J Am Soc Nephrol 10: 51–61, 1999 The Dysregulated Phenotype: A Novel Concept in the Pathogenesis of Collapsing Idiopathic Focal Segmental and HIV-Associated Nephropathy

LAURA BARISONI,* WILHELM KRIZ,† PETER MUNDEL,† and VIVETTE D’AGATI* *Department of , Columbia University College of Physicians and Surgeons, New York, New York; and †Department of and , University of Heidelberg, Heidelberg, Germany.

Abstract. are highly differentiated, postmitotic cells, human . In and membranous whose function is largely based on their complex cytoarchitec- glomerulopathy, all mature podocyte markers were retained at ture. The differentiation of podocytes coincides with progres- normal levels despite severe and foot process fu- sive expression of maturity markers, including WT-1, CALLA, sion; no cell proliferation was observed. In contrast, in collaps- C3b receptor, GLEPP-1, podocalyxin, and synaptopodin. In ing idiopathic FSGS and HIV-associated nephropathy, there collapsing forms of focal segmental glomerulosclerosis was disappearance of all markers from all collapsed glomeruli (FSGS), including idiopathic FSGS and HIV-associated ne- and of synaptopodin from 16% of noncollapsed glomeruli. phropathy, podocytes undergo characteristic, irreversible ultra- This phenotypic dysregulation of podocytes was associated structural changes. This study analyzes the expression pattern with cell proliferation in both diseases. It is concluded that the of the above differentiation markers and of the proliferation loss of specific podocyte markers defines a novel dysregulated marker Ki-67 in collapsing idiopathic FSGS and HIV-associ- podocyte phenotype and suggests a common pathomechanism ated nephropathy compared with minimal change disease, in collapsing FSGS, whether idiopathic or HIV-associated. membranous glomerulopathy, as well as normal adult and fetal

Podocytes are highly specialized cells whose functions include associated with a loss of proliferative activity and progressive support of the glomerular , synthesis of glomerular expression of distinctive podocyte markers, including the , and regulation of glomerular permselec- Wilms’ tumor WT-1, CALLA (common acute lympho- tivity. These complex functions depend on a highly differen- blastic leukemia antigen), C3b receptor, GLEPP-1 (glomerular tiated and unique cytoarchitecture. Based on their cytoarchi- epithelial protein-1), podocalyxin, and synaptopodin. tecture, podocytes may be divided into three structurally and WT-1 is a critical protein in nephrogenesis, as indicated by functionally different segments: cell body, major processes, total failure of metanephric development in WT-1-deficient and foot processes. This segmentation of podocytes is also mice (4). Early in glomerular development, WT-1 is widely observed at the level of the cytoskeleton. Microtubules and expressed in glomerular progenitor cells, but becomes re- intermediate filaments predominate in cell bodies and major stricted to podocytes as the matures. WT-1 is a zinc processes. The foot processes contain an -based contrac- finger transcription factor that downregulates proliferation (5), tile apparatus, which is linked to the GBM at focal contacts by but may also be necessary to maintain the mature podocyte ␣ ␤ an 3 1-integrin complex (reviewed in reference (1)). phenotype (6). The expression of these specialized cellular features is de- On their apical surface podocytes are equipped with a neg- velopmentally regulated. As the developing glomerulus passes atively charged which plays a dual role in perm- from the vesicle stage via the S-shaped body stage to the selectivity and maintenance of foot process cytoarchitecture. loop stage and the mature glomerulus, podocytes Podocalyxin, the major sialoprotein of this glycocalyx, first acquire their characteristic complex cell architecture including appears in the S-shaped body stage of the developing glomer- foot processes and slit diaphragms (2,3). This maturation is ulus (7). In addition to podocytes, podocalyxin is also ex- pressed by endothelial cells (8). GLEPP-1, a podocyte-specific protein tyrosine phosphatase, is another integral apical mem- Received June 15, 1998. Accepted August 24, 1998. Parts of this work have been presented in abstract form at the 30th Annual brane protein that first appears in the S-shaped body stage. It Meeting of the American Society of , San Antonio, Texas, No- has been proposed that signal transduction through GLEPP-1 is vember 2–5, 1997. involved in regulation of foot process dynamics, cell differen- Correspondence to Dr. Peter Mundel, Division of Nephrology, Albert Einstein tiation, and contact inhibition (9). College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: 718-430-3158; Fax: 718-430-8963; E-mail: [email protected] A convergence has been demonstrated between antigenic determinants of B lymphocytes and leukemic cells on the one 1046-6673/1001-0051$03.00/0 Journal of the American Society of Nephrology hand and podocytes of fetal and adult kidney on the other. Copyright © 1999 by the American Society of Nephrology Acquisition or loss of these hematopoietic antigens has been 52 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 51–61, 1999 correlated with the various stages of differentiation Materials and Methods and development (10). Some of these antigens, such as CD35, We selected 28 cases from the archives of the are expressed only transiently during the early stages of glo- Laboratory at Columbia Presbyterian Medical Center, including 10 merulogenesis, whereas others such as CALLA and C3b re- cases of collapsing idiopathic FSGS, eight cases of HIV-associated ceptor, whose expression first appears in the S-shaped body nephropathy, five cases of minimal change disease, and five cases of stage, are maintained in podocytes of the mature glomerulus membranous glomerulopathy. Normal fetal and adult kidney were (10,11). used as controls. Synaptopodin represents a novel proline-rich actin-associ- Three-micrometer-thick sections from each biopsy were stained with hematoxylin and eosin, periodic acid-Schiff, trichrome, and ated protein of telencephalic dendrites and glomerular podo- silver methenamine. Focal segmental and global glomerular sclerosis cytes that may play a role in the actin-based shape and motility as well as glomerular collapse were evaluated and expressed as of podocyte foot processes (12). During kidney develop- percentage of affected glomeruli per total number of glomeruli in each ment, synaptopodin first appears at the capillary loop stage. biopsy for each category. Sclerosis was defined as an expansile scar Thus, the expression of synaptopodin coincides with the for- of the glomerular tuft characterized by increased matrix, with or mation of foot processes (13). Because of its association with without associated hyalinosis and adhesion to Bowman’s capsule. a highly developed cytoskeletal architecture, synaptopodin is Collapse was defined as an implosive wrinkling and retraction of the an important marker of the mature podocyte phenotype. glomerular basement membrane, with resulting narrowing or obliter- In glomerular diseases with massive proteinuria, podocytes ation of capillary lumina and hypertrophy of overlying extracapillary undergo dramatic structural alterations. In some conditions, cells (16). such as minimal change disease, proteinuria and podocyte cellular alterations are reversible, whereas in other conditions, such as focal segmental glomerulosclerosis (FSGS), they are not. Among the many forms of FSGS (14), the glomerular To characterize the expression profiles of podocyte marker degeneration in collapsing idiopathic glomerulosclerosis and in glomerular development and in mature kidney, sections of normal HIV-associated nephropathy differs from that of other forms fetal and adult human kidney were studied by immunohistochemistry using the full panel of podocyte-specific antibodies (Table 1). In by its more severe clinical presentation, prognosis, and mor- addition, the proliferative activity of podocytes was analyzed using an phologic features. In these two diseases, the glomerular alter- antibody directed to the cell cycle protein Ki-67. Serial 3-␮m-thick ations are characterized by segmental or global tuft collapse, sections of formalin-fixed paraffin-embedded tissue were obtained and most striking, the crowding of epithelial cells on the outer from the cases listed above. The sections were rehydrated in a graded aspect of the tuft. It also differs in its lack of synechiae, at least series of , blocked with 10% normal serum for polyclonal in the early stages. It has been suggested that a primary antibodies in 1% bovine serum and 10% normal horse serum podocyte injury plays a central role in the pathogenesis of for monoclonal antibodies in 1% bovine , and stained certain forms of FSGS (15), although the nature of this inju- using avidin-biotin immunoperoxidase technique with monoclonal rious factor remains to be discovered. antibodies against GLEPP-1 (reference 9; courtesy of R.C. Wiggins, The current study was designed to investigate to what extent Ann Arbor, MI), podocalyxin (courtesy of R.C. Wiggins), synaptopo- the morphologic alterations seen in collapsing idiopathic glo- din (12,13), and Ki-67 (Clone MIB1, Dako), and a polyclonal anti- serum against WT-l (C19, Santa Cruz Biotechnology). Sections were merulosclerosis and HIV-associated nephropathy reflect an predigested by microwaving for 25 min and then incubated overnight alteration of the mature podocyte phenotype. Based on the loss with synaptopodin (1:20), GLEPP 1 (1:10), podocalyxin (1:10), Ki-67 of all specific structural features as well as the loss of specific (1:500), and WT-1 (1:200). Horse anti-mouse IgG and goat anti-rabbit markers of differentiated podocytes, we propose a new patho- IgG (both from Dako) were used as secondary antibodies (1:100) for genetic mechanism of a dysregulated podocyte phenotype for 30 min. Sections were incubated for 30 min with avidin-biotin com- both collapsing idiopathic glomerulosclerosis and HIV-associ- plex (1:10; Vector) and developed with diaminobenzidine as chromo- ated nephropathy. gen.

Table 1. “Expression of Ki-67 and podocyte maturity markers in glomerular diseases with and in normal adult human kidneya”

Segmental and Group Proteinuria Global Ki-67 WT-1 GLEPP-1 Podocalyxin Synptopodin CALLA C3b Receptor (g/24 h) Collapse/Sclerosis

HIV-AN 7.0 70 ϩϪ Ϫ Ϫ Ϫ Ϫ Ϫ Collapsing FSGS 13.9 57 ϩϪ Ϫ Ϫ Ϫ Ϫ Ϫ MCD 12.3 0 Ϫϩ ϩ ϩ ϩ ϩ ϩ MGN 7.0 0 Ϫϩ ϩ ϩ ϩ ϩ ϩ NAK 0 0 Ϫϩ ϩ ϩ ϩ ϩ ϩ

a WT-1, Wilms’ tumor protein-1; GLEPP-1, glomerular epithelial protein-1; CALLA, common acute lymphoblastic leukemia antigen; HIV-AN, HIV-associated nephropathy; FSGS, focal segmental glomerulosclerosis; MCD, minimal change disease; MGN, membranous glomerulopathy; NAK, normal adult human kidney. J Am Soc Nephrol 10: 51–61, 1999 The Dysregulated Podocyte Phenotype 53

The percentage of glomeruli with one to five cells expressing Ki-67 not shown). Thus, the expression of podocyte markers in was calculated in each category of disease. The percentage of non- developing human kidney is consistent with previously de- sclerotic/noncollapsed glomeruli with decreased expression of three scribed expression patterns of these proteins in developing maturity markers (GLEPP-1, podocalyxin, synaptopodin) was also rodent kidney (reviewed in reference (1)). calculated in each category. Frozen sections were obtained from fetal human kidney, adult Dysregulation of Podocyte Phenotype in Collapsing normal kidney, and from 10 of the above cases, including membra- nous glomerulopathy (three cases), minimal change disease (one Idiopathic FSGS and HIV-Associated Nephropathy case), collapsing idiopathic FSGS (two cases), and HIV-associated Versus Minimal Change Disease and Membranous nephropathy (four cases). Sections were fixed in acetone and incu- Glomerulopathy bated with monoclonal antibodies to CALLA (1:20; Innovex Bio- We next analyzed the expression of the cell cycle marker sciences) and C3b receptors (1:100; Accurate Chemical Scientific Ki-67 and of the above podocyte markers in renal biopsies Corp.) for 1 h. The immunolabeling was visualized with the same from collapsing idiopathic glomerulosclerosis and HIV-asso- immunoperoxidase technique described above. ciated nephropathy (HIV-AN) compared with two other glo- merular diseases associated with morphologic alterations of Transmission Electron Microscopy podocytes and nephrotic syndrome, i.e., minimal change dis- Specimens for transmission electron microscopy were fixed with ease (MCD) and membranous glomerulopathy (MGN). 2.5% glutaraldehyde in -buffered saline (PBS), washed with Proteinuria was in the nephrotic range (Ͼ3.0 g/24 h) in 27 of

PBS, and incubated with OsO4 for 60 min. The sections were coun- 28 patients at the time of the biopsy. Proteinuria was more terstained with 1% tannic acid in PBS for 15 min, dehydrated in a severe in patients with collapsing idiopathic glomerulosclerosis graded series of ethanol (30, 50, 70, 95, and 100%), and finally (mean 13.9 g/24 h) and MCD (mean 12.3 g/24 h) compared embedded in Epon 812 according to standard procedures. Ultrathin with HIV-AN (mean 7.0 g/24 h) and MGN (mean 9.8 g/24 h). sections were cut with a Reichert Jung Ultracut E ultramicrotome and The biopsies from patients with MCD revealed no labeling studied under a Philips EM 301 electron microscope. of Ki-67, indicating the absence of cell cycle-engaged podo- cytes (Figure 2a). The staining profile of the podocyte marker Results proteins in MCD (Figure 3, c, e, and g) was virtually identical Expression of the Cell Cycle Marker Ki-67 and of to that of normal adult kidney, with retention of all markers Mature Podocyte Marker Proteins during Renal studied. This persistence of the mature podocyte phenotype Development and in Normal Adult Human Kidney was observed despite severe foot process effacement and high During ontogeny, podocytes develop from simple epithelial levels of proteinuria. cells of the S-shaped body, where they divide avidly. Later, In MGN, another form of idiopathic nephrotic syndrome during the transition to the capillary loop stage, the cells stop associated with severe morphologic alterations of podocytes dividing and transform into the mature mesenchymal-like cells including foot process fusion, the findings were similar to with the characteristic pattern of primary and foot processes. those observed in MCD. No proliferating podocytes were Expression of Ki-67 was found in up to 90% of cells detected with Ki-67 immunostaining. The expression pattern of comprising the glomerular vesicles, the earliest stage of glo- mature podocyte markers was comparable to that seen in merular development, with progressive reduction in the per- normal adult kidney. The single exception was loss of mature centage of Ki-67-expressing cells in the course of glomerular podocyte markers overlying a lesion of segmental sclerosis in maturation (Figure 1a). In the normal adult kidney, no Ki-67- a single glomerulus from one of the five cases studied (data not expressing podocytes were observed. WT-1 was weakly ex- shown). pressed in all cells of renal vesicles, showed increased staining The results in collapsing idiopathic FSGS and HIV-AN are during the S-shaped body stage, and became restricted to virtually identical, both clinically and morphologically, and podocytes in the capillary loop stage of development (Figure therefore will be described together. In agreement with previ- 1b) and in the adult kidney, where strong nuclear labeling of ous studies (16–18), changes in podocytes appear to crucially podocytes was found. underlie glomerular degeneration in these diseases. Podocytes The expression of C3b receptor (data not shown), GLEPP-1 lose many of their specific attributes including cellular archi- (Figure 1c), and podocalyxin (Figure 1d) commences at the tecture, cell cycle characteristics, and specific nuclear, cyto- early S-shaped body stage, increases thereafter, and persists at plasmic, and membrane proteins. In contrast to MCD and high levels in the adult podocytes. Podocalyxin was also MGN, in both collapsing idiopathic glomerulosclerosis and strongly expressed in all vascular endothelia including glomer- HIV-AN, Ki-67-positive cells were seen in regions where the ular endothelial cells. The expression of CALLA (data not number of epithelial cells on the outer aspect of the tuft was shown) and synaptopodin (Figure 1e) was first seen at the obviously increased (Figure 2b). Eleven percent of glomeruli transition from the late S-shaped body stage to the capillary in collapsing idiopathic FSGS and 10% of glomeruli in loop stage. Synaptopodin staining was restricted to the basal HIV-AN showed from one to five podocytes with nuclear portion of the podocytes, where foot processes form. In con- staining for Ki-67. We observed extensive loss of all podocyte trast, GLEPP-1 and podocalyxin were found in a polarized markers (CALLA, C3b receptor, GLEPP-1, podocalyxin, syn- manner at the apical surface of the podocytes. C3b receptor and aptopodin, and WT-1) in all glomeruli with collapsing sclerosis CALLA showed no preferential intracellular distribution (data (Figure 2D; Figure 3, d, f, and h). In segmentally affected 54 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 51–61, 1999

Figure 1. Expression of podocyte markers during normal human glomerulogenesis. (a) In the S-shaped bodies, most of the cells show nuclear positivity for the proliferation marker Ki-67. At the capillary loop stage, the absence of Ki-67 expression is consistent with entry of podocytes into G0 (resting phase) of the cell cycle. (b) WT-1 is weakly expressed in all cells in the early stages, but later becomes restricted to podocytes. (c) The tyrosine phosphatase GLEPP-1 is focally expressed in an apical localization during the S-shaped body stage and becomes restricted to podocytes in the capillary loop stage and in maturing glomeruli. (d) Podocalyxin has a similar distribution pattern as GLEPP-1, but in addition to podocytes is also found in endothelial cells. (e) The expression of synaptopodin commences at the transition from the S-shaped body to the capillary loop stage and coincides with podocyte foot process formation. S, S-shaped body stage; C, capillary loop stage; M, maturing glomerulus. J Am Soc Nephrol 10: 51–61, 1999 The Dysregulated Podocyte Phenotype 55

Figure 2. Inverse expression of Ki-67 and WT-1 in minimal change disease (MCD) and HIV-associated nephropathy (HIV-AN). (a and c) MCD. (b and d) HIV-AN. In MCD, no Ki-67-positive cell cycle-engaged cells are seen (a), and all podocytes express WT-1 (c) at levels comparable to normal adult kidney. In contrast, in HIV-AN proliferation of cells in the urinary space is revealed by Ki-67 immunoreactivity (b). The same glomerulus as in b shows complete loss of WT-1 expression (d). Magnification, ϫ 260. glomeruli (26% of glomeruli in collapsing idiopathic FSGS peared, whereas staining of endothelial cells was clearly main- and 38% in HIV-AN), this loss was restricted to collapsed tuft tained (Figure 3f). The results of the immunohistochemical areas, where the typical crowding of epithelial cells on the studies are summarized in Table 1. outer tuft surface was seen. In globally affected glomeruli (31% of glomeruli in collapsing idiopathic FSGS and 32% in Ultrastructural Alterations in Collapsing Forms of HIV-AN), the loss was total. However, a reduction in synap- Glomerulosclerosis topodin expression (in contrast to the other markers) was also The loss of specific podocyte markers in these two diseases frequently observed in unaffected portions of the tuft of seg- was accompanied by characteristic structural changes, among mentally injured glomeruli, or even in glomerular profiles which the most prominent and characteristic were tuft collapse without any visible collapse or other histologic abnormalities and the accumulation of epithelial cells on the outer aspect of (Figure 4, b and d). In collapsing idiopathic FSGS and HIV- the glomerular basement membrane (GBM) in the collapsed AN, 16% of the histologically unaffected (i.e. nonsclerotic and areas (Figure 5). In most of the affected glomeruli the tuft noncollapsed) glomeruli showed marked reduction of synap- collapse was global, but some glomerular profiles were con- topodin expression versus only 3% in collapsing idiopathic sistently encountered in which the injury was restricted to a FSGS and 4% in HIV-AN for GLEPP-1 and 0% in both portion of the tuft. The collapse was associated with a wrin- categories for podocalyxin. With respect to the staining pattern kling and progressive thickening of the GBM and the disap- of podocalyxin, an interesting observation was made. Glomer- pearance of capillaries (Figure 5). Normally, the GBM is ular profiles with global collapse were frequently found in clearly separated from adjacent portions of the GBM either by which podocalyxin staining of podocytes had totally disap- the mesangium along its inner aspect or by podocytes along its 56 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 51–61, 1999

Figure 3. Differential expression of mature podocyte markers in MCD and HIV-AN. (a, c, e, g) MCD. (b, d, f, h) HIV-AN. (a and b) Silver methenamine. In MCD, the normal pattern of the glomerular capillaries is well preserved (a), whereas in HIV-AN the glomerular tuft shows global collapse (b). The podocytes overlying the collapsed capillaries are hypertrophied. (c and d) GLEPP-1 is expressed in MCD at levels comparable to that of normal adult kidney (c), but has almost completely disappeared from the podocytes in the collapsed tuft (d). (e and f) In MCD, podocalyxin is expressed in podocytes and endothelial cells (e). In HIV-AN, podocalyxin disappears from podocytes, but is preserved in glomerular endothelial cells (f). (g and h) Synaptopodin is strongly positive in MCD (g) but is completely lost from podocytes in glomeruli with collapsing sclerosis (h). Magnification, ϫ 260. J Am Soc Nephrol 10: 51–61, 1999 The Dysregulated Podocyte Phenotype 57

Figure 4. Loss of synaptopodin in noncollapsed glomeruli. (a and c) Normal adult human kidney. (b and d) Noncollapsed glomeruli of collapsing idiopathic focal segmental glomerulosclerosis. Low-power view showing strong global distribution of synaptopodin immunoreac- tivity in all glomeruli (a). Synaptopodin is localized in podocyte foot processes along the glomerular basement membrane (c). (b) In the top left corner, there is a noncollapsed glomerulus with marked reduction of synaptopodin expression (arrow). Loss of synaptopodin staining is also seen overlying a lesion of segmental sclerosis in the bottom glomerulus (arrowheads) (d). Most of the podocyte foot processes have lost synaptopodin expression, although no wrinkling or collapse of the glomerular basement membrane is observed. These findings suggest that the dysregulation of the mature podocyte phenotype precedes the collapse of the tuft. Magnification: ϫ100 in a and b; ϫ1000 in c and d. outer aspect. In the collapsed glomeruli, this separation is lost formed extracellular matrix material (Figure 6b). Intermediates locally at both sites followed by a merging of folded segments between a normal podocyte and the fully dysregulated pheno- of GBM, producing an irregular and “anastomosing” GBM type point to a gradual loss of characteristic cellular features, network (Figure 5). which is different from what is usually seen when podocytes The second prominent structural change is the “crowding” (e.g., in secondary forms of glomerular sclerosis) undergo (probably hyperplasia) of epithelial cells in Bowman’s space simplification and degeneration (19,20). In addition to the loss (Figure 5). In early stages of glomerular collapse, these cells of foot processes, these cells lose their ultrastructurally visible can be identified as podocytes or podocyte-like cells by their actin cytoskeleton (Figure 6c). In early stages of cell injury attachment to the outer aspect of the GBM; the visceral and (i.e., partially injured cells with maintenance of some of their parietal cells are still separated by a urinary space. In later primary and foot processes), patches of the actin cytoskeleton stages, such a space has disappeared rendering a distinction are seen in the basal cytoplasm of the “fused” cell areas (Figure between parietal and visceral epithelial cells impossible. Areas 6b). However, we never observed the rearrangement of the with loss of cells leading to a peripheral exposure of naked actin cytoskeleton into a continuous cytoskeletal mat (as occurs GBM areas or tuft adhesions to Bowman’s capsule were not in MCD and MGN). In contrast, the cytoskeleton disappears observed. leading eventually to the phenotype of pale, seemingly “emp- The cells that finally fill the entire Bowman’s space do not ty” cells adhering smoothly to the GBM (Figure 6, b and c). possess structural characteristics of the developed podocyte At some sites, these epithelial cells contain numerous elec- cell architecture. They are large, pale polygonal cells smoothly tron-dense, membrane-bound droplets (generally interpreted as adhering to the GBM (Figure 6a) or to intervening newly the result of extensive protein at sites of protein 58 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 51–61, 1999

Figure 5. Transmission electron microscopy (TEM) morphology of collapsing glomerulopathy: HIV-AN. Overview of an entire glomerular profile (a). An accompanying drawing illustrates some details (b). The collapsed tuft area inside the glomerular basement membrane (GBM) is shown in dark gray, the area outside the GBM filled with cells is shown in pale gray, and remnants of a urinary space are displayed in white. A hatched line indicates the (greatly thickened) parietal basement membrane. Most of the “crowded” cells on the outer aspect of the GBM are of a pale undifferentiated type. A minor fraction of cells darker in appearance contains abundant lysosomal elements (absorption droplets; arrows). The pale cell type includes parietal (P) cells, one of which is undergoing mitosis (asterisk), and cells adhering to the GBM (stars). Cells in between appear to be simply piled up on top of others, thus being without any contact to a basement membrane. A distinction between these different cell types based on cytologic criteria is not possible. Note the dramatic wrinkling of the GBM resulting in an anastomosing pattern (arrowheads). TEM, ϫ1500. J Am Soc Nephrol 10: 51–61, 1999 The Dysregulated Podocyte Phenotype 59

leakage through the glomerular filter), consistent with hyper- plasia of the endocytotic vesicular apparatus (Figure 5).

Discussion The results of the present study provide direct support for the concept that collapsing forms of FSGS (both idiopathic and related to HIV ) have a common pathomechanism leading to a dysregulated podocyte phenotype. This concept is based on three lines of evidence. (1) Both diseases showed identical changes in podocyte phenotype as reflected by the loss of all specific podocyte markers examined. (2) In both diseases, a comparable number of podocytes were recruited into the cell cycle. (3) The morphologic alterations of podocyte cytoarchitecture were indistinguishable between collapsing id- iopathic glomerulosclerosis and HIV-AN. These findings are in sharp contrast to those obtained from the other two diseases studied, MCD and MGN, where severe podocyte damage also occurs. Our data are consistent with a report that in MCD and MGN expression of GLEPP-1 is preserved despite intracellular redistribution (21). Unlike in secondary forms of FSGS (19,20), in collapsing forms of FSGS the key event is not the loss of podocytes themselves but particular alterations of the podocyte phenotype that include loss of molecular markers and mature structural features. In collapsing forms, although podo- cyte detachment is common, we never observed segments of “naked” GBM in the periphery of the glomerular tuft. In contrast, the periphery of the collapsed GBM areas is com- pletely covered by dysregulated epithelial cells. This lack of “naked” GBM areas may account for the absence of segmental synechiae typically seen in secondary forms of FSGS (19,20). The dramatic structural changes as well as the complete loss of specific podocyte markers in collapsing idiopathic FSGS and HIV-AN raise the question whether these cells are indeed podocytes. Since these cells are located on the outer surface of the GBM, it is reasonable to assume that these cells originate from podocytes, but exhibit a severely dysregulated phenotype (22). Most convincing in this respect is the loss of WT-1 expression in collapsing forms of FSGS. In contrast, in glo- merular diseases with reversible podocyte alterations but pre- served phenotype, such as MCD and MGN, expression of WT-1 remains unchanged. The loss in collapsing sclerosis of WT-1, which is present during nephrogenesis from the very beginning of podocyte ontogeny, makes it very difficult to assign these cells the status of “dedifferentiated” podocytes, but lends strong support to the concept of a dysregulated Figure 6. Changes in podocyte cell architecture in HIV-AN. Changes phenotype. Because in the normal adult kidney, podocytes are in podocyte phenotype as seen in three different stages of injury: early the only WT-1-expressing cells (23), it has been suggested that (a), intermediate (b), and severe (c). Podocytes in areas with wrinkled WT-l might act as a repressor of proliferation in mature podo- GBM (asterisks) lose their foot processes, resulting in direct attach- cytes (5,23,24). Therefore, the observed activation of cell pro- ment of podocyte cell bodies to the GBM. Remnants of the actin- liferation in the dysregulated podocytes might be the conse- based cytoskeleton of podocyte foot processes are encountered in the basal cytoplasm of adhering portions of the podocyte cell body quence of the loss of WT-1. Alternatively, the loss of WT-1 (arrows in a and b) associated with enlarging areas devoid of basal may be responsible for the dysregulation of the podocyte cytoskeleton (arrowheads in a and b). In the late stages (c), pale phenotype in collapsing forms of FSGS, including the disap- epithelial cells with few organelles smoothly adhere to the GBM; no pearance of podocyte processes and of synaptopodin, whose cytoskeleton is detected. Podocyte surfaces facing the urinary space expression both in vivo and in vitro is associated with the undergo “microvillous transformation” (star in a). TEM: ϫ3700 in a; formation and maintenance of processes (6,13). In recent in ϫ5100 in b; ϫ4100 in c. vitro studies of human and rodent podocytes, we were able to 60 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 51–61, 1999 demonstrate that (like during development) WT-1 is expressed phenotype as in the human disease (32). It is intriguing to by both proliferating and nonproliferating cultured podocytes, speculate that podocyte viral infection and resulting alter- and therefore seems more important for maintaining a certain ations of podocyte gene expression may underlie the ob- degree or potential of podocyte differentiation rather than served phenotypic dysregulation in human HIV-AN as well. regulating podocyte proliferation per se (6,25). At present, the pathogenesis of collapsing idiopathic glomer- There is an ongoing debate in the literature as to whether ulosclerosis remains unknown, although possible viral media- podocytes in the mature glomerulus are capable of undergoing tors have been proposed (17). The striking morphologic and cell division (19,26–28). In the collapsing forms of FSGS, the phenotypic similarities between HIV-AN and collapsing idio- “crowding” of cells in Bowman’s space, together with the pathic FSGS raise the question whether the altered podocyte increased number of cycling cells clearly demonstrated in the gene expression in collapsing idiopathic FSGS may also be due present study, provides strong evidence that podocytes do to a viral infection. This hypothesis is further supported by de indeed proliferate in association with an altered cellular phe- novo occurrence of collapsing idiopathic FSGS in immunosup- notype. This dysregulated phenotype may reflect a state of pressed renal transplantation patients (33) and by epidemio- misdirected cell activation culminating in apoptosis (our un- logic data. Similar to HIV-AN, collapsing idiopathic glomer- published observations) or may result in activation of pathways ulosclerosis first appeared as a recognizable entity in the early leading to sustained cell proliferation. 1980s in our own institution, and its incidence progressively One hallmark of collapsing FSGS is the fact that the struc- increased with a peak in the early 1990s, suggesting possible tural changes observed in the dysregulated podocyte phenotype environmental factors (16). Moreover, the strong predomi- are fundamentally different from the well established podocyte nance in African-Americans points to a genetic predisposition lesions seen in MCD and MGN. Although the foot processes for the development of collapsing forms of glomerulosclerosis disappear in all of these conditions, this is achieved by quite (16,18). different means. In MCD and MGN, the effacement of foot In summary, we have demonstrated a novel, common patho- processes is associated with a reorganization of the actin cy- toskeleton, whereas the cell body and primary processes re- mechanism for collapsing idiopathic glomerulosclerosis and main unaffected. In this reorganized actin cytoskeleton, we HIV-associated nephropathy. A primary injury of the podocyte found undiminished levels of synaptopodin. These findings are leading to dysregulation of the cellular phenotype appears to corroborated by a study reporting the preservation of synap- mediate the glomerular tuft collapse in both conditions. The topodin (previously termed pp44) expression in diseases with availability of an HIV-transgenic mouse model (31) mimicking reversible foot process alterations (29). In contrast, in collaps- the human disease (32) should allow further avenues to identify ing forms of FSGS, the disappearance of primary processes factors responsible for altered podocyte gene expression in and foot processes is associated with a loss of cytoskeletal collapsing forms of glomerulosclerosis. It will be of particular elements, including the disappearance of synaptopodin. interest to unravel the cellular signaling pathways that mediate One central question in collapsing forms of FSGS is podocyte dysregulation with the hope of designing therapeutic whether the podocyte damage is primary to the collapse of interventions to block their activation. the tuft or a consequence of the collapse. Three lines of evidence support the hypothesis that a severe and direct injury of the podocyte represents the primary injury, which Acknowledgments then to the collapse of the tuft. First, as shown in this This study was supported by grants from the National Kidney study, synaptopodin is markedly reduced in noncollapsed Foundation (Research Fellowship to Dr. Barisoni) and by the glomeruli; podocalyxin, which is expressed by both podo- Deutsche Forschungsgemeinschaft (Drs. Kriz and Mundel). The cytes and endothelial cells, is selectively lost from podo- antibodies against GLEPP-1 and podocalyxin were the generous cytes but is retained by the endothelial cells of the affected gift of Roger C. Wiggins (University of Michigan, Ann Arbor, MI). glomeruli. Second, in the glomerulus podocytes are the only We thank Bruni Ha¨hnel, Hiltraud Hosser, Llewellyn Ward, and cells that produce vascular endothelial growth factor Liqun Chen for their expert technical assistance, as well as Ingrid Ertl and Rolf Nonnenmacher for skillful photographic work and (VEGF). Kitamoto and coworkers have recently shown that artwork. application of a neutralizing antibody to VEGF in newborn mice may effectively block the development of the glomer- ular capillary tuft (30). It will be interesting to investigate References whether the dysregulation of the podocyte phenotype leads 1. Mundel P, Kriz W: Structure and function of podocytes: An to a loss of VEGF production, which in turn causes tuft update. Anat Embryol 192: 385–397, 1995 collapse. Third, strong support for the concept that the 2. Reeves W, Caulfield JP, Farquhar MG: Differentiation of epi- collapse is secondary to a podocyte damage comes from a thelial foot processes and slits. Lab Invest 39: 90–100, transgenic mouse model of HIV-associated nephropathy 1978 carrying a construct lacking the gag and pol genes but 3. Reeves WH, Kanwar YS, Farquhar MG: Assembly of the glo- retaining the other HIV regulatory elements (31). 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