Some ultrastructural aspects of endemic nephropathy of the Balkans*

Bull. Org. mond. Sante 1972, 47, 37-48 Bull. Wld Hlth Org. Some ultrastructural aspects of endemic nephropathy of the Balkans * I. MORARU,1 P. NICOLESCU, E. MANDACHE, C. ZOSIN, N. MANESCU, & A. SCHWARZKOPF Two renal biopsies selected from a large number of specimens taken by renal puncture from patients suffering from endemic nephropathy were studied by electron microscopy. Ultrastructural lesions were seen as electron-dense deposits in the walls of the glomerular capillaries, sometimes associated with glomerular proliferative processes. Such lesions, which are probably the early stages of this obscure renal disease, suggest that an auto- immune mechanism may be involved. Around the year 1957, investigators in Yugoslavia, Bulgaria, and Romania described, more or less simul- taneously, a disease now known as endemic nephro- pathy of the Balkans (Zosin, 1967). Subsequent clinical, anatomical, and pathological studies, car- ried out in the three neighbouring countries, have provided some information on this disease. The results and theoretical conclusions already reported (Craciun et al., 1965; Bruckner et al., 1965; Craciun & Ro,culescu, 1965) have revealed a general con- sensus of opinion concerning the clinical aspects and histopathology of this obscure disease. Opinions dif- fer, however, about the physiopathological mecha- nism, and especially the etiology (Markovic & Lebedev, 1965; Tonea, 1966, 1967; Buli0, 1967; Mustata, 1969; Georgescu et al., 1969). The geographical character of the disease is evi- dent, and the groups most affected are middle-aged females (Zosin, 1967). The familial character is probably linked with proximity in the home (Bruck- ner & Lazarescu, 1967). The clinical picture is poor in symptoms and the onset is insidious (lumbar pains, pollakiuria, dysuria, slight albuminuria, anae- mia, oedema, and arterial hypertension). The dis- ease undergoes a long evolution (10-15 years) and ends in renal insufficiency (Tancev, 1963; Bruckner et al., 1965; Zosin et al., 1966; Bruckner, 1967; Puhlev, 1967). * From the Victor Babes Institute of Pathology and Medical Genetics, Bucharest, and the ITIrd Medical Clinic, Timisoara, Romania. This investigation received financial support from the World Health Organization. 1 Professor, Department of Electron Microscopy and Immunology, Victor Babes Institute. The histological lesions have formed the subject of many studies (Hall et al., 1965; Craciun et al., 1965; Puhlev, 1967; Bruckner et al., 1967; Para- schiv et al., 1967; Markovi6, 1968; Craciun & RoSculescu, 1970). Zosin et al. (1966) and Georgescu (1967) described the light microscopic appearance of the lesions, basing their analysis on many cases. They emphasized the diversity and lack of specificity in the histological picture, and the variety in the extent of the lesions within the same kidney. From studies of renal biopsies taken from apparently healthy inhabitants of the endemic area, they were able to differentiate the pathological aspects of advanced cases from those found in the early stages of the disease. In both types of case the glomerular lesions exhibited various aspects of glomerulitis (intracapillary, extracapillary, lobular or total scleros- ing, etc.); lesions of the tubules were more pro- nounced in the advanced cases. However, there is no general agreement about interpreting the evolu- tion of these lesions. Contrary to the opinion of some authors (Hall et al., 1965; Puhlev et al., 1965; Markovic & Lebedev, 1967), Zosin et al., (1966) and Georgescu (1967) supplied arguments to lend sup- port to the hypothesis of initial glomerular and sub- sequent tubular lesions. Detailed studies of these aspects have been obstructed by the absence of clin- ical or laboratory tests, the diagnosis being more difficult to establish in the initial stages of the dis- ease. If to the diagnostic difficulties the possibility of other pathogenic renal factors is added, bearing in mind the long evolution of endemic nephropathy of the Balkans, then the question arises of the origin 2871 37- 4 I. MORARU ET AL. of certain microscopic or rare ultrastructural fea- tures of the lesions reported by Georgescu et al., (1969) for example. Craciun & Rosculescu (1965), considering the glo- merular lesion as the initial lesion, suggested the possible intervention of certain autoaggressive pro- cesses in the pathogenesis of this nephropathy; such a mechanism has been increasingly implicated dur- ing the last few decades in the genesis of glomerulo- nephritis. Autoimmunity has also been implicated in the self-maintenance and chronic transformation of glomerulonephritis. Numerous experimental data lend support to these assumptions. Unanue & Dixon (1967) reported immunological mechanisms in the following experimental forms: nephrotoxic serum nephritis, nephritis induced by soluble antigen- antibody complexes, and nephritis induced by immu- nization with renal antigens. Gang & Kalant (1970) and Gang et al. (1970) studied the relationship be- tween structural alterations of the glomerular base- ment membrane and the onset of albuminuria in nephrotoxic serum nephritis. Their results suggest the existence of two mechanisms responsible for the production of proteinuria. The interaction between nephrotoxic antibodies and the glomerular basement membrane might result in a decrease of the phospho- lipids and a change at the molecular level in the conformation of the basement membrane proteins, which would increase their permeability to macro- molecules and result in albuminuria. Subsequently, intervention of the polymorphonuclear leucocytes (PMN) leads, following hydrolytic degradation of the basement membrane, to the filtration of serum albumins. Cochrane et al. (1965) experimentally established the role of PMN as mediators in lesions of the glomerular basement membrane. MATERIALS AND METHODS The study material was collected by renal biopsy from several patients from the endemic foci (region of Banat) who had a clinical symptomatology that tallied with the advanced phase of endemic nephro- pathy of the Balkans. After light and electron microscopic examinations, 2 cases were selected that showed a wide variety of lesions. The kidney fragments were fixed in 4% glutaral- dehyde adjusted to pH 7.4 with 0.1 M phosphate buffer at a temperature of 4°C. After the specimens had been washed with phosphate buffer solution for 24 hours they were again fixed in 1% osmium tetroxide buffered to pH 7.4. The material was embedded in Epon 812 and sections were cut with an LKB ultramicrotome. Thick sections stained with toluidine blue were used to select fields of study. Fine sections (about 50-70 nm) were con- trast stained with bis(acetato)dioxouranium (" uranyl acetate ") and lead citrate, and examined by means of a Zeiss EM 9A electron microscope. RESULTS Light microscopy Thick sections of all the specimens obtained by biopsy were examined and glomerular lesions of different types and intensities were found. About half the glomeruli exhibited various degrees of atro- phy up to complete sclerosis, some of them still containing cellular residues which, examined under the electron microscope, proved to be derived from the visceral epithelium. Other glomeruli exhibited partial sclerozing glomerulitis with permeable capil- lary lobules. Finally, a certain proportion of the glomeruli with a maintained cellular structure pre- sented lesions of the component elements of the capillary walls. These walls were thickened, and the reduced capillary lumen contained few erythrocytes (a sign of intracapillary glomerulitis). The convoluted tubules also presented epithelial lesions (dystrophic, atrophic, and infrequently hyper- trophic lesions of the epithelium of the proximal tubules). In the interstitium, apart from signs of sclerosis, inflammatory lymphohistiocytic cellular elements were also noted. Electron microscopy The changes appearing in the capillary wall in the intracapillary glomerulitis lesions first studied showed a pathological process affecting particularly the endocapillary cells (endothelium and mesangium) and the lamina densa (Fig. 1 and 2). The glomerular capillary walls appeared to be thickened on account of endothelial alterations, especially a thickening of the glomerular basement membrane. Signs of mem- branous glomerulitis, and to a lesser extent of proliferative glomerulitis, were encountered. Deposits of impressive size were formed in the lamina densa (Fig. 6 and 7). The localization of these deposits, which as a rule were less electron- dense than the basement membrane, was mostly intramembranous or subendothelial. The appear- ance of the deposit was sometimes nonhomogenous and granular (Fig. 5 and 6). Their formation was 38 - 4, A Li- .. V. . p '46i,'Tf.'~2 ; L;v4'jS 0i, T't~~S AMW.W.'% ."' Fig. 1. Glomerular capillary loops with thickened walls and narrowed lumina. D, massive subendothelial deposits; P, epithelial cells; E, endothelial cells; L, capillary lumen; Bm, base- ment membrane (x 4600). *4 JO 1*.t gp'"A ORL t _" mA Ai,i d 4 S ; x- ..... £',,_, A .1" . .. i4 ~ .s: .iA . 4 ;-14 -RI "':.. Sk -4r vi .".W t p danh_ ~ winm 4AL Fig. 2. Part of the glomerular capillary wall. E, endothelium; P, epithelium; Bm, basement membrane; D, deposits; L, lumen; F, intracellular filaments (x 17 220). .. Ir I ' , .t. .' r- 'f-'04 :. -,. . '. V..X (7. ,, .-r, u .! w4 t. * ;t', n' .,G ise .: k. 44 4'. 4;. . Jr - . k.- A FLm Fig. 3. Part of the glomerular capillary wall with electron-dense deposits and endothelial ballooning. E, endothelium; P, epithelium; N, epithelial cell nucleus; L, lumen; D, suben- dothelial deposits; BM, basement membrane; EB, endothelial ballooning; FP, fused pedicels(x 16 800). t "4"t6 " -# _i. A 'e" 7 1% fi F4.qw..s , y. i)A I. J$Ht1I. ' Fig. 4. Part of the glomerular capillary wall exhibiting granular, filamentous, and subendo- thelial deposits. RBC, red blood cell (x 16 800). I _vm MY: lj P 7 Fig. 5. Glomerular capillary loop exhibiting subendothelial deposits with various stuctures and densities (x 15 600). I I Ir -I, X 2^ 16, ,- I.':3 z~~~~~~~~~~~~~~~~~~~~~~~~ __;_2#., v f i i^ Fig. 6. Glomerular capillary loop with abundant subendothelial deposits (D) and endothelial ballooning (EB); E, endothelium (x 18200). A J t E-A '. "', ir, t "1,-14 ;b, i. APJ LIZ 1. "i, '% . 110% i4..4W-1 . .i. % ;.. -., . , t. .6 .A- ;,"'AMIEko 1 . PW. :. ., 'P. -., mk. '.,-Lab? I, ': w," ,I iU V ;- . - .1.rt -Ps,lFu 7-W k -~~~~~~~~~1 Fig. 7. Capillary wall with intramembranous and subendothelial deposits of different density (x 16 200). 'o,I .,I .- ULTRASTRUCTURAL STUDIES ON ENDEMIC NEPHROPATHY not uniform and sometimes subendothelial humps were developed (Fig. 4). When the deposit was intramembranous, two electron-dense layers were maintained on the two sides (subepithelial and sub- endothelial), recalling the basement membrane (Fig. 4). Thus, these intramembranous deposits appeared to be formed by cleavage of the lamina densa. The capillary endothelium underwent hypertrophy. The most frequent lesion was a discontinuous thick- ening of the endothelium that protruded into the capillary and narrowed the lumen still more. Apart from the thickening, there were frequent zones of ballooning of variable size in the endothelium (Fig. 3). Some of the thickened endothelia had a filamentous structure (Fig. 2). Zones with fused epithelial cell pedicles were also seen (Fig. 3). DISCUSS[ON The difficulty of identifying lesions and their characteristics in the extensive pathological picture of endemic nephropathy of the Balkans is reflected in the attempts to establish the site of the initial lesions (i.e., glomerulus or tubule). The investiga- tions carried out in Yugoslavia (Hall et al., 1965; Markovic & Lebedev, 1967) support the assumption that this nephropathy is primarily a tubulointer- stitial disease. The Romanian investigators maintain, on the basis of morphological arguments, the prior- ity of glomerular lesions. Zosin et al. (1966), in stu- dies of renal biopsies from many patients in the endemic region who had no clinical signs of renal disease, found that glomerular lesions were more frequent than tubular lesions. The ultrastructural findings reported here corroborate the clinical and experimental data and support the view that the glomerulus is the initial site of attack. The tubular lesions would therefore be a consequence of the glomerular lesions. The ultrastructural studies of proliferative glo- merulitis show the occurrence of frequent membra- nous glomerulitis alterations and relatively reduced endothelial thickenings. However, lesions of true proliferative glomerulitis were also observed. The main lesions in membranous glomerulitis are depo- sits, most of which are localized intramembranously. Their density to electrons is inferior to that of the basement membrane, and they have a finely granular appearance. The fibrinoid character of the lesions agrees with the descriptions given by Burkholder et al. (1970) of mixed glomerulonephritis. The size of the deposits suggests that they may have been formed after disturbances in the filtration properties of the glomerular basement membrane. It is sug- gested that after injury, the lamina densa becomes more permeable to macromolecules because of a decrease in the phospholipid component. A con- sequence of this change would be that low-molecular- weight plasma proteins could cross the lamina, which would represent a barrier only to heavy macromole- cules. The latter would be stopped favouring the formation of deposits. One consequence of the for- mation of deposits is that the lumina of glomerular capillaries become narrowed; endothelial and mes- angial alterations contribute in an equal measure to this narrowing. Our observations showed slight endothelial thickenings with numerous zones of bal- looning. Constriction of the capillary lumina by these two factors is at times so marked that red blood cells are unable to circulate; erythrocytes trapped in the lumen can be seen in sections. This phenomenon reduces the oxygen supply and the resulting hypoxia is likely to aggravate the already- formed lesions. Renal involvement with a long evolution during the course of endemic nephropathy seems to be caused by a progressive impairment and gradual exclusion of the renal function of the nephrons, the phenomenon of summation occurring with time. This suggests the action of a common etiological mechanism continuing throughout the whole course of the disease, either in its initial form or associated with, or continued by, maintenance immunological phenomena. This view is supported by the simul- taneous existence of glomerular lesions in various stages: membranous, proliferative, and sclerosing. These findings show that the " glomerular disease " affects only some of the glomeruli at first, and later, as the lesions progress, extends to other glomeruli. This successive and continuous evolution finally results in renal insufficiency, when the number of functional nephrons falls below a certain limit. Therefore, the ultrastructural lesions may be in- cluded in the initial sequence of aggression against the nephrons, either as a result of the initial mecha- nism of the disease or of the automaintenance pro- cesses. Correlation of the electron microscopic find- ings with clinical and experimental data on the pathology of glomerulonephritis shows that similar immunopathological mechanisms interfere in the maintenance and chronic evolution of endemic nephropathy of the Balkans. 47 48 I. MORARU ET AL. RtSUMt QUELQUES ASPECTS DES LESIONS RtNALES AU MICROSCOPE ELECTRONIQUE DANS LA NEPHROPATHIE ENDEMIQUE DES BALKANS La nephropathie endemique des Balkans est une affec- tion renale chronique, a evolution lente, qui est observ6e dans certaines regions de Yougoslavie, de Roumanie et de Bulgarie. On n'est pas arrive a ce jour a elucider son etiologie et sa pathogenie. Le materiel etudie ici a e choisi parmi une serie de biopsies renales prelevees chez des sujets presentant les signes cliniques de la maladie. L'examen des coupes au microscope electronique montre la presence de dep6ts opaques aux 6lectrons dans les parois des capillaires glo- m6rulaires, avec epaississement de la couche sous-endo- theliale et lesions de glomerulite. Ces modifications patho- logiques sont consider6es comme correspondant aux stades precoces de la maladie et plaident en faveur de l'hypothese qui situe le debut des alterations renales au niveau des glomerules. Selon les auteurs, il est probable que les lesions consta- tees soient dues a l'intervention de certains mecanismes d'auto-immunit6 declenchant la maladie et responsables de son evolution chronique. REFERENCES Bruckner, I. (1967) Arch. Un. med. Balkan., 5, 565-568 Bruckner, I. & Lazarescu, R. (1967) Med. Interna (Buc.), 19, 259-267 Bruckner, I. et al. (1965) [A clinical study of nephropathy of an endemic character in the People's Republic of Romania.] In: International Symposium on Endemic Nephropathy, Sofia, p. 25 Bruckner, I. et al. (1967) Med. Interna (Buc.) 8, 153-165 Bulic, F. (1967) Med. Interna (Buc.), 19, 277-280 Burkholder, P. M. (1970) Lab. Invest., 23, 459-479 Cochrane, C. G. et al. (1965) J. exp. Med., 122, 99 Craciun, E. et al. (1965) [Morphological observations con- cerning endemic nephropathy] In: International Sym- posium on Endemic Nephropathy, Sofia, p. 87 Cracuin, E. C. & Ro§culescu, I. (1965) A hypothetical etiology of endemic nephropathy. In: International Symposium on Endemic Nephropathy, Sofia, p. 93 Cracuin, E. C. & Ro~culescu, I. (1970) Amer. J. Med., 49, 774-779 Gang, N. F. & Kalant, N. (1970) Lab. Invest., 22, 531 Gang, N. F. et al. (1970) Lab. Invest., 23, 150-157 Georgescu, L. (1967) Med. Interna (Buc.), 19, 301-323 Georgescu, L. et al. (1969) Timi4oara med., 14, 345 Hall, P. W. et al. (1965) Amer. J. Med., 39, 210 Markovic, B. (1968) J. Urol. Nephrol., 74, 843 Markovic, B. & Levedev, S. (1965) Presse mid., 73, 401 Markovic, B. & Lebedev, S. (1967) Klin. Med. (Wien), 45, 91 Mustata, N. et al. (1969) Viata med., 16, 977 Paraschiv, D. et al. (1967) Med. Interna (Bue.), 19, 313-323 Poukhlev, A. et al. (1965) Arch. Un. mid. Balkan., 3, 559-564 Puhlev, A. (1967) Med. Interna (Buc.), 19, 293-300 Tancev, J. (1963) Med. Interna (Buc.), 15, 1313 Tonea, T. (1966) Med. Interna (Buc.), 18, 149-158 Tonea, T. (1967) Med. Interna (Buc.), 19, 333-340 Unanue, E. R. & Dixon, F. J. (1967) Experimental glo- merulonephritis: immunological events andpathogenetic mechanisms. In: Dixon, F. J. & Humphrey, J., ed., Advances in immunology, New York, Academic Press, vol. 6, p. 1 Zosin, C. (1967) Med. Interna (Buc.), 19, 269-275 Zosin, C. et al. (1966) Sem. Hop. Paris, 42, 194