Podocytes undergo phenotypic changes and express macrophagic-associated markers in idiopathic collapsing glomerulopathy
1998; Elsevier BV; Volume: 53; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1755.1998.00845.x
ISSN1523-1755
AutoresJ Bariéty, Dominique Nochy, Chantal Mandet, C. Jacquot, Denis Glotz, A Meyrier,
Tópico(s)Abdominal vascular conditions and treatments
ResumoPodocytes undergo phenotypic changes and express macrophagic-associated markers in idiopathic collapsing glomerulopathy. Collapsing glomerulopathy (CG), a severe form of focal segmental glomerulosclerosis (FSG), is characterized by tuft retraction and consolidation in numerous glomeruli and changes in podocyte morphology and topography. Other glomeruli are less affected. Collapsing glomerulopathy is also characterized by tubulointerstitial atrophy and fibrosis. The pathophysiology of the glomerular and tubulointerstitial lesions is poorly understood. We studied renal tissue of five Black and three White patients, all human immunodeficiency virus (HIV) negative, with nephrotic syndrome, renal failure, and histological evidence of CG. Immunohistochemistry identified normal podocyte phenotypes by podocalyxin, vimentin and complement receptor 1 (CR1) labeling. Three monoclonal antibodies were used to further characterize podocyte epitopes: anti-CD68 clone KP1, anti-CD68 clone PG-M1 and anti-M130 clone M18 (Ber-MAC3). Light microscopy of collapsed glomeruli showed podocyte swelling, vacuolization, multinucleation, “cobblestone-like” alignment around the glomerular tuft, and pseudo-crescent formation in Bowman's space. In collapsed glomeruli, podocalyxin, vimentin and CR1 labeling tagged both normal and vacuolated podocytes still attached to the GBM, but labeling was not found in cobblestone-like podocytes or in podocytes detached from the GBM. Conversely, numerous podocytes undergoing detachment and shedding into Bowman's space expressed macrophagic-associated epitopes. Cells with macrophagic-associated epitopes clumped in cystically dilated tubules and were aligned in tubules of smaller caliber. Their appearance was that of viable cells. There was no morphologic indication that these cells expressing macrophage-associated antigens originated from outside the glomeruli or outside the tubules. We conclude that in CG podocytes detach from the GBM, lose their normal podocytic phenotype and acquire macrophage differentiation antigens. The presence of cells with such antigens in tubular lumens suggests that detached metaplastic podocytes progress along the tubule or, alternatively, that CG tubular cells also undergo metaplastic changes into macrophage-like cells. Podocytes undergo phenotypic changes and express macrophagic-associated markers in idiopathic collapsing glomerulopathy. Collapsing glomerulopathy (CG), a severe form of focal segmental glomerulosclerosis (FSG), is characterized by tuft retraction and consolidation in numerous glomeruli and changes in podocyte morphology and topography. Other glomeruli are less affected. Collapsing glomerulopathy is also characterized by tubulointerstitial atrophy and fibrosis. The pathophysiology of the glomerular and tubulointerstitial lesions is poorly understood. We studied renal tissue of five Black and three White patients, all human immunodeficiency virus (HIV) negative, with nephrotic syndrome, renal failure, and histological evidence of CG. Immunohistochemistry identified normal podocyte phenotypes by podocalyxin, vimentin and complement receptor 1 (CR1) labeling. Three monoclonal antibodies were used to further characterize podocyte epitopes: anti-CD68 clone KP1, anti-CD68 clone PG-M1 and anti-M130 clone M18 (Ber-MAC3). Light microscopy of collapsed glomeruli showed podocyte swelling, vacuolization, multinucleation, “cobblestone-like” alignment around the glomerular tuft, and pseudo-crescent formation in Bowman's space. In collapsed glomeruli, podocalyxin, vimentin and CR1 labeling tagged both normal and vacuolated podocytes still attached to the GBM, but labeling was not found in cobblestone-like podocytes or in podocytes detached from the GBM. Conversely, numerous podocytes undergoing detachment and shedding into Bowman's space expressed macrophagic-associated epitopes. Cells with macrophagic-associated epitopes clumped in cystically dilated tubules and were aligned in tubules of smaller caliber. Their appearance was that of viable cells. There was no morphologic indication that these cells expressing macrophage-associated antigens originated from outside the glomeruli or outside the tubules. We conclude that in CG podocytes detach from the GBM, lose their normal podocytic phenotype and acquire macrophage differentiation antigens. The presence of cells with such antigens in tubular lumens suggests that detached metaplastic podocytes progress along the tubule or, alternatively, that CG tubular cells also undergo metaplastic changes into macrophage-like cells. Focal segmental glomerulosclerosis is a nonspecific glomerular lesion1.Cameron J.S. 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Focal glomerular sclerosis in nephrotic patients: An electron microscopic study of glomerular podocytes.Kidney Int. 1975; 7: 111-122Abstract Full Text PDF PubMed Scopus (138) Google Scholar. Podocyte changes consisted of cytoplasmic degeneration and detachment of visceral epithelial cells from basement membranes with filling of the resulting space by cell debris and extracellular matrix. Ten years later, Schwartz and Lewis shed further light on the cellular lesion of FSG25.Schwartz M.M. Lewis E.J. Focal segmental glomerular sclerosis: The cellular lesion.Kidney Int. 1985; 28: 968-974Abstract Full Text PDF PubMed Scopus (129) Google Scholar. Their description showed segmental proliferation, hypercellularity in Bowman's space and reactive changes in the podocytes. Electron microscopy revealed cytoplasmic inclusions, absorption droplets within hypertrophied visceral epithelial cells and separation of some of these podocytes from the glomerular basement membrane (GBM). This work highlighted the key role of podocyte injury in the genesis of capillary tuft collapse followed by sclerosis. Several further observations made on human renal biopsies26.Verani R.R. Hawkins E.P. Recurrent focal segmental glomerulosclerosis. A pathological study of the early lesion.Am J Nephrol. 1986; 6: 263-270Crossref PubMed Google Scholar, 27.SCHWARTZ M.M. Korbet S.M. Rydell J. Borok R. Genchi R. Primary focal segmental glomerular sclerosis in adults: Prognostic value of histologic variants.Am J Kidney Dis. 1995; 25: 845-852Abstract Full Text PDF PubMed Scopus (80) Google Scholar, 28.Schwartz M.M. KORBET S.M. Primary focal segmental glomerulosclerosis: Pathology, histological variants, and pathogenesis.Am J Kidney Dis. 1993; 22: 874-883Abstract Full Text PDF PubMed Scopus (86) Google Scholar, 29.Yoshikawa N. Hiroshi I. Akamatsu R. Hazikano H. Okada S. Matsuo T. 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MEYER T.W. Rennke H.G. Glomerular hypertrophy and epithelial cell injury modulate progressive glomerulosclerosis in the rat.Lab Invest. 1989; 60: 205-218PubMed Google Scholar, 32.NAGATA M. Schärer K. Kriz W. Glomerular damage after uninephrectomy in young rats. I. Hypertrophy and distortion of capillary architecture.Kidney Int. 1992; 42: 136-147Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 33.Nagata M. KRIZ W. Glomerular damage after uninephrectomy in young rats. II. Mechanical stress on podocytes as a pathway to sclerosis.Kidney Int. 1992; 42: 148-160Abstract Full Text PDF PubMed Scopus (226) Google Scholar confirmed that the podocyte is the primary culprit in the development of FSG. Primary collapsing glomerulopathy is a particular form of FSG that has recently elicited considerable interest. Collapsing FSG is not a new entity but its incidence has increased over the last twenty years34.Valeri A. Barisoni L. Appel G.B. Seigle R. D’AGATI V. Idiopathic collapsing focal segmental glomerulosclerosis: A clinicopathologic study.Kidney Int. 1996; 50: 1734-1746Abstract Full Text PDF PubMed Scopus (237) Google Scholar, 35.Detwiler R.K. Falk R.J. Hogan S.L. Jennette J.C. Collapsing glomerulopathy: A clinically and pathologically distinct variant of focal segmental glomerulosclerosis.Kidney Int. 1994; 45: 1416-1424Abstract Full Text PDF PubMed Scopus (267) Google Scholar, 36.Weiss M.A. Daquioag E. Margolin E.G. Pollack V.E. Nephrotic syndrome, progressive irreversible renal failure, and glomerular “collapse”: A new clinicopathologic entity?.Am J Kidney Dis. 1986; 7: 720-728Abstract Full Text PDF Scopus (152) Google Scholar, leading some nephrologists to evoke a possible infectious/viral etiology34.Valeri A. Barisoni L. Appel G.B. Seigle R. D’AGATI V. Idiopathic collapsing focal segmental glomerulosclerosis: A clinicopathologic study.Kidney Int. 1996; 50: 1734-1746Abstract Full Text PDF PubMed Scopus (237) Google Scholar. The podocyte lesion that characterizes FSG, including collapsing glomerulopathy28.Schwartz M.M. KORBET S.M. Primary focal segmental glomerulosclerosis: Pathology, histological variants, and pathogenesis.Am J Kidney Dis. 1993; 22: 874-883Abstract Full Text PDF PubMed Scopus (86) Google Scholar, 30.Korbet S.M. SCHWARTZ M.M. LEWIS E.J. Recurrent nephrotic syndrome in renal allografts.Am J Kidney Dis. 1988; 11: 270-276Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 34.Valeri A. Barisoni L. Appel G.B. Seigle R. D’AGATI V. Idiopathic collapsing focal segmental glomerulosclerosis: A clinicopathologic study.Kidney Int. 1996; 50: 1734-1746Abstract Full Text PDF PubMed Scopus (237) Google Scholar, consists of swelling, vacuolization, protein storage and lysosomal multiplication. Such modified podocytes line up around the tuft and assume a “cobblestone” appearance. Visceral epithelial cells detach from the underlying GBM, and the resulting space, filled with new matrix material, appears as a clear halo by light microscopy. In collapsing FSG numerous podocytes clump in Bowman's space, forming a pseudo-crescent. In the most severely damaged glomeruli the whole tuft collapses. In other, less retracted glomeruli adhesions or “synechia” form between peripheral loops of the tuft and Bowman's capsule. The podocytes and the opposing epithelial cells of Bowman's capsule are pushed aside by synechia, leading to the typical lesion of FSG. These glomerular lesions are accompanied by tubulointerstitial wasting, comprising tubular cell atrophy, tubular dilatation and the presence of large proteinaceous tubular casts. The cause of such devastating podocyte aggression and of the accompanying tubulointerstitial damage is still unknown. We thus undertook a study of primary collapsing glomerulopathy, focusing on podocytes. We show, for the first time in human disease, that when no longer tethered to the GBM, or when assuming the dysmorphic appearance of “cobblestones,” podocytes are shed into the urinary space, lose their specific markers and acquire macrophagic-related epitopes. These findings are substantiated by experiments using rat models of glomerular cell culture, which have demonstrated that podocytes can change into antigen-presenting cells37.Mendrick D.L. Kelly D.M. RENNKE H.G. Antigen processing and presentation by glomerular visceral epithelium in vitro.Kidney Int. 1991; 39: 71-78Abstract Full Text PDF PubMed Scopus (35) Google Scholar or into macrophagic cells38.Orikasa M. Iwanaga T. Takahashi-Iwanaga H. Kozima K. Shimizu F. Macrophagic cells outgrowth from normal rat glomerular culture: Possible metaplastic change from podocytes.Lab Invest. 1996; 75: 719-733PubMed Google Scholar. In addition, we show that cells with macrophagic-like phenotypes are also found in the tubular lumens. We studied the renal tissue of eight patients, four males and four females, who were referred to our Nephrology Unit for nephrotic range proteinuria with renal failure in seven and a rapid course to ESRD in three Table 1. The main data regarding these patients are summarized in Table 1. Five of them were Black. Their mean age at the time of renal biopsy was (mean ±SD) 42 ± 11.7 years (range 28 to 57 years). All were human immunodeficiency virus (HIV) negative on two tests. None of them was a user of illicit drugs. Renal tissue samples were obtained percutaneously in six patients. In one, samples were obtained surgically, and in another, whose collapsing glomerulopathy had relapsed after renal transplantation and who returned to maintenance hemodialysis, transplantectomy provided the whole kidney for histologic examination. None of the samples contained less than eight non-obsolescent glomeruli. A typical picture of collapsing glomerulopathy was found in all patients.Table 1Main clinical data in 8 patients with collapsing glomerulopathy The renal samples were processed for light microscopy and immunofluorescence using standard techniques. Immunofluroescence was performed using antisera directed against IgG, IgA, IgM, C3, C1q, fibrinogen, albumin (Behringwerke, Marburg, Germany) and κ and λ light chains (Dakopatts A/S, Glostrup, Denmark). Podocytes were identified by presence of the glycocalyx sialoglycoprotein podocalyxin, of vimentin, a protein of the podocyte cytoskeleton39.Vasmant D. Maurice M. Feldmann G. Cytoskeleton ultrastructure of podocytes and glomerular endothelial cells in man and in the rat.Anat Rec. 1984; 210: 17-24Crossref PubMed Scopus (58) Google Scholar,40.Ronco P.M. Ardaillou N. Verroust P. Lelongt B. Pathophysiology of the podocyte: A target and a major player in glomerulonephritis.Adv Nephrol. 1994; 23: 91-131PubMed Google Scholar, and of complement receptor 1 (CR1)41.Kazatchkine M.D. Fearon D.T. Appay M.D. Mandet C. Bariéty J. Immunohistochemical study of the human glomerular C3b receptor in normal kidney and in seventy-five cases of renal disease. Loss of C3b receptor in focal hyalinosis and in proliferative nephritis of systemic lupus erythematosus.J Clin Invest. 1982; 69: 900-912Crossref PubMed Scopus (139) Google Scholar. These three phenotypes were characterized using two anti-podocalyxin monoclonal antibodies (mAbs) raised against human podocalyxin identified by Western blot, a kind gift of Dr. Pierre Ronco (INSERM U 489, Hôpital Tenon, Paris, France), an anti-human mAb (Dakopatts) and a mouse anti-human C3b receptor (CR1, clone J3D3)42.Cook J. Fischer E. Boucheix C. Mirsrahi M. Jouvin M.H. WEISS L. Jack R.M. KAZATCHKINE M.D. Mouse monoclonal antibodies to the human C3b receptor.Mol Immunol. 1985; 22: 531-539Crossref PubMed Scopus (47) Google Scholar, a kind gift of Dr. Michel Kazatchkine (INSERM U 430, Hôpital Broussais, Paris, France). To further characterize the phenotypic expression of swollen, vacuolated, cobblestone-like and/or detached podocytes, we used three anti-human mAbs with different specificities for macrophage-associated epitopes: an anti-CD68 clone KP1 (Dakopatts), an anti-CD68 clone PG-M1 (Dakopatts) and an anti-human macrophage clone M 18 (Ber-MAC3; Dakopatts). For KP1 and PG-M1, 4-μm thick paraffin sections of renal tissue were fixed in Bouin solution or in formalin. Slides were deparaffinized and rehydrated. Immunohistochemical staining was performed by two methods: the ABC method using avidin-biotin system with alkaline phosphatase enzyme (Vector Laboratories, Burlingame, CA, USA) and the streptavidin-biotin peroxidase system (Biogenex). Ber-MAC3 identification was carried out on cryostat sections, using the ABC method described below. Immunostaining was performed by the ABC alkaline phosphatase procedure (Vector Laboratories) involving: (1) blocking with normal horse serum diluted 1/20 for 20 minutes; (2) 30 minute incubation with primary mouse anti-human mAbs (anti-vimentin, anti-CR 1 or anti-human macrophage markers KP-1, PGM-1 and M 18); (3) after washing in PBS buffer, pH 7.4, 30 minute incubation with biotinylated secondary antibody, a horse anti-mouse mAb; and (4) after washing, 30 minute incubation with a preformed avidin-biotinylated alkaline phosphatase complex. The indicator system was developed with Vector Red substrate (Vectastain, ABC Kits; Vector Laboratories). Slides were counterstained with hematoxylin. Double labeling was performed using the ABC alkaline phosphatase procedure, comprising six steps for the first labeling: (1) digestion by pronase for 10 minutes at room temperature; (2) blocking for 20 minutes with normal horse serum diluted 1/10; (3) 30 minutes incubation with a mouse anti-human podocalyxin mAb; (4) after washing in PBS buffer, pH 7.4, 30 minute incubation with the biotinylated horse anti-mouse Mab; (5) after washing, 30 minute incubation with a preformed avidin-biotinylated alkaline prosphatase complex; (6) after washing, development of the indicator system on the Vector Blue substrate. The procedure was the same for the second labeling, except that in Step 3 the anti-CR 1 mAb (clone J3D3) or the anti-CD68 (clone PG-M1) was used, and in Step 6 the indicator system was developed with Vector Red substrate. The following negative control experiments were carried out on paraffin and cryostat sections: (1) replacement of the primary mAb with antibody-diluting buffer; (2) replacement of the primary mAb by mouse anti-cytomegalovirus mAb, clone EB (Clonatec Biosoft, Paris, France); (3) replacement of the primary mAb by normal mouse serum. By light microscopy, collapsing glomerulopathy was observed on all specimens Figure 1. Most, but not all glomeruli showed segmental or global collapse of the glomerular capillary walls, podocyte vacuolization and multinucleation of some podocytes. In a majority of glomeruli cobblestone-like podocytes were aligned around the collapsed glomerular tuft and still adhering to the underlying glomerular basement membrane (GBM). Typical lesions of FSG, characterized by increased extracellular matrix proteins, hyalinosis and capsular adhesions, were observed in numerous, incompletely collapsed glomeruli. In some glomeruli, the mono- or multinucleated visceral epithelial cells formed pseudo-crescents, and many contained hyaline protein resorption droplets. The pseudo-crescents differed from true crescents, as they were adjacent to the tuft and not to Bowman's capsule, they were composed of round cells, and they were not surrounded by extracellular matrix material. The visceral epithelial cells forming these pseudo-crescents had the appearance of living cells. Some podocytes were detached from the underlying GBM and were drifting in Bowman's space. Bowman's capsules were intact, with no gaps or perforations and no evidence of cell trafficking from or to the interstitium. Numerous microcystic dilated tubules were filled with proteinaceous casts. Marked dilation affected other tubules throughout the cortex and the medulla. By immunofluorescence, the hyaline droplets within hypertrophied podocytes were strongly labeled with albumin and IgA, indicating protein phagocytosis. Three podocytic markers were studied: vimentin, podocalyxin, and CR1. Basically, the three markers diclosed similar labeling pattern changes according to podocyte attachment to, or detachment from the underlying GBM. Anti-vimentin antibody variably labeled podocytes according to the severity of lesions. When glomerular tufts or lobules were little affected, the labeling pattern was normal. When still attached to the tuft, vacuolated podocytes had a vimentin-positive cytoplasm. Cobblestone-like and completely detached podocytes were vimentin negative Figure 2. Examination of nephron segments further downstream, from Bowman's space to tubular lumens, showed that cells in the urinary space were vimentin negative. In relatively preserved glomeruli, podocalyxin labeling was normally distributed. Podocyte immunostaining circled the tuft lobules and tagged the capillary endothelium. In glomeruli containing vacuolated podocytes still attached to the GBMs, podocalyxin surrounded and circled the bullous vacuoles. In more altered glomeruli, with a consolidated tuft surrounded with cobblestone-like podocytes, podocalyxin labeling was negative, except in a few remaining lobules with no cobblestone-like alignment and having open capillaries. In consolidated glomeruli where podocytes were completely detached, podocalyxin labeling marked only the capillary endothelium Figure 3 and 4.Figure 4Patient number 1 showing anti-human podocalyxin. Close-up of part of the glomerular tuft, at the urinary pole of the tuft (compare with Figure 5 and 6). The glomerular tuft is completely collapsed. The anti-podocalyxin antiserum labels capillary endothelial cells. Podocytes are podocalyxin negative (×400).View Large Image Figure ViewerDownload (PPT) CR1 labeling also differed according to podocyte attachment to, or detachment from, the glomerular tuft. Normal, podocytes were invariably labeled by CR1. Swollen podocytes still anchored to the GBM and having a “bullous” appearance were CR1 positive. Cobblestone-like podocytes lining the glomerular tuft and detached podocytes had lost this labeling Figure 5. We particularly focused on the podocytes remaining attached to, or in the process of detachment from the GBM, and on the detached cells free in Bowman's space or in the tubular lumen Figure 6, 7 and 8.Figure 7Patient number 8, showing antibody against human CD68, clone PG M1. Three atrophic tubules with microcystic changes. The lumen of the upper tubule is almost occluded by a clump of macrophage-like cells. Their nuclei are apparently normal, giving the impression that these cells are viable. The two other tubules are considerably dilated, with a microcystic appearance. Their epithelium is flattened and the lumen is entirely filled with large proteinaceous casts. No CD68 labeled cells are present in the infiltrate that occupies the peritubular interstitium (×300).View Large Image Figure ViewerDownload (PPT)Figure 8Patient number 7, showing antibody against human CD68, clone PG M1. The lumen of a small caliber tubule, presumably distal, shows a line of macrophagic cells. They seem to progress in single-file, following the urinary flow. A nearby small inflammatory infiltrate is devoid of any macrophage-like cellular elements (×400).View Large Image Figure ViewerDownload (PPT) Regarding glomerular cells, similar results were observed using three different mAbs recognizing macrophage-associated epitopes: anti-CD68 clone KP1, anti-CD68 clone PG-M1 and anti-human M 130, M 18 (Ber-MAC3). Tissue preservation was better in paraffin than in cryostat sections. This applied to KP1 and PG-M1, as the M 18 (Ber-MAC3) epitope was no longer expressed after fixation and thus could only be studied on cryostat sections. Cytoplasmic labeling appeared smooth and homogeneous with anti-CD68 clone PG-M1 whereas it had a granular character with anti-CD68 clone KP1. There was no correlation between the severity of podocyte vacuolization, cobblestone-like alignment along the outer aspect of the glomerular lobule and detachment into Bowman's space on the one hand, and the number of detached podocytes with a CD68 labeling. Podocytes still present within the glomerular tuft or those covering the outer aspect of the GBM were KP1, PG-M1 and M 18 negative Figure 6. Once detached from the GBM and free within Bowman's space they expr
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