Abstract
Marmosets (Platyrrhini: Callitrichidae) are small New World monkeys well suited for laboratory research because of their close relationship to man and their low maintenance costs. Marmosets have a low immunological competence compared to other primate species which may be due to defective B-cell activity.8,9 When kept in captivity, marmosets are very susceptible to several disorders such as wasting marmoset syndrome (WMS)4,13, jejunitis and colitis5, colon carcinoma1, and nephritis3,15, characterized as IgM-nephropathy2. Furthermore a high incidence of subclinical chronic urinary tract disease in a colony of marmosets at the German Primate Center (GPC) was reported.7
For further information on chronic glomerulopathy in marmosets triple diagnostic (light microscopy, immunofluorescence microscopy, and electron microscopy) was done and similarities to IgA-nephropathy found. The degree of glomerular IgA-deposition was correlated to hematuria and proteinuria.
Methods
The kidneys of the 127 marmosets and tamarins from the GPC were investigated for histologic lesions, IgA- and IgM-deposits. All animals were submitted by other departments for routine necropsy after natural death or following sacrifice. The control group consisted of 13 Old World monkeys (5 Macaca mulatta, 5 Papio hamadryas, 1 Macaca fascicularis, 1 Papio sphinx, 1 Presbytis entellus).
The light microscopical and immunopathological techniques applied to the renal tissues have been previously in principle described.2,3 Because there are no antisera to marmoset immunoglobulins available, fluorescin-labeled polyclonal antisera to human IgA and IgM (Dako, Hamburg) were used. These antibodies were checked for cross-reactivity with agarose diffusion techniques.11 According to the results of the cross-reactivity tests, working dilutions of 1:20 (in phosphate buffered saline) for the antiserum to human IgA and 1:50 for the antiserum to human IgM were used. The histological lesions and glomerular deposits were graded from “negative” to “weakly positive” and “moderate to heavy positive.”
For electron microscopy, 1 mm3 fragments of kidney tissue were immediately placed in 2,5% glutaraldehyde, post-fixed in phosphate buffered 1% osmium tetroxide, dehydrated in graded alcohols and propylene oxide and Epon embedded. Sections were cut on an ultramicrotome (Leica, Bensheim) and stained with uranyl acetate and lead citrate. For electron microscopy a transmission electron microscope EM 10 (Zeiss, Oberkochen) was used.
Hematuria and proteinuria were determined by puncturing the separated bladder during necropsy, and the bladder urine was checked for blood and protein with test-sticks (Boehringer, Mannheim).
Statistical evaluation: Statistically significant differences between the different monkey groups (p<0,5) were evaluated by one-way-analysis of variances to compare more than 2 groups followed by Dunn’s test for pairwise multiple comparison procedures.
Results
Histology
Typical signs of nephropathy in marmosets were: increased mesangium (cells and matrix), adhesions between Bowman’s capsule and glomerular tufts, glomerulosclerosis, segmental sclerosis and interstitial fibrosis often accompanied by tubulointerstitial nephritis. According to these criteria 36 of 127 animals showed distinctive signs of mesangioproliferative glomerulonephropathy (mesGN).
Electron Microscopy
In kidneys with minor glomerular IgA-deposits frequently thickened peripheral capillary basement membranes and scattered electron dense deposits “humps” subepithelial in the peripheral capillary walls were found. In kidneys with heavy glomerular IgA-deposits, large electron dense deposits in the mesangial and paramesangial areas were present.
Immunopathology
Compared to previous descriptions2,3 more frequent and more prominent IgA deposits were found in the mesangial areas. Twenty-nine of the 127 marmosets showed moderate to heavy granular IgA-deposits in the glomerulum, mostly in the mesangium and rarely along the glomerular tufts. Moderate to heavy IgM-deposits were found in the glomeruli of 56 of the 127 marmosets of the same group in mesangial pattern.
In contrast in 13 Old World monkeys, no animal showed signs of chronical nephropathy or moderate to strong IgA-deposits. In 4 of 13 of this group moderate IgM-deposits were found, but in minor amounts and along the capillary loops and not in the glomerular mesangium.
Hematuria and Proteinuria
Thirty-one marmosets of the German Primate Center, previously checked for glomerular IgA-deposits were tested for hematuria and proteinuria, and the results compared with the graduation of nephropathy. The marmosets were graded into three groups: those without glomerular IgA-deposits (group I, n=7), those with slight (group II, n=15) and those with strong glomerular IgA-deposits (group III, n=9).
Significant differences in proteinuria were found between the three groups by one-way-analysis of variances and between group I and group II by pairwise multiple comparison. Also, significant differences in hematuria were found between the three groups by one-way-analysis of variances and between group I and group II and between group I and group III.
Discussion
Using triple-diagnostic procedures6 (histology, immunopathology, electron microscopy) in the present study, the immune-mediated nephropathy in marmosets was found to be immune-complex-glomerulopathy (IC-GP) showing striking similarities to IgA-nephropathy (Berger’s disease) in humans. The pathology of disease consists of mesangial proliferation, segmental sclerosis, interstitial fibrosis and tubulointerstitial nephritis and electron-dense deposits and is very similar to human IgA-nephropathy. Because of the weak cross-reactivity of the antibody to human IgA with marmosets IgA, for immunofluorescence technique a 1:20 dilution of antibody instead of 1:50 in previous studies was used and thereby IgA was found more frequently and more prominent in marmoset glomeruli than previously described.2,3 Considering the weak cross-reactivity of the antibody with marmoset IgA even a higher incidence and more prominent IgA-deposits in marmoset glomeruli might be expected using an antibody to marmoset IgA. IgM was a frequent finding in the glomeruli of marmosets with IC-GP, but occasionally it was also found in minor amounts and different pattern in the kidneys of monkeys from other primate species not suffering from the disease.
The marmosets of the German Primate Center (GPC) showed a spontaneous onset of disease and clinical features typical for Berger’s disease like hematuria and proteinuria. There was a good correlation between hematuria and proteinuria and the glomerular IgA-deposition in marmosets suffering from IC-GP. The IC-GP in marmosets might be a good and suitable animal model for Berger’s disease. Chronic tubulointerstitial nephritis and glomerulopathy in marmosets were reported several times.2,3,14,15 Given the diagnostic obstacles, requiring special histological staining methods (Movat, PAS-McManus, immunofluorescence techniques) and therefore a higher incidence of IC-GP in marmoset colonies might be expected.
The diet in captivity as well as intestinal lesions, infections and their immunologic and metabolic situation were considered as a possible reason for enteritis and nephropathy in marmosets.2 Also a certain role of gliadin from gluten in etiology and pathogenesis of IC-GP as well as in WMS in marmosets was assumed.12 Their low immunological competence might be important for the pathogenesis of immune complex glomerulopathy in marmosets as is assumed the disease in humans.10
There are further studies necessary about the occurrence and genetics of IC-GP in marmoset colonies. Having a suitable animal model now, there might be better chances to get information about the still enigmatic etiology and pathogenesis of Berger’s disease in humans.
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