 |
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
|
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
University Hospital Broussais and INSERM U 430, Paris, France.
Correspondence to Dr. Alain Meyrier, Service de Néphrologie, Hôpital Broussais, 96 rue Didot, 75674 Paris Cedex 14, France. Phone: 33143958190; Fax: 33143958188; E-mail: alain.meyrier{at}brs.ap-hop-paris.fr
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Relapse of primary FSGS on transplanted kidneys presents the opportunity for one to study FSGS with the assurance of dealing with patients with the primary rather than secondary form of FSGS. It thus offers a privileged model for studying the early and later events that characterize this glomerulopathy, which has no equivalent in experimental animals (8). The present study was based on six cases of primary nephrotic FSGS that relapsed after renal transplantation and allowed immunohistochemical phenotypic identification of glomerular cells and of free migrating cells. Some were found to undergo phenotypic changes suggesting transdifferentiation (3,9,10,11), a phenomenon that has been defined as "a dynamic interplay between positive and negative regulatory molecules" in cells, such that their state of differentiation is characterized by a certain plasticity that may be molded by the environment (12).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Controls consisted of sound parts of three kidneys removed for renal carcinoma in patients with no proteinuria and of renal biopsies from three nephrotic patients with minimal change disease (MCD). One sample was a biopsy of the donor kidney in patient 1, minutes after unclamping the renal vessels. Three normal fetal kidneys (30 wk) were also used as controls, with glomerulogenesis spanning renal vesicle, S-shaped, capillary, and maturing stages.
Technical Methods
Standard Pathology. The renal samples were processed for light
microscopy and immunofluorescence (IF) using standard techniques
(3).
Immunohistochemistry. Podocyte phenotypes were characterized using an anti-human podocalyxin monoclonal antibody (mAb; MLC48A8, a gift of Pierre Ronco, M.D., INSERM U 489, Hôpital Tenon, Paris, France), an antisynaptopodin mAb, clone G1D4 (Progen Biotechnik, Heidelberg, Germany), an anti-glomerular epithelial protein-1 (anti-GLEPP-1) mAb (Biogenex, San Ramon, CA), an anti-C3b receptor mAb (complement receptor-1 [CR-1], clone J3D3) (13,14) (a gift of Michel Kazatchkine, M.D., INSERM U 430, Hôpital Broussais, Paris, France), an anti-Wilm's tumor protein-1 (WT-1) polyclonal antibody (pAb; C19, Santa Cruz Biotechnology, Santa Cruz, CA), and an anti-vimentin mAb (Dakopatts, Trappes, France).
Cytokeratin (CK) polypeptides were labeled by AE1/AE3 (Dakopatts), C2562 (Sigma Aldrich Chimie, St. Quentin Fallavier, France), CK22 (Biomeda, Foster City, CA), and AEL-KS2 (Argen-Biosoft, Varhilles, France) mAb. Also used were mAb with different specificities for macrophage-associated epitopes: an anti-CD68 mAb, clone PGM1 (Dakopatts); an anti-macrophage HAM56 mAb (Dakopatts); and an anti-86KD protein mAb, clone 25F9 (Valbiotech, Paris, France). Anti-HLA-DR (CR3/43; Dakopatts) and anti-CD16 (GRM1) mAb (Harlan Sera-Lab, Loughborough, UK) were used as activation markers of macrophagic cells (15).
Immunostaining was performed by the ABC alkaline phosphatase (Vector Laboratories, Burlingame, CA) or peroxidase procedure as follows: (1) digestion by pronase (anti-CR-1, PGM1, HAM 56, AE1/AE3, C 2562, CK22, and AEL-KS2 anti-CK mAb) or microwave processing for 5 min in ethylenediaminetetraacetate buffer (pH 8), 0.1 M (WT-1, synaptopodin, and GLEPP-1), (2) blocking with normal horse or goat serum diluted 1:20 for 20 min, (3) 30-min incubation with the primary anti-human mAb or pAb, (4) after washing in TRIS buffer (pH 7.4), 30-min incubation with biotinylated secondary antibody, a horse anti-mouse Ab to detect the mAb or a goat anti-rabbit Ab to detect the pAb, and (5) after washing, 30-min incubation with a preformed avidin-biotinylated alkaline phosphatase or streptavidin peroxidase complex. The indicator system was developed with Vector Red, aminoethylcarbazol, or diaminobenzidine substrates. Slides were counterstained with hematoxylin.
Negative procedures were (1) replacement of the primary mAb with antibody-diluting buffer; (2) replacement of the primary mAb by anticytomegalovirus mAb, clone E13 (Clonatec Biosoft, Paris, France) or replacement of the primary pAb by an anti-von Willebrand factor pAb (Dakopatts); and (3) replacement of the primary mAb by normal mouse serum or replacement of the primary pAb by normal rabbit serum.
Study of Cell Apoptosis and Proliferation. The in situ DNA nick end labeling (TUNEL) technique (16) was used to detect apoptosis. Paraffin-embedded sections were processed in a microwave oven (750 W) for 5 min, three times in citrate buffer (2 x SSC, pH 6.2). A TUNEL kit (ApoTag, Oncor, Gaithersburg, MD) was used according to the standard procedure recommended by the manufacturer. The procedures were revealed using peroxidase and aminoethylcarbazol substrate.
Cell proliferation was assessed by proliferating cell nuclear antigen (PCNA) immunohistochemistry (17). It was carried out using the PC 10 mAb (Dakopatts), diluted 1:200. Antigen retrieval was done in a microwave oven for 5 min in ethylenediaminetetraacetate buffer 0.1 M (pH 8). The intensity of microwave oven treatment necessary for specific labeling without background or nonspecific labeling was obtained in positive control tissues, fixed and processed in the same conditions, i.e., intestinal epithelium for PCNA and reactive lymph nodes for TUNEL.
Cell Counts. Quantitative assessment of the glomerular lesions and of cell labeling was performed at x 16 magnification by scanning the whole renal tissue sections. The total number of glomeruli showing each type of lesion (cellular, sclerotic, ischemic, and obsolescent) was counted. Regarding cell labeling, (TUNEL, PCNA, and CD68), the total number of positive cells was counted in Bowman's space and along the external aspect of the tuft. Cell density was obtained by dividing the total cell number by the total number of glomeruli. The total number of positive free cells that drifted in the tubular lumens was counted. To compare samples of variable sizes, this number was divided by the total number of glomeruli.
Confocal Laser Microscopy. Confocal laser microscopy was used to specify epitope colocalization on three transplanted kidneys removed surgically. For this purpose, in each case, three sets of slides were tagged with one antibody, or with another antibody, or with both antibodies. Digitized images captured with one or the other antibody made a clear distinction between the two epitopes in each pair.
For double-labeled IF, deparaffinized sections were first incubated with a primary mAb (AE1/AE3 anti-CK, or anti-CD68 PGM1, or anti-podocalyxin) and then with the biotinylated anti-mouse IgG as described above. Subsequently, the sections were incubated with streptavidin-cyanin-2 (Amersham, Les Ulis, France) at a dilution of 1:400 for 30 min. After rinsing in phosphate-buffered saline (PBS) the sections were incubated with the complementary primary antibody (AE1/AE3 anti-CK, or anti-CD68 PGM1, or anti-podocalyxin mAb) so that the three following couples were obtained: anti-podocalyxin + AE1/AE3 anti-CK, anti-podocalyxin + anti-CD68, and AE1/AE3, anti CK + anti-CD68. After the sections were rinsed in PBS buffer, they were incubated with an anti-mouse IgG antibody tagged with cyanin-3 (Amersham), at a dilution of 1:400 for 30 min. The sections were rinsed in PBS buffer and mounted in Immu-Mount (Dakopatts). They were examined using a confocal microscope Leica TCS SP (Leica Microsystems, Heidelberg, Germany) powered with an argonkrypton laser beam, with excitation at 488 nm for cyanin-2 (detection, 500 to 550 nm) and at 568 nm for cyanin-3 (detection, 580 to 680 nm).
Morphology: Definitions and Cell Markers
Glomerular Lesions. The lesions observed by light microscopy fell
into four subsets defined as follows.
Cellular Variant. This variant was segmental and occasionally global in the tuft. Some podocytes were swollen and vacuolated and contained protein reabsorption droplets. Others appeared as large round cells that contained a voluminous nucleus, occasionally multinucleated. Pseudocrescents adjacent to the tuft and not to Bowman's capsule were composed of large cells that were not surrounded by extracellular matrix. The capillaries underlying the podocyte lesions were often collapsed.
Synechiae between the tuft and Bowman's capsule were rare but, when present, helped distinguish this variant from the typical appearance of "collapsing glomerulopathy." Detached large round cells could be found free in Bowman's space and in the tubular lumens. In noncollapsed loops, foam cells, hyaline deposits, and cell debris occasionally were found. Part or all of the tuft was covered by an alignment of cobblestone-like cells.
Scar Variant. Sclerosis appeared as a localized scar of the tuft surrounded by cuboid cobblestone epithelial cells that differed from the swollen or large round cells observed in cell FSGS. The localized scars within the glomerular tuft were not always associated with hyalinosis and/or with adhesions to Bowman's capsule. Apart from synechiae, scars were covered by an alignment of cobblestones that adhered to the underlying glomerular basement membrane (GBM) and to that of Bowman's capsule. The space formed between two adhesions was lined by a layer of continuous cells with no demarcation between parietal and visceral epithelium, yielding the appearance of a pseudotubule. Once again, large round cells could be found free in Bowman's space.
Ischemic Glomeruli. Glomerular ischemia was characterized by retraction toward the hilum of a paucicellular tuft, with widening of Bowman's space and thickening and splitting of Bowman's capsule. Epithelial cells that covered the tuft were rare and did not show hypertrophy, vacuolization, or cobblestone alignment. Detached cells were not present in Bowman's space.
Glomerular Obsolescence. The diagnosis of end-stage FSGS reasonably still could be made on globally sclerotic glomeruli given the presence of hyaline and lipid deposits within the tuft and of some pseudotubules that still partitioned Bowman's space.
Immunohistochemical Markers. Podocytes. Podocytes were identified (Table 2) by six epitopes: podocalyxin (18), synaptopodin (19), GLEPP-1 (20,21), WT-1 (22,23), CR-1 (13,14), and vimentin (18,24,25).
|
CK Markers. CK polypeptides were labeled by AE1/AE3 C2562 (used as a marker of parietal epithelial cells [PEC]) (26), CK22, and AEL-KS2 mAb.
Macrophages. Macrophage-associated epitopes studied were an anti-CD68 mAb, clone PGM1; an anti-macrophage HAM56 mAb for the detection of monocytes and macrophages; and an anti-86KD protein mAb, clone 25F9 that recognizes mature macrophages but not monocytes (27). Anti-HLA DP-DQ-DR (CR3/43) and anti-CD16 (GRM1) mAb were used as activation markers of macrophagic cells (15).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Light Microscopy
The changing distribution of the different types of glomerular lesions over
the course of successive biopsies and nephrectomies is shown in
Table 2. A majority of
specimens showed a mixture of the cellular variant of FSGS, with segmental or
global distribution in the glomerular tufts, the scar-variant of FSGS, along
with ischemic, obsolescent, and normal glomeruli. The interpretation of the
lesions was not biased by histologic changes suggesting rejection. Of note,
silver methenamine staining did not show gaps in the glomerular or tubular
basement membranes or in Bowman's capsule. In the seven controls, the renal
tissue was normal.
Immunohistochemistry
Controls. Podocyte markers used were expressed by fetal podocytes
from the stage of S-shaped or capillary loop stage onward, in normal adult
kidneys and in kidneys with MCD (Table
3). In the fetus, PEC also expressed podocalyxin and GLEPP-1. In
adult control kidneys, occasional PEC in some glomeruli expressed GLEPP-1,
usually at the hilar reflection. The mAb used to characterize macrophagic
cells did not tag any titular nephron cells or cells free in the urinary
space.
|
In the fetus, the anti-CK mAb stained neither podocytes nor PEC, with the exception of very rare PEC that were labeled with C2562, CK22, and AEL-KS2. The tubular cells were labeled as in adult controls. In adult controls, the AE1/AE3 mAb labeled no podocytes but did faintly label very rare PEC. The CK22, AELSK2, and C2562 mAb were uniformly negative on podocytes. They tagged a number of PEC in a mostly segmental fashion. AE1/AE3 labeled only the distal tubules. The other mAb strongly tagged collecting ducts, distal tubules, and the thin limbs of Henle's loops. The proximal tubules were also, although less frankly, positive. The anti-CK mAb thus could be arranged in order of progressively broader positivity, both in the fetal and adult kidneys, as shown in Table 3: AE1/AE3, CK22, C2562, AEL-KS2. Note that although PEC stained progressively more extensively with successive anti-CK mAb, normal podocytes were negative for all.
On TUNEL and PCNA preparations, no cell nuclei were labeled in the glomerular cells or in the cells found within the tubular lumens. Regarding TUNEL, in lymph node sections (control tissue), positive nuclei were observed only in reactive germinal centers. Concerning PCNA, in intestinal epithelium (control tissue), positive nuclei were observed in the proliferative part of this tissue, i.e., in the bottom and the lower part of Lieberkühn's crypts.
Patients with Relapse of FSGS on Transplanted Kidneys. Podocyte Markers: Podocalyxin, Synaptopodin, and GLEPP-1. The results were identical with the three markers. Normal or large vacuolated podocytes attached to the GBM were labeled. The cobblestones were negative (Figure 1A). In the cellular variants of FSGS, most podocytes were not tagged. Some large round cells that were attached to the tuft or free in Bowman's space (Figure 1, B through D) and in the tubular lumens (Figure 1, B and C) were strongly positive. Very few PEC were also labeled with podocalyxin and GLEPP-1. Podocalyxin was expressed on the glomerular and extraglomerular endothelial cells.
|
WT-1. WT-1 nuclear tagging was negative on swollen and vacuolated podocytes, on the cobblestone-like cells (Figure 2A), and on the large round cells that were free in Bowman's space and in tubular lumens.
|
Vimentin. Podocytes that were still attached to the tuft were vimentin positive. Cobblestone-like cells were negative. Some of the hypertrophic-hyperplastic large round cells that formed part of the pseudocrescent that was still in contact with the GBM or detached from the tuft strongly expressed vimentin (Figure 2B). A few parietal epithelial cells were faintly labeled. Some epithelial tubular cells and arterial myocytes were tagged.
CR-1. Swollen vacuolated podocytes that kept contact with the GBM were CR-1 positive. Cobblestone-like cells that lined the glomerular tuft and detached large round cells had lost this labeling (not shown).
CK Labeling. Podocytes and PEC were often tagged by these mAb in FSGS kidneys. All of the anti-CK mAb labeled diffusely (Figure 2C) or sparsely (Figure 2D) cells (presumably podocytes) that clumped at the periphery of the tuft in retracted glomeruli, even where no synechiae were found and in areas where PEC did not express CK. Cobblestones that surrounded the synechiae and formed pseudotubules often were tagged by the anti-CK mAb (Figure 3A). Nevertheless, these cobblestones occasionally were not labeled (Figure 3B) by those mAb (C2562, CK22, and AEL-KS2) that usually strongly tag PEC. The large round cells found in Bowman's space and in tubular lumens rarely expressed CK epitopes (Figure 2D).
|
Macrophagic Epitope Labeling. PGM1 (Figure 3, C and D), HAM56 (Figure 4A), and CR3/43 (Figure 4B) labeled the same cells. Cells that were tagged with these mAb were especially numerous in cell FSGS. These large round cells were aligned at the periphery of the glomerular tuft and free in Bowman's space and in the tubular lumens (Figure 3D). In contrast, cobblestones were not labeled. Spindle-shaped interstitial cells were labeled, without evidence of trafficking through tubular or GBM. Some sparse PEC also expressed the macrophagic CD68 epitope.
|
The number of glomerular CD68-positive cells varied from case to case, with a density from 0 in paucicellular forms to 4.31 in highly cellular variants. The same was true regarding the tubular lumens, in which the density was 0.12 to 21.52. In the three cases in which early biopsies were available, no positive cells were observed before day 20 posttransplantation. In the six cases studied, CD68-positive cells were detected in the tubular lumens at least in one or several tissue samples during the follow-up period. Regarding glomeruli, CD68-positive cells were observed in five of the six cases.
A marker of macrophage maturation, 25F9, labeled some tubular cells, whereas others were entirely 25F9 negative (Figure 4C). Within some lumens, 25F9 labeled large round cells, some of which were apparently free, whereas others seemed to replace some of the tubular epithelial cells (Figure 4D).
Anti-CD16, another marker of activated macrophages, also tagged a few large round cells within the tubular lumens (Figure 4E). 25F9 and CD16 were invariably negative on interstitial infiltrates.
Confocal Laser Microscopy
Double IF labeling with the AE1/AE3 anti-CK mAb and the anti-human
podocalyxin mAb showed that some free cells that drifted in tubular lumens
coexpressed CK and podocalyxin (Figure
5A). No such coexpression was found on glomerular cells.
|
Double IF labeling with the antipodocalyxin and the PGM1 (anti-CD68) mAb showed that some cells that were free in the tubular lumens (Figure 5B) and in Bowman's space (Figure 5C) were tagged by PGM1 but also that some of these cells were tagged by both antibodies, indicating co-localization of podocyte and macrophagic epitopes.
Double IF labeling with the AE1/AE3 (anti-CK) and the PGM1 (anti-CD68) mAb showed that some podocytes that were still attached to the tuft strongly expressed AE1/AE3 CK, whereas others, whether attached to the tuft or free in Bowman's space, expressed CD68 (Figure 6A). However, it was also apparent that podocytes that were still attached to the glomerular tuft coexpressed AE1/AE3 CK and CD68 (Figure 6B). A few Bowman's capsule PEC also coexpressed AE1/AE3 CK and CD68. The flow of cells migrating from Bowman's space into the glomerular outlet and progressing along the proximal tubule strongly expressed CD68 (Figure 6C). In other tubular sections, some of the free cells coexpressed AE1/AE3 CK and CD68 (Figure 6D).
|
Assessment of Apoptosis and Proliferation
Apoptosis (TUNEL). No positive cells were found in the adult control
kidneys. In the patients' kidneys, the density of intratubular cell nuclei
labeling was variable (density 0 to 0.85). Very rare positive cell nuclei were
found in some Bowman's spaces (Figure
7) and on the external aspect of the glomerular tufts (density 0
to 0.20). Overall, apoptosis was inconstantly observed as compared with the
number of CD68-positive cells, as shown by assessing the following ratio:
Density of TUNEL-positive cells:Density of CD68-positive cells, respectively
in the glomeruli and within the tubular lumens, a ratio that yielded 0 to 0.08
and of 0 to 0.15, respectively.
|
Cell Proliferating Activity (PCNA). In control kidneys, very rare tubular epithelial cell nuclei were positive, and no podocytes were tagged. In the patients' kidneys, intratubular cells were labeled (density, 0 to 0.85). Very rare positive cell nuclei (density, 0 to 0.11) were found in some Bowman's spaces and on the external aspect of the glomerular tufts (Figure 8). Overall, proliferating activity was highly variable. The ratio Density of PCNA-positive cells:Density of CD68-positive cells, respectively in the glomeruli and in the tubular lumens, yielded a figure of 0 to 0.53 and of 0 to 0.07, respectively.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Relapse of FSGS Is Characterized by Striking Epithelial Cell
Phenotypic Changes Suggesting Transdifferentiation
Loss of Normal Podocyte Epitopes. We and others have shown that the
peculiar entity described as "primary collapsing glomerulopathy"
(3,4,30)
and HIV-associated nephropathy
(4) is characterized by
podocyte phenotypic dysregulation. In this study, we showed that, similarly,
in transplanted kidneys with relapse of classical FSGS, the expression of
specific podocyte markers, including podocalyxin, synaptopodin, GLEPP-1, WT-1,
CR-1, and vimentin was often but not constantly lost on swollen, large round
cells located along the outer aspect of the tuft or free in Bowman's space. In
fact, that some of these large cells still adherent to the tuft or free in
Bowman's space or drifting within the tubular lumens were indeed podocytes was
demonstrated by their strong expression of podocalyxin, synaptopodin, and
GLEPP-1. Conversely, expression of podocalyxin, synaptopodin, GLEPP-1, WT-1,
CR-1, and vimentin was not found on cobblestones.
Acquisition of CK Epitopes. Labeling by AE1/AE3 anti-CK mAb of cells that clumped at the periphery of the tuft and on cobblestone-like cells that surrounded the synechiae and formed pseudotubules suggests that in FSGS these cells undergo transdifferentiation. This interpretation is substantiated by the fact that AE1/AE3 mAb did not label (or very faintly labeled) podocytes or PEC in normal adult control kidneys. Labeling of cobblestone-like and pseudotubular epithelial cells by C2562, CK22, and AEL-KS2 might suggest that these cells originate from the PEC of Bowman's capsule, as these antibodies marked most of the PEC in adult control kidneys. This is consistent with the hypothesis that migration of PEC onto areas of the tuft where podocytes have disappeared might be an explanation for cobblestone and pseudotubule formation, as others have suggested (31,32,33). However, this hypothesis does not adequately explain the presence of CK-positive cells on the glomerular tuft in the absence of synechiae, in particular in collapsed glomeruli and in areas where PEC did not express CK.
Acquisition of Macrophagic Epitopes. A number of large round cells located on the tuft or drifting free in Bowman's space and in tubular lumens expressed both podocyte and macrophagic epitopes, and particularly two epitopes that are recognized by the PGM1 and the HAM56 mAb. That these cells that express epitopes specific for the monocyte/macrophage lineage were endowed with macrophagic attributes was shown by expression of 25F9, which characterizes macrophage maturation, and moreover by expression of HLA-DR and CD16, which characterize cell activation.
We consider it most unlikely that these cells were in fact true bone marrow-derived macrophages that had gained access to the urinary space from the circulation. By silver methamine staining, no gaps could be found in Bowman's capsule or in glomerular or tubular basement membranes. By contrast, small, spindle-shaped macrophages that infiltrated the renal interstitium were not labeled by 25F9 and CD16. Therefore, acquisition of macrophagic epitopes on the large round cells described above suggests that they had undergone transdifferentiation.
The best argument for a process of cell transdifferentiation occurring in the glomerular epithelial cells stems from confocal laser microscopy. With this technique, we found coexpression of (1) POD + AE1/AE3, (2) POD + CD68, and (3) CD68 + AE1/AE3 on cells that drifted in Bowman's space and within the tubular lumens.
The origin of these transdifferentiated cells might be interpreted in several ways. (1) Cells that coexpress CD68 or AE1/AE3 CK and podocalyxin are transdifferentiated podocytes that have acquired two new epitopes, or, alternatively, (2) cells that coexpress CD68 and AE1/AE3 CK originate from PEC or from tubular epithelial cells as well as from podocytes. In fact, some cells of the tubular epithelium occasionally expressed 25F9, an epitope of mature macrophages.
Transdifferentiation of podocytes into macrophagic cells has an experimental counterpart. Orikasa et al. (34) cultured podocytes from whole glomeruli in the rat. Between 3 and 11 d of culture, these detached visceral epithelial cells lost both the electron microscopic appearance and some immunohistochemical markers of normal podocytes and acquired morphologic and functional characteristics of macrophages.
Transdifferentiation in the Glomeruli Is Accompanied with Expression
of Proliferation and Apoptosis Markers
Our results confirm that in normal human glomeruli, the number of
proliferating cells identified by PCNA
(35), as well as the number of
apoptotic cells identified by TUNEL
(36), is nil. By contrast, on
allografts undergoing relapse of FSGS, these markers were significantly
present. Barisoni et al.
(4), in idiopathic collapsing
glomerulopathy, similarly found that the cell cycle marker Ki-67 was expressed
in regions where the number of epithelial cells on the outer aspect of the
tuft was obviously increased. The term hyperplasia has been used by
several authors regarding cell FSGS
(28). This fits with our
observations of cell proliferation demonstrated by PCNA and suggests, although
without conclusive morphologic evidence, that, contrary to what has been
repeatedly shown in the rat
(31,32,33),
the human podocyte is likely to undergo cell division. Finally, the
demonstration by PCNA and TUNEL that podocytes attached to the tuft as well as
cells free in Bowman's space and in some tubular lumens suggests that relapse
of primary FSGS is characterized by an increased cell turnover.
In summary, this study showed that relapse of FSGS, which most likely reflects the initial phenomenon that occurs in this variety of idiopathic nephrotic syndrome, is characterized by glomerular visceral epithelial cell dysregulation, or more specific, transdifferentiation. In the cellular variant of FSGS, most podocytes had lost their specific epitopes. This was also true in the so-called scar lesion, where the cobblestone-like epithelial cells did not express podocyte epitopes. Conversely, visceral glomerular epithelial cells acquired CK epitopes that are not expressed in the normal fetal and adult glomeruli. Immunohistochemical and confocal microscopy findings strongly suggested that in primary FSGS, some podocytes, occasionally some parietal epithelial cells, and possibly some tubular epithelial cells undergo a process of transdifferentiation with acquisition of epitopes that are characteristic of activated macrophages.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Since this article was accepted for publication, two important articles that shed new light on the issue of podocyte proliferation in FSGS and in collapsing glomerulopathy (CG) have been published. Barisoni and coauthors (Barisoni L, Mokrzycki M, Sablay L, Nagata M, Yamase H, Mundel P: Podocyte cell cycle regulation and proliferation in collapsing glomerulopathies. Kidney Int 58: 137-143, 2000) showed that loss of cyclin kinase inhibitors p27 and p57 leading to expression of cyclin A may account for the activation of podocyte proliferation in CG. Likewise, Shankland and coauthors (Shankland SJ, Eitner F, Hudkins KL, Goodpaster T, D'Agati V, Alpers CE: Differential expression of cyclin-dependent kinase inhibitors in human glomerular disease: Role in podocyte proliferation and maturation. Kidney Int 58: 674-683, 2000) studied the Cip/Kip family of cyclin-dependent kinase inhibitors p21, p27, and p57 along with the proliferation marker Ki-67. They showed that in proliferating areas of the tuft, both in cell FSGS and in CG, loss of expression of p27 and of p47 and acquired expression of p2 are correlated with expression of Ki-67.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  J. Achenbach, M. Mengel, I. Tossidou, I. Peters, J.-K. Park, M. Haubitz, J. H. Ehrich, H. Haller, and M. Schiffer Parietal epithelia cells in the urine as a marker of disease activity in glomerular diseases Nephrol. Dial. Transplant., October 1, 2008; 23(10): 3138 - 3145. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. D. Van Huyen, L. Cheval, M. Bloch-Faure, M. F. Belair, D. Heudes, P. Bruneval, and A. Doucet GDF15 Triggers Homeostatic Proliferation of Acid-Secreting Collecting Duct Cells J. Am. Soc. Nephrol., October 1, 2008; 19(10): 1965 - 1974. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. S. Thorner, M. Ho, V. Eremina, Y. Sado, and S. Quaggin Podocytes Contribute to the Formation of Glomerular Crescents J. Am. Soc. Nephrol., March 1, 2008; 19(3): 495 - 502. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Letavernier, P. Bruneval, C. Mandet, J.-P. D. Van Huyen, M.-N. Peraldi, I. Helal, L.-H. Noel, and C. Legendre High Sirolimus Levels May Induce Focal Segmental Glomerulosclerosis De Novo Clin. J. Am. Soc. Nephrol., March 1, 2007; 2(2): 326 - 333. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Albaqumi, T. J. Soos, L. Barisoni, and P. J. Nelson Collapsing Glomerulopathy J. Am. Soc. Nephrol., October 1, 2006; 17(10): 2854 - 2863. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Bariety, C. Mandet, G. S. Hill, and P. Bruneval Parietal Podocytes in Normal Human Glomeruli J. Am. Soc. Nephrol., October 1, 2006; 17(10): 2770 - 2780. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Aouam, R. Tissier, P. Bruneval, C. Mandet, A. Berdeaux, and B. Ghaleh Preconditioning of salvaged myocardium in conscious rabbits with postinfarction dysfunction Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H2763 - H2769. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J.M. Coward, R. R. Foster, D. Patton, L. Ni, R. Lennon, D. O. Bates, S. J. Harper, P. W. Mathieson, and M. A. Saleem Nephrotic Plasma Alters Slit Diaphragm-Dependent Signaling and Translocates Nephrin, Podocin, and CD2 Associated Protein in Cultured Human Podocytes J. Am. Soc. Nephrol., March 1, 2005; 16(3): 629 - 637. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Meyrier Nephrotic focal segmental glomerulosclerosis in 2004: an update Nephrol. Dial. Transplant., October 1, 2004; 19(10): 2437 - 2444. [Full Text] [PDF]
|
|
|
|
|
|
B. Smeets, N. A.J.M. Te Loeke, H. B.P.M. Dijkman, M. L.M. Steenbergen, J. F.M. Lensen, M. P.V. Begieneman, T. H. van Kuppevelt, J. F.M. Wetzels, and E. J. Steenbergen The Parietal Epithelial Cell: A Key Player in the Pathogenesis of Focal Segmental Glomerulosclerosis in Thy-1.1 Transgenic Mice J. Am. Soc. Nephrol., April 1, 2004; 15(4): 928 - 939. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. Patek, S. Fleming, C. G. Miles, C. O. Bellamy, M. Ladomery, L. Spraggon, J. Mullins, N. D. Hastie, and M. L. Hooper Murine Denys-Drash syndrome: evidence of podocyte de-differentiation and systemic mediation of glomerulosclerosis Hum. Mol. Genet., September 15, 2003; 12(18): 2379 - 2394. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Bariety, G. S. Hill, C. Mandet, T. Irinopoulou, C. Jacquot, A. Meyrier, and P. Bruneval Glomerular epithelial-mesenchymal transdifferentiation in pauci-immune crescentic glomerulonephritis Nephrol. Dial. Transplant., September 1, 2003; 18(9): 1777 - 1784. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Bariety, P. Bruneval, G. S. Hill, C. Mandet, C. Jacquot, and A. Meyrier Transdifferentiation of Epithelial Glomerular Cells J. Am. Soc. Nephrol., June 1, 2003; 14(90001): S42 - 47. [Full Text] [PDF]
|
|
|
|
 |
|
 T. B. Huber, H. C. Reinhardt, M. Exner, J. A. Burger, D. Kerjaschki, M. A. Saleem, and H. Pavenstadt Expression of Functional CCR and CXCR Chemokine Receptors in Podocytes J. Immunol., June 15, 2002; 168(12): 6244 - 6252. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||
