Morphogenesis of Axial Sclerosis and Fibrosis Following Proliferative Glomerulitis and Their Significance for the Nephrotic Syndrome

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1 Tohoku J. exp. Med., 1978, 124, Morphogenesis of Axial Sclerosis and Fibrosis Following Proliferative Glomerulitis and Their Significance for the Nephrotic Syndrome TERUO FUJIMOTO Department of Pathology,* Osaka City University Medical School, Osaka 545 FuJIMOTO, T. Morphogenesis of Axial Sclerosis and Fibrosis Following Proliferative Glomerulitis and Their Significance for the Nephrotic Syndrome. Tohoku J. exp. Med., 1978, 124 (3), Mode of development of axial sclerosis and fibrosis of glomerular loops following proliferative glomerulitis was studied on the basis of renal biopsies. The analyses of biopsy specimens revealed that mild proliferative glomerulitis with preservation of capillary structure is followed by axial sclerosis or sclerosis of mesangial area of glomerular loops, whereas marked or severe proliferative glomerulitis characterized by disorganization of glomerular architecture due to proliferation of local fixed cells with reticular arrangement, chiefly of endothelial origin, following histolysis of loop walls results in crowding of proliferated cells accompanied by formation of fibers along them toward axis of the loop (axial fibrosis) and recanalization at its periphery. It was also clarified that when cell proliferation and following axial fibrosis of glomerulus are pronounced, lobulation of glomerular loops associated with further formation of centrolobular nodules is brought about, which is the lesion characteristic of lobular glomerulonephritis. Some clinical features concerning these processes were presented and discussed, and significance of glomerular lesions here presented for the nephrotic syndrome was considered. glomerulosclerosis; glomer ulonephritis; nephrotic syndrome; renal biopsy; histogenesis For the past 25 years our knowledge of the pathology of nephrotic syndrome has been greatly amplified by numerous workers, employing continued improvements in technique, such as applications of PAS and PAM staining methods, electron microscopy, fluorescent antibody technique, etc. to the analyses of kidney specimens, especially those obtained at percutaneous biopsies as well as introducing experimental pathological studies into the understanding of the pathologic processes. These studies have revealed the histologic characteristics of glomerular changes which are contributory to the nephrotic syndrome, i.e., minimal changes, Received for publication. August 23, This study was supported by a Research Grant from the Intractable Diseases Divi sion, Public Health Bureau, Ministry of Health and Welfare, Japan, and this paper was presented in part at the Annual Meeting of the Ministry of Health and Welfare Nephrotic Syndrome Research Committee, Japan, February 9, 1977, Tokyo. * Director: Prof. T. Fujimoto. Mailing address: T. Fujimoto, M.D., Department of Pathology, Osaka City University Medical School, Asahi-machi, , Abeno-ku, Osaka, 545 Japan. 285

2 286 T. Fujimoto proliferative, membranous, lobular, membranoproliferative, and sclerosing glomerulonephritis, diabetic glomerulosclerosis, glomerular amyloidosis, bilateral renal vein thrombosis, etc. (Allen 1951, 1955; Jones 1957; Burch et al. 1962; Mandalenakis et al. 1971). But, histogenetical observation or understanding of these glomerular lesions from the standpoint of glomerular functional structure and its alterations has not been sufficiently obtained so far. It is the purpose of this paper to present the mode of development of axial sclerosis and fibrosis of glomerular loops following proliferative glomerulitis clarified by analyses of biopsy specimens and consider the significance of these histologic manifestations for the nephrotic syndrome. MATERIALS AND METHODS The materials consisted of 184 biopsied cases showing generalized occurrence of axial sclerosis and/or axial fibrosis of glomerular loops. One hundred and sixty-three cases (102 males and 61 females) of them were selected from 274 biopsied cases of nephrotic syn drome (proteinuria of more than 3.5 g/day, hypoproteinemia of less than 6.0 g/100 ml, serum total cholesterol level of more than 250 mg/100 ml in adults and children of school age and more than 220 mg/100 ml in low-aged children, and edema) of the hospitals to which members of the Ministry of Health and Welfare Nephrotic Syndrome Research Committee were attached (Group 1), and the remaining 21 cases (13 males and 8 females) were collected from 45 cases having nephrotic syndrome out of 500 renal biopsy cases of Miwa Medical Clinic of the Chiba University Hospital from 1958 to 1964 (Group 2) (Table 1). Renal biopsy specimens of these examined cases were formalin-fixed, embedded in paraffin, cut at 3 to 4 Đm, and treated with hematoxylin and eosin (HE) stain, PAS stain, and occasionally with PAM stain. RESULTS Axial sclerosis. Glomerular loops showed preservation of capillary structure, but the lumens were dilated, containing a moderate number of erythrocytes. An increase of mesangial matrix (sclerosis) was characteristically recognized, which was occasionally associated with crowding of fixed cells due to contraction of the tissue (Figs. 1 and 2). Swelling or slight proliferation of glomerular endothelial cells was visible not infrequently. In some instances proliferation of glomerular epithelial cells was moderate. In a few cases having repeated biopsies, it was clarified that such glomerular changes as described above were lesions subsequent to mild proliferative glomerulitis with comparatively well preserved structure, which was characterized by edematous swelling of loop walls, especially of mesangium, some endothelial and occasional mesangial cell proliferation and/or accumulation of neutrophils and monocytes in capillary lumens of glomerular loops. Axial fibrosis. Glomerular loops demonstrated disorganization of the archi tecture, which was the most characteristic and fundamental feature of this lesion. In the axial portion of loop, which was not restricted to the original mesangium, there appeared crowding of proliferated cells chiefly of endothelial origin, accompanied by formation of fibrils or fibers along these cells and an increase of some matrix, toward axis of the loop, whereas blood spaces were recognizable in

3 Axial Sclerosis and Fiborsis in Nephrotic Syndrome 287 Fig. 1. Axial sclerosis of glomerulus. PAS stain Osaka Univ. Pediatr. P year-old female. Fig. 2. Axial sclerosis of glomerulus with crowding of some fixed cells. PAS stain Osaka Univ. 1st Med. Clin year-old male. the peripheral portion (Fig. 3). It was conspicuous and worthy of note that the proliferated cells crowding toward axial portion were connected by their cytoplasmic processes and arranged in reticular pattern, sometimes showing small spaces between them (Fig. 3). The peripheral blood spaces contained various numbers of erythrocytes. Glomerular epithelial cells exhibited swelling or some proliferation not infrequently. In a very small number of cases of serial

4 288 T. Fujimoto Fig. 3. Axial fibrosis of glomerulus. Some small spaces between proliferated cells are visible (indicated with arrows). PAS stain Hirosaki Univ. 2nd Med. Clin. B year-old male. Fig. 4. Lobulation of glomerulus. PAS stain Nagoya Univ. Branch Med. Clin year-old female. biopsies it was confirmed that the initial stage of the lesions presented here was characterized by transformation of the glomerular capillary and mesangial tissue into an enmeshed or reticular structure due to proliferation of local fixed cells, chiefly of endothelial origin, following histolysis which was more conspicuous in the axial side of glomerular loop (Fig. 5). In the exaggerated state of axial fibrosis crowding

5 Axial Sclerosis and Fibrosis in Nephrotic Syndrome 289 Fig. 5. Proliferative glomerulitis. HE stain Chiba Univ. Miwa Med. Clin. B year-old male. Fig. 6. Lobulation of glomerulus with some hyalinization of centrolobular nodules. PAS stain Chiba Univ. Miwa Med. Clin. B year-old male. of the proliferated cells toward axis was outstanding and lobular appearance of glomerular loops became manifest (lobulation) (Figs. 4 and 6). In the advanced lesion the crowded cells decreased in number and hyalinizing process took place in the centrolobular areas (Fig. 6). In these glomerular lesions peripheral wall or its basement membrane was not thickened but single-contoured generally. Some clinical features. Outline of the cases including age at renal biopsies,

6 290 T. Fujimoto period between onset of symptoms and biopsy, relation to Ellis' typing of nephritis, etc. is demonstrated in Table 1. In 163 cases of Group 1 responses to drug therapy (including that of steroids and immunosuppressants) were followed up by the members of the Ministry of Health and Welfare Nephrotic Syndrome Research Committee for the periods of more than 2 years in 22 cases, from 1 to 2 years in 28 cases, and less than 1 year in 113 cases, and the outcome of the patients including these data which were available from the records attached to biopsy specimens is shown in Table 2. The patterns of responses to drug therapy accord ing to the Committee (Ueda 1976) are as follows: TABLE 1. Outline of the cases TABLE 2. Outcome of The figures express the numbers of patients.

7 Axial Sclerosis and Fibrosis in Nephrotic Syndrome 291 Complete remission... no demonstrable proteinuria Incomplete remission I...proteinuria less than 1 g/day Incomplete remission U... proteinuria more than 1 g/day Worsening In this Table 2 histologic characteristics of glomerulonephritis are expressed by the following terms : Healing: Glomeruli show generalized occurrence of axial sclerosis or axial fibrosis, and capillaries contain a moderate number of erythrocytes, but otherwise renal tissues are normal. Prolonged: Glomeruli show generalized occurrence of axial sclerosis and/or axial fibrosis, and blood capillaries contain some erythrocytes. Proteinaceous exudations into Bowman's capsular spaces, or some intracapsular hemorrhages are shown. Atrophy and sclerosis of a small number of proximal convoluted tubules are occasionally visible. Recurrent: Besides widespread development of axial sclerosis and/or axial fibrosis, recent proliferative glomerulitis is demonstrated. Chronic: Recent proliferative glomerulitis, glomerular hyalinization, capsular changes, various phases of urinary tubular changes, etc. are recognizable besides widely distributed axial sclerosis and/or axial fibrosis. Arteriolar changes are shown not infrequently. Lobular: Glomeruli show generalized enlargement with lobulation and centrolobular nodules, cellular to hyalinized, and peripherally arranged blood spaces. Items of cases of lobular glomerulonephritis are shown in Table 3. DISCUSSION Allen (1951, 1955) applied the term "chronic lobular glomerulonephritis" to a specific histologic lesion which was always associated with a nephrotic syndrome complicated by hypertension and often by renal insufficiency, hematuria, etc. the patients (Group 1)

8 292 T. Fujimoto TABLE 3. Lobular glomerulonephritis The histologic lesion was characterized by transformation of glomerular loops into several acidophilic nodules surrounded by dilated capillaries. Allen (1955) offered his impression that lobular glomerulonephritis represents a more fulminant variant of membranous glomerulonephritis in which the wall of many of the glomerular capillaries have undergone marked fibrinoid swelling and even coalescence, and in which exudative and proliferative glomerulonephritis have often been super imposed. But, it must be pointed out that before Allen there had been some contributions to the understanding of this lesion. Already in 1936 Kimmelstiel and Wilson mentioned that in the subchronic stage, intracapillary glomerulo nephritis was frequently complicated by the nephrotic syndrome and characterized histologically by occurrence of a peculiar hyalinization of glomerulus which accentuated its lobulation in the same way as in intercapillary glomerulosclerosis. Ellis (1942) described that in several instances of type 2 nephritis there was a slight proliferative glomerulitis with a tendency to increased lobulation of the tufts, which became more definite with a rather longer duration, leading finally to a picture characterized by focal deposition of hyaline material at the center of lobules of glomerular tufts. Bell (1950), considering glomerulus of the human kidney as a simple contortion of capillaries lacking any stromal elements, pointed out as follows: "The basement membrane of the outer surface of the capillaries is intact but the inner layers are split into numerous pieces which appear as hyaline fibers. At the lesion progresses the hyaline fibers increase in number and become

9 Axial Sclerosis and Fibrosis in Nephrotic Syndrome 293 fused to form a solid mass in the center of the lobule. The capillaries of the lobule are completely rearranged; the lobule shows central hyaline mass and the blood circulates at its periphery." This was the glomerular change which he called chronic latent glomerulonephritis. He also stated that such chronic proliferative glomerulonephritis with lobulation often corresponded clinically to lipoid nephrosis of mixed type. Jones (1957), believing the nature of glomerulo nephritis simply as pericapillaritis of glomerulus, considered that the glomerular lesion in chronic lobular glomerulonephritis in adults is an exaggerated form of mesangial scar, which is minimal in nephrotic children and becomes moderate in older children and young adults with nephrotic syndrome. Mandalenakis et al. (1971) pointed out in a case, 16-year-old male, which had serial renal biopsies that in the initial biopsy there was minimal sclerotic nodule formation, but sequential biopsy studies revealed appearance of well-defined nodules which were pronounced at autopsy. Through their studies including this case they considered that mesangial hypercellularity appears to precede nodule formation, although varying degrees of mesangial proliferation persisted in the presence of well-formed nodules. Allen (1955) also pointed out that in the days of antibiotic therapy chronic membranous and lobular glomerulonephritis were seen less frequently at autopsy, while there were observed a great many instances of intermediate as well as final stages in the transformation of chronic membranous and lobular glomerulonephritis into sclerosing glomerulonephritis. Burch et al. (1962), in the pathologic classifica tion of so-called primary form of nephrotic syndrome, presented a category, sclerosing glomerulonephritis, which is characterized by diffuse and generalized glomerular disease with partial or complete hyalinization and obliteration. Indeed there have been many literatures available as to nephrotic syndrome, but even those based on sequential biopsy studies have not sufficiently elucidated a precise mode of development of glomerular changes characteristic of lobular glomerulonephritis as yet. And, differentiation between sclerosis which is characterized by increase of fibers and ground substance of the tissue associated with little proliferation of fibroblastic cells, and fibrosis subsequent to disorganiza tion of tissue architecture accompanied by mesenchymal cell proliferation has not strictly been fulfilled. Fujimoto (1954a) studied structural changes of glomeruli in Masugi nephritis induced in rabbits by injection of anti-rabbit-kidney duck serum, and ascertained the mode of development of glomerular changes in the same individual animals on the basis of consecutive biopsies. The typical glomerular changes appeared simultaneously with the onset of albuminuria, and were characterized by dilatation and elongation of capillaries, proliferation of local fixed cells with reticular arrangement chiefly of endothelial origin, and anemia of glomerular loops, i.e., transformation of glomerular capillary and intercapillary connective tissue into enmeshed structure, or disorganization of glomerular architecture following histo lysis. The subsequent course was characterized by total or partial (axial) fibrosis (fibrosis in organisation) of such a new structural material. Succeeding to

10 294 T. Fuiimoto this report he (1954b) presented a description of autopsied cases of acute diffuse glomerulonephritis having typical glomerular changes entirely comparable in their histogenetical characteristics to those of Masugi nephritis. It was worthy of note that a glomerulonephritis characterized by generalized occurrence of marked axial fibrosis, simulating lobular glomerulonephritis, of only 23 days duration of symptoms which developed in a 19-month-old girl was described in this paper. Fujimoto and Akashi (1955) also described the edematous-sclerosing processes in glomeruli revealed by histopathological study of Masugi nephritis in rabbits employ ing repeated biopsies. According to the study the initial stage of the processes was characterized by dilatation of capillaries, swelling and some proliferation of endothelial cells, accumulation of a few neutrophils and mononuclears in capillary lumens, anemia, and edematous swelling of capillary walls, and the subsequent stage by diffuse (axial and peripheral) or axial sclerosis (fibrosis in situ) in the swollen capillary walls. Human glomerulonephritis showing glomerular lesions comparable to those of Masugi nephritis was also described in the same paper. In a presented case, 23-year-old male, having glomerulonephritis with generalized development of axial sclerosis of glomeruli, the duration of symptoms was only 11 days when autopsied. These modes of development of glomerular changes in Masugi nephritis were later confirmed by histopathological analyses employing electron microscopy as well (Fujimoto et al. 1964). The present study based on renal biopsies including some sequential observa tions revealed that mild proliferative glomerulitis with preservation of capillary structure is followed by axial sclerosis of glomerular loops, whereas marked or severe proliferative glomerulitis characterized by disorganization of glomerular architecture following its histolysis results in axial fibrosis which occurs in the newly formed structure and is accompanied by recovery of glomerular blood circulation due to recanalization in the periphery of the structure. Accordingly, strict differentiation between axial sclerosis and axial fibrosis of glomeruli is eagerly required. Axial fibrosis is not a mere exaggerated state of axial sclerosis. As already mentioned, fibrosing process in the axial fibrosis is not restricted to the original mesangial areas, which is the reason why the author prefers to call the process "axial" fibrosis. Development of axial sclerosis or fibrosis seems to be largely conditioned by the fact that edematous swelling or histolysis in the initial stage of proliferative glomerulitis is likely to take place in the mesangial and adjacent tissue. Moreover, requirement of the body to regain glomerular capillary circulation and physiologic state of the filtration membrane may also contribute to a recovery of glomerular functional structure. It was further clarified that the characteristic feature of glomeruli in lobular glomerulonephritis is an exaggerated state of axial fibrosis, in which various phases of the serial pathologic processes, i.e. from marked cellular proliferation, fibrosis to hyalinization of centrolobular nodules of glomeruli are demonstrated. As shown in Table 1, the majority of cases of axial sclerosis (72 out of 80 cases) corresponded to Ellis' type 2 nephritis and the remaining 8 cases did to Ellis' type

11 Axial Sclerosis and Fibrosis in Nephrotic Syndrome 295 1, whereas axial fibrosis was recognized in 11 cases of Ellis' type 1 nephritis and 28 cases of Ellis' type 2. As to lobular glomerulonephritis shown in Table 3, 2 cases fell into Ellis' type 1 nephritis and 4 did into Ellis' type 2. The result was different from that pointed out by Ellis (1942). According to Table 2, 57 out of 76 cases over 15 years of age showed prolonged glomerulonephritis with axial sclerosis of glomeruli, which resulted in complete remission (21 cases), incomplete remission I (17 cases), incomplete remission II (6 cases), and worsening (8 cases), while in 11 out of 18 cases under 15 years of age 8 cases showed complete remission and one case incomplete remission I. Twenty-five out of 39 patients over 15 years of age showed prolonged glomerulonephritis with axial fibrosis of glomeruli, which terminated in complete remission (9 cases), incomplete remission I (8 cases), incomplete remission U(4 cases), and worsening (one case), whereas 6 out of 13 cases under 15 years of age, even developing recurrent glomerulonephritis with axial fibrosis of glomeruli, resulted in complete remission in all 6 cases. In 2 out of 39 cases over 15 years of age having axial fibrosis and 4 out of 13 cases under 15 years of age with the same lesion there developed lobular glomerulonephritis, which terminated in incomplete remission I (one case) and worsening (one case) in the former, and worsening (3 cases) in the latter. Twelve out of 15 cases over 15 years of age which had prolonged glomerulonephritis with combination of axial sclerosis and axial fibrosis resulted in complete remission (4 cases), incomplete remission I (one case), incomplete remis sion U (2 cases), and worsenting (3 cases), while 2 cases under 15 years of age consisted of one case of prolonged glomerulonephritis terminated in complete remission and one case with the course which was not fully followed up. The glomerular lesions here presented, irrespective of alterations of glomerular functional structure, are characterized by preservation of glomerular circulation to a certain extent, which appears one of the requisites for the occurrence and persistence of nephrotic syndrome. Accordingly, increased permeability of the glomerular loop wall or filtration membrane due to these glomerular lesions, in the case of generalized occurrence, seems to play an important role in the disease processes. Elucidation of the structural characteristics and various other properties of the filtration membrane responsible for its increased permeability awaits further efforts. As to the outcome of the disease processes it might be said through the present study in the following ways: First, these glomerular lesions tend to appear most frequently in prolonged glomerulonephritis, but nevertheless the disease is liable to healing or complete remission to some extent especially in the patients under 15 years of age. Secondly, recurrent glomerulonephritis with generalized occurrence of axial sclerosis and/or axial fibrosis has a tendency to termination in complete remission in the patients under 15 years of age, whereas this is not the case in the patients over 15 years of age. And thirdly, in the case of chronic glomerulonephritis with nephrotic syndrome, even when most of the functioning glomeruli show axial sclerosis and/or axial fibrosis, recovery of the disease is not expected.

12 296 T. Fujimoto Acknowledgment I am grateful to members of the Ministry of Health and Welfare NephrotieSyndrome Research Committee, Japan, Emeritus Prof. S. Miwa and Dr. K. Hirose, China university,. Chiba, and Prof. S. Tojo, Tsukuba University, Ibaragi, for supplying :the renal biopsy specimens with attached clinical data used in this study. References 1) Allen, A.C. (1951) The Kidney. Medical and Surgical Diseases. 1st Ed., Grune and Stratton, New York, pp ) Allen, A.C. (1955) The clinicopathologic meaning of the nephrotic syndrome. Amer. J. Med., 18, ) Bell, E.T. (1950) Renal Diseases. 2nd Ed., Lea and Febiger, Philadelphia, pp ) Burch, R.R., Pearl, M.A. & Sternberg, W.H. (1962) A clinicopathological study of the nephrotic syndrome. Ann. intern. Med., 56, ) Ellis, A. (1942) Natural history of Bright's disease. Clinical, histological and experimental observations. I. Nephritis. Lancet, 1, ) Fujimoto, T. (1954a) Histopathologic study of Masugi nephritis. The mode of development of the glomerular changes. Acta path. jap., 4, ) Fujimoto, T. (1954b) Histopathologic study of diffuse glomerulonephritis. The mode of development of the glomerular changes. Acta path. jap., 4, ) Fujimoto, T. & Akashi, S. (1955) Mode of development of the edematous-sclerosing processes in glomeruli. Histopathologic study of Masugi nephritis. Osaka City med. J., 2, ) Fujimoto, T., Okada, M., Kondo, Y. & Tada, T. (1964) The nature of Masugi nephritis. Histo and immunopathological studies. Acta path. jap., 14, ) Jones, D.B. (1957) Nephrotic glomerulonephritis. Amer. J. Path., 33, ) Kimmelstiel, P. & Wilson, C. (1936) Intercapillary lesions in the glomeruli of the kidney. Amer. J. Path., 12, ) Mandalenakis, N., Mendoza, N., Pirani, C.L. & Pollak, V.E. (1971) Lobular glomeru lonephritis and membranoproliferative glomerulonephritis. A clinical and pathologic study based on renal biopsies. Medicine, 50, ) Ueda, Y. (1976) Nephrotic syndrome in Japan. Jeep. J. Med., 15,