 7 weeks of the initial immunization, test mice had IgG1 deposited in a linear pattern along the GBM only. Two to 3 weeks later, the other IgG subgroups were detected along the GBM, and all subgroups were found on the TBM. IgG1 deposits on the GBM were generally linear from the time of their detection until the time when the animal was killed (as late as 25 weeks). In contrast, the other subgroups gradually assumed a granular pattern of GBM distribution, often superimposed on a linear background. The transition to granularity occurred first with IgG2B and IgG3, but it was also noted later with IgG2A. Since the eluate of nephropathic kidneys contained antibody activity to normal GBM and TBM primarily of the IgG1 subgroup, the other subgroups may have been directed to an abnormal type of GBM. Alternatively, the other subgroups may have combined with an unidentified antigen to produce soluble immune complex disease with deposits in the lamina rara externa; however, the absence of ultrastructural electron-dense deposits and the periodicity of the deposition both argue against these hypotheses. It is also possible that the other subgroups were directed against fixed GBM antigens located in the lamina rara externa or at the epithelial slit pores. This has recently been postulated to be the mechanism of GBM deposits in autologous immune complex disease in rats (Van Damme et al., 1976). This appears unlikely here, since no antibody activity to proximal tubule brush border was present in test sera or eluate. The most plausible explanation for the GBM beading in our test mice would be an alteration in the metabolism of the epithelial cells with an elaboration of excess amounts of lamina rara externa or a decrease in the turnover of this layer. The morphologically abnormal lamina rara externa may have been antigenically abnormal, leading to the formation of antibodies with specificity for this substance, separate from antibody to normal GBM. This would explain the relative decrease in IgG2A, IgG2B and IgG3 binding to normal GBM in vivo in normal mice given eluate intravenously, the presence of relatively large amounts of IgG subgroups in test animals, and the coincidental presence of beading in these subgroups. Differences in anti-GBM antibody specificity has been demonstrated in man (McCoy et al., 1976).
Our ultrastructural studies support the idea that abnormal GBM metabolism was responsible for the
granular appearance seen by immunofluorescence. Early lesions consisted of segmental thickening of the GBM, followed later by extremely regular, periodic out-pouching of the lamina rara externa involving all loops. Since the three layers comprising the GBM in mice are so distinct, it was easy to demonstrate that the beads
were
limited
to
the lamina rara externa with a normal, intact lamina densa and lamina
rara interna. The extensions of lamina rara externa were homogeneous and of the same density as normal externa. we found slight abnormalities suggesting non-homogeneity in some beads, but this was not usual. A somewhat similar lesion has been reported in guinea-pigs (Couser et al., 1975) but was less was associated with some abnormalities of the lamina densa and involved sub-
Occasionally,
diffuse, epithelial GBM expansions of a characteristically 'moth-eaten' appearance. Further, the ultrastructural abnormality in guinea-pigs was not associated with any granularity by immunofluorescence. Lack of complement fixation by immunoglobulin deposits in autoimmune nephropathy has been reported in guinea-pigs (Couser et al., 1973), but not in mice. Our mice had large quantities of all IgG subgroups along the GBM and TBM. Even though IgG2A and IgG2B have complement-fixing ability (Herzenberg & Herzenberg, 1973), they did not fix complement in this model. There was in vitro fixation of human C-3 by mesangial deposits in all animals. Fixation of the immunoglobulin to antigens in the
GBM and TBM may have altered the immunoglobulins rendering them incapable of C-3 fixation. A likely explanation would be spatial limitation with an inadequate density of individual IgG molecules. Fixation of human complement along the GBM and TBM of normal human kidney after layering with test sera containing circulating antibodies strongly supports this hypothesis, as does fixation of C-3 by the rabbit anti-mouse IgG1 after its attachment to the linear GBM-TBM deposits. A similar explanation could account for discrepancies in studies of C-3 fixation in the human kidney by IgG subgroups (Lewis, Burch & Schur, 1970; McPhaul & Dixon, 1971). Measurement of albumin excretion has been considered more sensitive than measurement of total more
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protein excretion in mice, but is allegedly a poor indicator of glomerular damage (Hoffsten, Hill & Klahr, 1975). The protein excretion rates of our mice was quite variable but, after the fifth week, test mice excreted more protein per 24 hr than controls (P< 0.05), while no abnormal proteinuria was noted in test females. This may be related to hormonal factors known to affect protein excretion in rodents (Bolton et al., 1976). There were no significant differences between test and control animals' BUNs. This probably resulted from the lack of glomerular proliferation and damage. Abnormal tubular function manifested as glycosuria or lysozymuria was not detected. Although test animals had greater proteinuria than controls, we were not able to demonstrate differences in mesangial uptake of colloidal carbon, in contrast to what has been shown in proteinuric rats (Hoyer et al., 1976). The mechanism of proteinuria in this model is not entirely clear. Complement has been shown to enhance protein excretion in other species with anti-GBM antibody disease (Unanue & Dixon, 1974), but is not involved in the proteinuria of guinea-pigs (Couser et al., 1973) or our mice. The onset of abnormal proteinuria in the guinea-pig is associated with the appearance of 'moth-eaten' expansions of the GBM ultrastructurally and loss of staining for sialoprotein (Couser et al., 1975). Despite proteinuria and ultrastructural changes of the GBM lamina rara externa in our mice, only minimal decreases in sialoprotein were detectable in a few animals. Alteration of the polyanionic sialoprotein coat covering the epithelial side of the GBM results in foot process obliteration, while reconstitution of the negative charge restores normal foot process morphology (Seiler, Venkatachalam & Cotran, 1975). Since the sialoprotein polyanionic coat appears intact in the present model, despite GBM changes, foot process obliteration and proteinuria, it is possible that mechanisms other than alteration of the sialoprotein charge are responsible for proteinuria. Circulating lymphokines have been incriminated in the production of proteinuria in certain cases of nephrotic syndrome in man (Lagrue et al., 1975; Shalhoub, 1974). The mechanism of GBM permeability alteration and proteinuria in autoimmune glomerulotubular nephropathy in mice has yet to be clarified. We would like to acknowledge the additional technical assistance of Mr M. Nickerson, Ms D. Shifflett and Mr R. Kirkpatrick; the secretarial assistance of Ms A. Brent and the editorial assistance ofMs M. Day.
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