Kidney biopsy in the hepatosplenic form of infection with Schistosoma mansoni in man*

Bull. Org. mond. Sante 1970, 42, 907-910 Bull. Wld Hlth Org. J Kidney Biopsy in the Hepatosplenic Form of Infection with Schistosoma mansoni in Man* L. CAETANO DA SILVA, M.D.,1 T. DE BRITO, M.D.,2 M. E. CAMARGO, M.D.,3 D. R. DE BONI, M.D.,4 J. D. LOPES, M.D.4 & J. GUNJI, M.D.4 A study of early glomerular lesions was made in 8 patients infected with Schistosoma mansoni but having no clinical evidence of renal disease. Electron-microscopy of renal biopsies showed the presence of electron-dense deposits in basement membranes and of laminated bodies near the mesangial cells. Immunofluorescence showed that the deposits corresponded to IgG in 8 cases and to IgM in 2 cases. These lesions are comparable with those found in the kidneys of patients with cirrhosis of the liver. During the last few years, there has been a growing impression among clinicians and pathologists that there is increased frequency of kidney diseases in patients with advanced forms of infection with Schistosoma mansoni. Light-microscopy studies of kidneys of schistosomiasis patients without clinical manifestations of renal disease disclosed local glomerular lesions, characterized by moderate proliferation of axial cells and focal thickening of the basement membrane (Andrade & Queiroz, 1968). In this paper, a study combining functional and microscopical observations with ultrastructural and immunofluorescence techniques on renal lesions in a small number of schistosomiasis patients is reported. Up to the present, except for a preliminary report by this group (Brito et al., 1969), no other reports of similar observations are known. MATERIALS AND METHODS Altogether, 8 patients with hepatosplenic schisto- somiasis and no clinical evidence of renal disease were studied. All ofthem had viable eggs in the stools, * From the Instituto de Medicina Tropical and the Hospital das Clinicas, University of Sao Paulo, Brazil. This work was supported by grants from the Instituto Brasileiro de Estudos e Pesquisas de Gastroenterologia and the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, and by grant No. DAHC 19-69-C-0014 from the US Army Research Office. Sec,ao de Hepatologia, Instituto de Medicina Tropical. 'Seccao de Anatomia Patologica, Instituto de Medicina Tropical. ' Sec,co de Imunologia, Instituto de Medicina Tropical. Hospital das Clinicas. except for patient No. 5 (F. A. M.) who had been treated with niridazole a year previously. After functional studies, including urine analysis, creatinine and phenolsulfonphthalein (PSP) clearance tests, a urine concentration test, blood-urea determi- nation and urography, kidney biopsies were per- formed on each patient. In 4 patients (No. 1, 2, 3 and 4) renal biopsies were performed before, and on the day immediately after, treatment with niridazole ended, to see whether renal changes are increased after massive deaths of worms. One healthy volun- teer was given the same drug treatment as the patients so that the possibility of the lesions being produced by the drug alone could be eliminated. Human kidney tissue was obtained by percutaneous renal biopsies and slices 0.3 mm-0.5 mm thick were cut with a razor-blade knife from different levels ofthe fragment and fixed for 2 hours at 5°C in 1% osmium tetroxide buffered to pH 7.4 with barbital acetate buffer. The slices were rapidly dehydrated in graded dilutions of ethanol, and were finally embedded in Araldyte resin. Thin sections were cut on a Porter- Blum microtome equipped with glass and diamond knives. The sections were double-stained, first in uranyl acetate (Luft, 1961) and then in lead citrate (Reynolds, 1963). The preparations were examined in an electron microscope (Zeiss EM 9). Fragments of kidney tissue not used for electron-microscopy were fixed in Helly's fluid, embedded in paraffin and stained with haematoxylin-eosin and periodic- acid-Schiff's reagent for light-microscopy. Thin sections of the material, also embedded in Araldyte, 2531 907_ 908 L. C. DA SILVA AND OTHERS RENAL FUNCTION IN PATIENTS WITH HEPATOSPLENIC FORMS OF SCHISTOSOMIASIS BEFORE AND AFTER TREATMENT WITH NIRIDAZOLE Creatinine Serum TConcentration Blo ra Serum proteins clearance (mra100 ml phaeninulo tests (specific Blood urea electrophoresisPatients (ml/min) blood) m phthalein ~f/ gravity of urine) (mg/'100 ml) (g/100 ml blood) Before After Before After Before After Before After Before After Albumin v-globulin 1. (O.P.R.) 88.2 86.7 0.70 0.80 65 65 1.026 1.026 25 26 2.98 3.40 2. (N.J.V.) 127.0 97.3 0.60 0.45 - - 1.026 1.024 23 24 3.25 4.40 3. (J.A. F.) 115.6 104.6 0.90 0.70 67 73 1.026 1.026 24 27 3.96 3.19 4. (H.M.J.) 118.0 98.0 0.70 0.80 86 83 1.027 1.026 25 34 2.76 1.69 5. (F.A.M.) - 107.0 - 1.10 - 70 - 1.026 - 50 3.14 1.94 6. (D.V.L.) a 99.0 - 0.50 - 87 - 1.025 - 27 - 2.87 1.68 7. (V.V.S.) 93.1 - 0.85 - 62 - 1.026 - 24 - 3.00 1.79 8. (S.E.S.) 118.8 - 0.05, - 66 - 1.030 - 33 - 3.17 3.39 9. (L.P.A.) b 120.3 96.6 0.70 0.80 78 75 1.030 1.031 16 20 3.23 1.82 a Previously submitted to splenorenal anastomosis. b Normal control. were stained with toluidine blue and examined by light-microscopy (Trump et al., 1961). For the fluorescent staining, biopsy fragments about 5 mm in length were immediately frozen in a liquid-nitrogen-isopentane mixture and kept at -20°C. Sections 4 ,u thick were cut in a cryostat and fixed on slides by drying. Direct staining was performed by incubating sections with diluted conjugates for 1 hour at 370C. Slides were washed for about 2 hours in several changes of a 0.1 5-M sodium chloride solution buffered to pH 7.2 with 0.01 M phosphate buffer, and mounted in alkaline glycerol at pH 8.0. The sections were examined under a binocular microscope provided with dark-field illumination, using x 20 dry and x 40 oil-immersion objectives, an HBO-200 lamp as the light source, a BG 12 filter as an exciter and a No. 50 filter (Zeiss) as a barrier. Conjugates were prepared by labelling the globulin fraction of rabbit immunosera with fluorescein isothiocyanate.1 A fluorochrome slow-addition dia- lysis technique (Clark & Shepard, 1963) was used, and the free fluorochrome was removed by gel- filtration in Sephadex G-25. Rabbits were immunized with chromatographically purified human IgG and 1gM. Immunosera were made specific by absorption 1 Crystalline, chromatographically pure isomer supplied by Baltimore Biological Laboratories, Md., USA. and gave immunoelectrophoretic precipitation lines only against the corresponding immunoglobulins. Anti-fl1c serum 2 was obtained in rabbits by injecting washed complexes of zymosan and fresh human serum, in a way similar to that described by Mardiney & Muller-Eberhard (1965). After absorption with human immunoglobulins, this antiserum produced only one immunoelectrophoretic precipitation line against human serum, corresponding to Pic. The fluorochrome : protein (F/P) weight ratios of the conjugates were between 10 and 15 and the conju- gates were diluted before use. For maximal reactivity, the dilutions contained not less than 1/4 precipitating unit per test sample, as defined by Beutner et al. (1967), and to avoid non-specific staining the concen- tration of fluorescein was not greater than 3 jug/ml. Controls for non-specific staining included the use of conjugates absorbed respectively with IgG, IgM or fresh human serum. Kidney sections obtained few hours post-mortem from a young accident victim were also included for comparison and showed no fluorescence with any of the conjugates. RESULTS Functional studies of the kidney disclosed no abnormalities before and after treatment with I Kindly provided by Professor C. Fava Netto, Faculty of Medicine, University of Sao Paulo. FIG. 1 ELECTRON-MICROGRAPH OF KIDNEY BIOPSY MATERIAL *v--~~~~~~~~~~~~~~~~~~~~~~~~~~N E -1#1_ LB Lari iviatecd body ovei muesanlqal maitrix (MM): FP unaltered foot process; arrows indicate small deposits. i, I p .: 1. . -t-, FIG. 2 ELECTRON-MICROGRAPH OF KIDNEY BIOPSY MATERIAL FP = Unaltered foot process; arrows indicate cleposits in the glomiierular basement milemlbrane close to a niesangial cell. FIG. 3 ELECTRON-MICROGRAPH OF KIDNEY BIOPSY MATERIAL 4N1 Arrows indicate deposits ini the glomerUlar basenient niemilbraneneear a niesangial cell (MC). There is slight fuLsion of the foot process (FP) n thiis area. FIG. 4 KIDNEY BIOPSY MATERIAL WITH FLUORESCENT STAINING Deposits of IgG aloicg the basement membrane; x 500. KIDNEY BIOPSY FIG. 5 MATERIAL WITH FLUORESCENT STAINING I)t1 rilori'' i I ! KIDNEY LESIONS IN HEPATOSPLENIC INFECTIONS WITH SCHISTOSOMA MANSONI niridazole (see accompanying table). The same was also true for urine analysis and urography. Light- microscopy showed essentially normal glomeruli, interstitial cells and tubules, though in a few glome- ruli there was a slight increase in the number of mesangial cells. Electron-microscopy showed hypertrophy and hyperplasia of mesangial cells. Both before and after treatment, electron-dense granular deposits, morphologically similar to those described in hepatic glomerulosclerosis (Fisher & Perez-Stable, 1968; Sakaguchi et al., 1965), were found in limited areas of the endothelial side of the basement mem- brane, usually near to or underneath mesangial cells (Fig. 1, 2, 3). Similar deposits were sometimes seen in the mesangial matrix as well as in limited areas of the mesangial cell cytoplasm. In no instance were depo- sits present on the epithelial side of the basement membrane like the " hump " described in acute glo- merulonephritis (Metcoff, 1967). Except for the depo- sits, the glomerular basement membrane appeared essentially normal. Rarely, the inner lamina rara was oedematous and with granular, faintly electron-dense material, resembling the subendothelial deposit of black particles described by Sakaguchi et al. (1965). Endothelial cells were unaltered and epithelial cells retained foot processes. Electron-dense linear areas were seen along the cytoplasmic border of the epithelial cells. Cytosomes were also observed to contain a mixture of oval structures with a well- defined contour and electron-dense material similar to the deposits described above. It was not possible to determine whether deposits were more frequent after treatment because of the limited number of glomeruli examined and the local nature of lesions in the glomeruli. Laminated bodies similar to those previously described in cirrhotic glomerulosclerosis (Sakaguchi et al., 1965) were also found nearby the mesangial cells, usually over the mesangial matrix (Fig. 1, 3). Immunofluorescence observations are in accord- ance with these ultrastructural findings. Confluent granular deposits of IgG along glomerular capillary walls were seen in all 8 cases. In 5 cases, anti-IgM staining was also employed and glomerular fluores- cence following the same pattern was obtained in only 2. Anti-flc staining wasperformed in 7 cases and was positive in all except 1 (Fig. 4, 5). Globulin-elution experiments were performed in cases No. 6 and No. 8 and previous treatment of sections with acid buffer caused the disappearance of the anti-IgM, but not of the IgG, staining in case No. 6, and of the IgG staining in case No. 8. No differences in staining were seen in sections treated with pH 7.2 buffer or with 2M NaCl. DISCUSSION Deposits were found in the kidneys ofpatients with hepatosplenic schistosomiasis without clinical evidence of renal disease. As shown by immuno- fluorescence, such deposits contain immunoglo- bulins and are located in the basement membrane near to mesangial cells. Laminated bodies were also observed close to the same cells. All these findings were previously described in kidney biopsies from patients with liver diseases (Sakaguchi et al., 1965), immunoglobulins being seen in the deposits but no antigen-antibody complexes could be demonstrated. In the present study, although complement was demonstrated in the sections, inflammation in the glomeruli was characterized mainly by hypertrophy and hyper- plasia of mesangial cells. Fisher & Perez-Stable (1968) noticed that all the patients with cirrhosis who exhibited renal insufficiency and pronounced intramembranous deposits had inversion of their serum albumin: gammaglobulin ratios. They speculated on the possibility that such deposits may represent protein trapped within the basement membrane. In the present studies, the fluorescence pattern of deposited immunoglobulins and complement is not suggestive of immunoreactivity of antibodies with a target-antigen at its histogenic site. The pattern rather suggests the deposition of immunocomplexes on the glomerular capillary walls. In order to show that immunocomplexes are involved in the patho- genesis of this disease, it is necessary to demonstrate the presence of S. mansoni antigens in the deposits. Such studies will be made at the Institute of Tropical Medicine, Sao Paulo. ACKNOWLEDGEMENTS The authors wish to acknowledge the technical assistance of Miss S. C. M. Felizetti and Miss C. Rebonato. 6 909 910 L. C. DA SILVA AND OTHERS RtSUMt BIOPSIE RtNALE DANS LA FORME HEPATOSPLtNIQUE DE L'INFECTION A SCHISTOSOMA MANSONI CHEZ L'HOMME Afin d'etudier les l6sions pr6coces du glom6rule renal dans la schistosomiase a Schistosoma mansoni, on a pra- tiqu6 des biopsies du rein chez 8 malades atteints de la forme hepatosplenique de l'affection mais ne presentant aucun signe clinique d'atteinte renale. L'examen au microscope 6lectronique a montre l'existence de depots opaques dans la membrane basale et de corps lamellaires a proximit6e des cellules du m6san- giome. Ces derni6res presentaient de l'hypertrophie et de I'hyperplasie. Chez 4 des malades, les biopsies ont ete effectu6es avant et imm6diatement apres un traitement par le niridazole pour v6rifier le r6le 6ventuel d'une destruction massive des parasites sur 1'evolution des lesions r6nales. Le petit nombre de glom6rules examin6s et le caractere local des l6sions n'ont pas permis de determiner si les depots etaient plus frequents apr6s le traitement. L'etude du materiel biopsique par immunofluorescence a montre que chez les 8 malades les d6pots correspon- daient a de l'immunoglobuline G et, dans 2 cas, a de l'immunoglobuline M. On a 6galement d6cele la presence de complement. Ce tableau evoque le depot d'immuno- complexes sur les parois capillaires glomerulaires. Les l6sions d6crites sont comparables a celles que l'on observe dans le rein des patients atteints de cirrhose hepatique. REFERENCES Andrade, Z. A. & Queiroz, A. C. de (1968) Rev. Inst. Med. trop. S. Paulo, 10, 36-40 Beutner, E. H., Holborow, E. J. & Johnson, G. D. 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