The Parietal Epithelial Cell: A Key Player in the Pathogenesis of Focal Segmental Glomerulosclerosis in Thy-1.1 Transgenic Mice
Bart Smeets*,
Nathalie A.J.M. Te Loeke*,
Henry B.P.M. Dijkman*,
Mark L.M. Steenbergen*,
Joost F.M. Lensen,
Mark P.V. Begieneman*,
Toin H. van Kuppevelt,
Jack F.M. Wetzels and
Eric J. Steenbergen*
Departments of *Pathology, Nephrology, and Biochemistry, University Medical Center Nijmegen, Nijmegen, the Netherlands.
Correspondence to Dr. B. Smeets, Department of Pathology, University Medical Center Nijmegen, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands. Phone: 31-24-3614326; Fax: 31-24-3540520; E-mail: b.smeets{at}pathol.umcn.nl
ABSTRACT. Focal segmental glomerulosclerosis (FSGS) is a hallmarkof progressive renal disease. Podocyte injury and loss havebeen proposed as the critical events that lead to FSGS. In thepresent study, the authors have examined the development ofFSGS in Thy-1.1 transgenic (tg) mice, with emphasis on the podocyteand parietal epithelial cell (PEC). Thy-1.1 tg mice expressthe Thy-1.1 antigen on podocytes. Injection of anti-Thy-1.1mAb induces an acute albuminuria and development of FSGS lesionsthat resemble human collapsing FSGS. The authors studied FSGSlesions at days 1, 3, 6, 7, 10, 14, and 21, in relation to changesin the expression of specific markers for normal podocytes (WT-1,synaptopodin, ASD33, and the Thy-1.1 antigen), for mouse PEC(CD10), for activated podocytes (desmin), for macrophages (CD68),and for proliferation (Ki-67). The composition of the extracellularmatrix (ECM) that forms tuft adhesions or scars was studiedusing mAb against collagen IV 2 and 4 chains and antibodiesdirected against different heparan sulfate species. The firstchange observed was severe PEC injury at day 1, which increasedin time, and resulted in denuded segments of Bowman’scapsule at days 6 and 7. Podocytes showed foot process effacementand microvillous transformation. There was no evidence of podocyteloss or denudation of the GBM. Podocytes became hypertrophicat day 3, with decreased expression of ASD33 and synaptopodinand normal expression of WT-1 and Thy-1.1. Podocyte bridgeswere formed by attachment of hypertrophic podocytes to PEC andpodocyte apposition against denuded segments of Bowman’scapsule. At day 6, there was a marked proliferation of epithelialcells in Bowman’s space. These proliferating cells werenegative for desmin and all podocyte markers, but stained forCD10, and thus appeared to be PEC. The staining properties ofthe early adhesions were identical to that of Bowman’scapsule, suggesting that the ECM in the adhesions was producedby PEC. In conclusion, the authors propose the following sequenceof events leading to FSGS lesions in the Thy1.1 tg mice: (1)PEC damage and denudation of Bowman’s capsule segments;(2) podocyte hypertrophy and bridging; and (3) PEC proliferationwith ECM production.
Focal segmental glomerulosclerosis (FSGS), a hallmark of progressiverenal disease, is one of the most common patterns of glomerularinjury encountered in human renal biopsies (1). In its classicalform, FSGS is characterized by mesangial sclerosis, obliterationof glomerular capillaries, formation of adhesions between theglomerular tuft and Bowman’s capsule, podocyte hypertrophy,hyalinosis, and intracapillary foam cells. More recently, variousmorphologic variants have been described (2). Notably, FSGSis merely a descriptive diagnosis and not a disease entity.Clinically, presentation of the patients varies widely (3).
FSGS was long considered to be the consequence of mesangialoverload leading to mesangial cell proliferation, mesangialcell injury, and increased production of extracellular matrix(ECM) (4–7). More recently, focus has turned on the visceralepithelial cell, the so-called podocyte. Seminal studies ofKriz et al. (8–17) in various rat models of FSGS haveproposed the following sequence of events that ultimately leadto FSGS: (1) podocyte injury resulting in podocyte loss withdenudation of the GBM; (2) adherence of the parietal epithelialcell (PEC) to the naked GBM; (3) formation of an adhesion ofthe GBM to Bowman’s capsule; (4) misdirected filtrationof proteins to the periglomerular and peritubular space. However,the studies by Kriz et al. were performed in animal models inwhich a common feature was glomerular hypertension with capillaryballooning and the development of hypocellular FSGS lesions,which resemble lesions observed in human secondary FSGS. Theirfindings may therefore not apply to FSGS in general. Recently,we have proposed the Thy-1.1 transgenic (tg) mice as a mousemodel to study proteinuria and FSGS (18). Thy-1.1 tg mice carrya mouse-human chimeric transgene that causes ectopic expressionof the mouse Thy-1.1 antigen on podocytes. Injection of mAbdirected against the Thy-1.1 antigen induces an acute albuminuriawithin 10 min and a dose-dependent development of FSGS within3 wk (19). FSGS lesions in this mouse model bear resemblanceto the collapsing variant of human FSGS.
In the present study, we have examined the histopathologic changesthat occur in the early stages of FSGS development. We particularlyfocused on the podocytes and PEC. Our results indicate thatPEC play the principal role in the formation of FSGS lesionsin this mouse model.
Animals
Heterozygous Thy-1.1 tg mice were kindly provided by Dr. D.J.Evans (T6, T-construct mice (20)). These mice were generatedby injecting a hybrid human-mouse Thy-1.1 gene into pronucleiof zygotes of Thy-1.2 CBA x C57BL/10 mice. These mice expressthe Thy-1.1 gene abnormally in podocytes, resulting in the presenceof the Thy-1.1 antigen on the podocytes. All mice were bredin our animal facility. Breading pairs consisted of a heterozygous(+/-) tg mouse and its non-tg (-/-) counterpart. The presenceof the tg was examined by PCR on genomic DNA from the tail,with a forward primer: 5'-CGCCTGAGTCCTGATCTCC-3' and a reverseprimer: 5'-AACCTGCATCTTCACTGGGT-3'. The presence of the tg resultedin a specific 834-bp amplicon. As a positive control for thepresence of amplifiable genomic DNA, a primerset for aminopeptidaseA (APA; EC 3.4.11.7), consisting of the forward primer: 5'-ACACAACCCCAGCTCCTTCC-3'and reverse primer 5'-TCTTCTGCAGCCTGGATCAC-3', was used. Theamplification of the APA gene with these primers resulted ina 367-bp amplicon.
Anti-Thy-1.1 mAb
For in vivo experiments, a mouse anti-mouse Thy-1.1 mAb (19XE5:subclass IgG3) was used. 19XE5 was generated in vitro, by hollowfiber culture, purified by protein-A column affinity chromatographyand concentrated (Nematology Department, Agriculture UniversityWageningen, The Netherlands). The mAb was decomplemented at56°C for 45 min and sterilized by passage through a sterile0.2-µm filter, and stored at -80°C.
Animal Experiments
Five-week-old Thy-1.1 tg mice received an intravenous injectionwith 1 mg anti-Thy-1.1 mAb (19XE5) in 0.1 ml 0.9% saline solution.Transgenic mice injected with 0.1 ml 0.9% saline solution alonewere used as controls. To evaluate the FSGS development, kidneysamples were collected at days 1, 3, 6, 7, 10, 14, and 21 afteranti-Thy-1.1 mAb injection.
Light Microscopy and Transmission Electron Microscopy
For light microscopy, kidney fragments were fixed in Bouin solution,dehydrated, and embedded in paraplast (Amstelstad, AmsterdamThe Netherlands). Four-micrometer sections were stained withperiodic acid-Schiff, and 2-µm sections with silver methenamine(21). At least 60 glomeruli per mouse were evaluated. We determinedthe percentage of glomeruli with a FSGS lesion (FGS score).Only lesions with an adhesion (matrix continuity between thetuft and Bowman’s capsule in the silver stain) were counted.The number of nuclei in Bowman’s space was determined(expressed as nuclei/glomerular cross section) and served asan objective measure for hyperplasia of glomerular epithelialcells.
For electron microscopy, we used immersion and perfusion fixedkidneys. For immersion fixation, small fragments of cortex werefixed in 2.5% glutaraldehyde dissolved in 0.1 M sodium cacodylatebuffer, pH 7.4, overnight at 4°C and washed in the samebuffer. To obtain perfusion fixed kidneys, anti-Thy-1.1-injectedmice were perfused retrogradely via the aorta. The animals wereflushed for 30 s with a buffered saline solution and perfusedwith a 2.5% glutaraldehyde dissolved in 0.1 M sodium cacodylatebuffer, pH 7.4, for 10 min. Perfusion was performed at a pressureof 120 mmHg. Small fragments of cortex were further fixed byimmersion in the same fixative overnight at 4°C and washedin 0.1 M sodium cacodylate buffer, pH 7.4. The tissue fragmentswere postfixed in Palade-buffered 2% OsO4 for 1 h, dehydrated,and embedded in Epon812, Luft procedure (Merck, Darmstadt, Germany).Ultrathin serial sections with 2-µm intervals were contrastedwith 4% uranyl acetate for 45 min and subsequently with leadcitrate for 5 min at room temperature. Sections were examinedin a Jeol 1200 EX2 electron microscope (JEOL, Tokyo, Japan).
Immunohistochemistry
Immunohistochemical staining was performed on kidneys, fixedin 4% buffered formaldehyde for 24 h and embedded in paraffin.Four-micrometer sections were incubated with rat rabbit anti-mouseKi-67 (Dianova Immundiagnostic, Hamburg, Germany), goat anti-CD10,and rabbit anti-WT-1 (Santa Cruz Biotechnology, Santa Cruz,CA). As secondary antibodies we used, a biotinylated goat anti-rabbitantibody for Ki-67 and WT-1, and a horse anti-goat for CD10(Vector laboratories Inc., Burlingame, CA). Detection was carriedout with vectastain ABC kit (Vector Laboratories, Burlingame,CA) with the use of peroxidase as label and diaminobenzidineas substrate. Quantification of the WT-1 and Ki-67-positivecells was performed with the Zeiss KS 400 image analysis system(Carl Zeiss b.v., Weesp, The Netherlands). Images were acquiredusing AxioPlan2 imaging microscope and AxioCam MRc digital ccdcamera (Carl Zeiss b.v., Weesp, The Netherlands). The outlineof individual glomeruli and segments of interstitium were interactivelydrawn using a graphic tablet. Positive nuclei in the selectedareas were interactively marked. A customized macro under KS400software (KS400 3.0) determined the area (mm2) of the selectedsegments, and the number of positive nuclei within this area.Approximately 30 glomeruli per mouse were counted in one cross-section.For staining of CD68 and desmin, we used 2-µm acetonefixed cryosections. The sections were incubated with rat anti-mouseCD68 (clone FA-11; Serotec, Oxford, UK), and rabbit anti-desmin(22). As secondary antibodies we used biotinylated rabbit anti-ratfor CD68 and biotinylated goat anti-rabbit antibody for desmin(Vector laboratories Inc., Burlingame, CA). Detection was carriedout with vectastain ABC kit for alkaline phophatase detection(Vector Laboratories, Burlingame, CA).
Immunofluorescence Microscopy
Kidney fragments were snap-frozen in liquid nitrogen, and 2-µmacetone fixed serial cryostat sections were used. Thy-1.1 wasdetected using a rat polyclonal anti-Thy-1 antibody (59AD2.2;IgG2a, (23)). As secondary antibody, we used a FITC-labeledsheep anti-rat antibody (Serotec, Oxford, UK). A double-immunolabelingtechnique was performed to determine the co-localization ofThy-1.1 and Ki-67 and of Thy1.1 and desmin. Thy-1.1 was stainedas described above. After washing with PBS, the sections wereincubated with rabbit anti-mouse Ki-67 (Dianova Immundiganostic,Hamburg Germany) or with rabbit anti-desmin (22) and detectedwith a goat anti-rabbit Alexa 568 antibody (Molecular ProbesInc, Leiden, The Netherlands). Synaptopodin was detected usingan anti-synaptopodin mAb (Progen Biotechnik, Heidelberg, Germany),followed by FITC-labeled sheep anti-mouse IgG1 (Nordic Immunologicals,Tilburg, The Netherlands). For detection of the podocyte, wealso have used ASD33, a rat monoclonal antibody, generated inour laboratory, which only reacts with the podocyte cell membrane(24). The identity of the antigen is unknown. For the detectionof ASD33, we have used FITC-labeled rabbit anti-rat antibody(Dako A/S, Glostrup, Denmark). Type IV collagen 2 and 4 chainswere detected with rat monoclonal antibodies directed againsthuman collagen (IV) 2 (H22) and human collagen (IV) 4 (H43),a gift from Dr. Yoshikazu Sado (Shigei Medical Research Institute,Okayama, Japan). Detection was done using goat anti-rat Alexa488/568 antibodies (Molecular Probes Inc, Leiden, The Netherlands).Nuclei were stained with TO-PRO-3 (Molecular Probes Inc, Leiden,The Netherlands), a monomeric cyanine nucleic acid stain. Differentheparan sulfate species were detected with various single chainantibodies, HS4C3 (25), HS4E4, and EV3C3 (26). The single chainantibodies were detected with a Cy3-labeled rabbit antibodydirected against the VSV-g epitope tag (Sigma-Aldrich, Zwijndrecht,The Netherlands). The sections were examined with a confocallaser-scanning microscope (CLSM) (Leica lasertechnik GmbH, Heidelberg,Germany).
Statistical Analyses
For multiple comparisons, ANOVA was used and post hoc analyseswere done with Tukey test. P < 0.05 was considered significant.All values are expressed as means ± SEM.
Histopathology of the Kidney
Thy-1.1 tg mice that received a single injection of 1 mg anti-Thy-1.1mAb developed FSGS lesions within 3 wk. The histopathology wasexamined by light microscopy at days 1, 3, 6, 7, 10, and 21(Figure 1). At day 1, we observed enlarged Bowman’s spacedue to collapse of the glomerular tuft, consistent with ischemia.No enlarged podocytes (hypertrophy) were observed. In contrast,PEC showed vacuolization and detachment. In tubular lumens weobserved many hyaline appearing protein casts. Proximal tubularepithelial cells showed damage similar to PEC (Figure 1A). Atday 3, glomeruli were not ischemic anymore, and the number oftubular protein casts had decreased. There was segmental hypertrophyof glomerular epithelial cells (Figure 1B). At days 6 and 7,the first FSGS lesions with tuft adhesions to Bowman’scapsule were observed. Affected glomeruli showed segmental collapseof the capillary tuft and a marked increase of epithelial cellsin Bowman’s space (Figure 1, C and D). It was shown thatthere was a significant overall increase of the number of nucleiin bowman’s space at day 7 (Table 1). Adhesions (continuityof ECM between the capillary tuft and Bowman’s capsule)were formed by thin strands of ECM that were located in betweenthe epithelial cells in Bowman’s space. Sporadically (notquantified) we observed endocapillary foam cells, an endocapillaryneutrophil or mononuclear cell, mild mesangial hypercellularity,and mild hyalinosis (Figure 1F). From a histologic point ofview, the FSGS lesions closely resembled the collapsing variantof human FSGS (27). The percentage of glomeruli with FSGS lesionswas scored at days 1, 3, 6, 7, 10, and 21 by light microscopy(Figure 1G). FSGS lesions, with tuft adhesions to Bowman’scapsule, could be observed in 11 ± 1% and 26 ±3% of the glomeruli at days 6 and 7, respectively. The percentageof glomeruli with FSGS lesions did not increase thereafter.However, at day 21, the lesions were more advanced, showinga high degree of ECM accumulation, which had culminated in theformation of segmental or even global scars (Figure 1E).
Figure 1. Histology of kidney sections of Thy-1.1 transgenic (tg) mice at days 1, 3, 6, 7, and 21 after anti-Thy-1.1 mAb injection, (A-E, respectively). (A) At day 1, light microscopy revealed vacuolization and detachment of PEC (arrow). In proximal tubules, many protein resorption droplets were observed (*). (B) At day 3, a pronounced hypertrophy of glomerular epithelial cells was observed (*). (C and D) At days 6 and 7, tuft adhesions were observed (arrows). Glomeruli showed segmental collapse of capillaries and an increased number of epithelial cells in Bowman’s space (*). (E) At day 21, glomeruli showed accumulation of ECM in Bowman’s space, with formation of segmental scars (arrow); unaffected glomerulus (*). Methenamine silver staining; original magnification, x400. (F) Mild hyalinosis (arrow) and a mitotic endocapillary cell (arrowhead) were occasionally observed. PAS staining; original magnification, x400. The percentage of glomeruli with FSGS lesions at days 1, 3, 6, 7, and 21 is depicted in panel G. * P < 0.05, *** P < 0.001 versus day 1.
Table 1. Number of nuclei in the urinary space in Thy-1.1 tg mice at days 1 and 7 after anti-Thy-1.1 mAb injection and in saline injected micea
EM analysis at days 1 and 3, using immersion fixed tissue, showedglobal podocyte alterations with foot process effacement andmicrovillous transformation. However, we did not observe podocytedetachment or areas of denuded GBM. In contrast to humans, mouseglomeruli frequently show prominent intraglomerular proximaltubular epithelial cells. These "glomerular proximal tubularcells" and PEC showed extensive nuclear activation, vacuolization,and widespread injury especially at day 1 (Figure 2A). As mostFSGS lesions develop around days 6 and 7, we performed extensiveEM analysis at this time point, using both immersion and perfusionfixed tissue. At days 6 and 7, in early lesions, PEC appearedactivated with enlarged nuclei and prominent nucleoli. The numberof PEC nuclei along Bowman’s capsule appeared increased,and mitotic figures were occasionally encountered (Figure 2, C and D).Detachment of PEC with naked BC was occasionally observed(Figure 2B). In affected glomerular segments, the podocyteswere markedly hypertrophic, showing extensive vacuolization,resorption droplets, and formation of pseudocysts (Figure 2D).In immersion-fixed tissue, Bowman’s space was absent andhypertrophic podocytes were positioned directly against PEC.In perfusion-fixed tissue, we frequently observed cell bridgesformed by a hypertrophic podocyte attached to a PEC (in ourhands perfusion fixation, as compared to immersion, leads tomore shrinkage of the glomerular tuft and flattening of thePEC, resulting in a prominent Bowman’s space). At theinterface between hypertrophic podocytes and PEC, junctionalcomplexes were occasionally observed (Figure 2E). In immersion-fixedtissue, we did occasionally see podocytes positioned directlyagainst denuded Bowman’s capsule (Figure 2B); in the perfusion-fixedtissue, optically empty space was always present and true attachmentcould not be confirmed. In more advanced lesions, Bowman’sspace was filled with an increased number of epithelial cells(both fixation techniques), and pericellular accumulation ofmatrix was observed (Figure 2F). Serial sections of single glomerulishowed that FSGS lesions did not develop exclusively close tothe urinary pole but were also encountered near the vascularpole (Figure 2C).
Figure 2. Electron microscopy. Injured glomeruli (days 1, 6, and 7) analyzed by transmission electron microscopy (TEM). (A) At day 1, PEC show widespread injury (insert). Epithelia of Bowman’s capsule constituting proximal tubular epithelial cells show extensive vacuolization of the cytoplasm (*). Podocytes showed foot process effacement (arrow) and microvillous transformation (arrowhead). (B) At days 6 and 7, segments of denuded Bowman’s capsule were observed (arrows). Podocytes positioned against the denuded capsule were observed, thus forming podocyte bridges between the GBM and the bare Bowman’s capsule (*). (C-E) At days 6 and 7, in early lesions, PEC appeared activated with enlarged nuclei and prominent nucleoli. The number of PEC nuclei along Bowman’s capsule appeared increased (). The hyperplasia of epithelial cells in Bowman’s space was also encountered near the vascular pole (vp). Podocytes were markedly hypertrophic showing extensive vacuolization, resorption droplets (rd), and formation of pseudocysts (*). In perfusion-fixed tissue, cell bridges formed by a hypertrophic podocyte attached to a PEC were observed (arrows). At the site of attachment between hypertrophic podocytes and PEC junctional complexes were occasionally observed (arrows in inserts 1 and 2). (F) Glomerular epithelial cells positioned in Bowman’s space showed pericellular accumulation of ECM (m). (A, B, and F: immersion-fixed; C-E: perfusion-fixed) Original magnifications: A, x1500; insert, x3000; B, x7500; C, x600; D, x500; E, x1000; insert 1, x12,000; insert 2, x20 000; F, x4000.
Podocyte Phenotype
To assess changes in podocyte phenotype during the developmentof FSGS, we studied the expression of podocyte-specific markers,WT-1, synaptopodin, ASD33 (an unidentified cell membrane antigen,strongly expressed on mouse podocytes), the Thy-1.1 antigen,Ki-67 as a marker of proliferation, desmin (associated withpodocyte dedifferentiation) (28), and CD68 (macrophage marker).At day 3, in the absence of sclerotic lesions, we already observedsome decrease in the expression of ASD33 and synaptopodin (datanot shown). At days 6 and 7, changes were even more prominent(Figure 3A). The decrease of ASD33 and synaptopodin expressionwas particularly observed in segments with a tuft adhesion ortuft collapse. During the first 7 d, we did not observe a changein the number of WT-1-positive cells. At day 21, we did observea significant decrease in number of WT-1-positive podocytescompared with days 1 and 6 (Table 2). At day 7, there was anincreased expression of desmin in affected glomeruli. Desminco-localized with the Thy-1,1 antigen and is expressed by podocytes(Figure 3B). There was no glomerular expression for CD68 (datanot shown).
Figure 3. Podocyte phenotype. (A) Immunofluorescence for podocyte markers (ASD33, synaptopodin, Thy-1.1) and an anti-heparan sulfate single chain antibody (HS4C3). Serial sections, from control and anti-Thy-1.1 mAb-injected mice (day 7). HS4C3 stains GBM and mesangium, showing normal tuft architecture in the control and a segmental lesion in Thy-1.1-injected mice (day 7, *). In control mice, ASD33, synaptopodin, and Thy-1.1 showed a homogeneous staining pattern along the GBM. There is a decrease in expression of ASD33 and synaptopodin in areas with a segmental lesion. There is still high expression of the Thy-1.1 antigen surrounding the collapsed segment (arrow). (B) IF for desmin and Thy-1.1. Section from an anti-Thy-1.1 mAb-injected mouse (day 7). Thy-1.1 shows a normal podocyte staining in the unaffected glomerulus (arrowhead) but an irregular pattern in an affected glomerulus (*). The affected glomerulus shows expression of desmin (*). Desmin and Thy-1.1 co-localize in the affected glomerulus (arrow). Original magnification, x500.
Table 2. Number of WT-1-positive cells per mm2 in Thy-1.1 tg mice at different days after anti-Thy-1.1 mAb injection and in saline injected micea
Characterization of the Proliferating Epithelial Cells
We have performed phenotypic analysis of proliferating epithelialcells in Bowman’s space to clarify their origin. Sectionswere stained for Ki-67. We observed an increase in the numberof proliferating cells, which peaked at day 3 for the interstitialcompartment and at day 7 for the glomerulus (approximately 5xbaseline levels). Thereafter, the number of Ki-67-positive cellsdeclined to baseline levels (Figure 4A). Approximately 50% ofproliferating cells were located in Bowman’s space; therest were located within the glomerular tuft. Proliferatingcells were often located adjacent to adhesions (Figure 5B).In addition, proliferating cells were often positioned alongBowman’s capsule and appeared to be PEC (Figure 4B). Bydouble immunofluorescence, we never observed co-localizationof Ki-67 and the podocyte specific Thy-1.1 antigen, arguingagainst the presence of proliferating podocytes. We did notobserve Thy-1.1 expression in areas with accumulations of epithelialcells in Bowman’s space (Figure 5A); however, in theseareas, we did observe expression of the neutral endopeptidaseCD10 (Figure 5D), a protein that, in glomeruli of control mice,is strongly expressed on PEC (Figure 5C).
Figure 4. Cellular proliferation in the glomerulus and interstitial compartment. (A) The number of Ki-67-positive proliferating cells in the glomerulus gradually increased and peaked at day 7. Thereafter, the number of Ki-67-positive cells declined to baseline levels. Peak proliferation of cells within the interstitial compartment was observed at day 3. (B) Immunohistochemistry for Ki-76. Immunoreactive nuclei stain brown. There is a high number of Ki-67-positive cells positioned along Bowman’s capsule (arrow). We also observed Ki-67 positive cells (arrowhead) within the glomerular tuft. Original magnification, x400; n = 4; ** P < 0.01 versus saline; *** P < 0.001 versus saline.
Figure 5. Phenotype of proliferating cells. Panel A shows immunofluorescence in an anti-Thy-1.1 mAb-injected mouse (day 7), for Thy-1.1 (green) and HS4E4, an anti-heparan sulfate antibody (red). Nuclei are stained with TO-PRO-3 (blue staining). Shown is a FSGS lesion with accumulation of cells in Bowman’s space (A, arrowhead). Thy-1.1 is present along the glomerular tuft, but there is no staining for Thy-1.1 within the lesion. HS4E4 stains Bowman’s capsule and tuft adhesions (A, arrow). Panel B shows immunofluorescence for collagen 2 (IV) (red) and Ki-67 (green). Collagen 2 (IV) is present in Bowman’s capsule, mesangium, and tuft adhesions (B, arrow); Ki-67-positive cells are located adjacent to tuft adhesions. Magnifications: A, x500; B, x600. Panels C and D show immunohistochemistry for CD10. Immunoreactive cells stain brown. In control mice, CD10 is strongly expressed on PEC within the glomerulus (C). In a FSGS lesion (day 7), epithelial cells in Bowman’s space strongly express CD10 (D). Original magnification, x400.
Characterization of ECM
The composition of the ECM that is present in the adhesionswas studied by staining serial sections with antibodies directedagainst the 2 and 4 chains of collagen IV. In addition, we haveused different anti-HS single chain antibodies that in controlmice predominantly stain Bowman’s capsule and not theGBM. The ECM forming tuft adhesions stained for collagen 2 (IV)and the anti-HS antibodies and were negative for collagen 4(IV) and HS4C3, an anti-HS antibody that stains GBM and mesangium.Thus, the staining properties of the newly formed ECM were identicalto those of Bowman’s capsule, arguing that this matrixwas produced by PEC rather than podocytes (Figure 6).
Figure 6. Assessment of the composition of the ECM. Immunofluorescence for collagen IV 2 and 4 (green) and HS4E4 (red) in serial sections of a glomerulus with adhesions (day 7). (A) Within the glomerulus, collagen 4 (IV) is expressed specifically in the GBM. (B) Collagen 2 (IV) is expressed in Bowman’s capsule, mesangium, and the tuft adhesions. (C and D) HS4E4 stains Bowman’s capsule and the accumulations of ECM, which form the tuft adhesions (arrows). (E) There was no co-localization of collagen 4 (IV) and heparan sulfate identified by HS4E4. (F) Collagen 2 (IV) shows co-localization with the HS4E4 heparan sulfate in Bowman’s capsule and the tuft adhesions. Original magnification, x500.
Our study indicates that PEC play an important role in the developmentof FSGS in the Thy-1.1 tg mouse. Although we also observed distinctchanges in the podocyte, we think that the involvement of thePEC is more prominent. In the early time points after inductionof albuminuria, we observed PEC injury, which was accompaniedby denudation of segments of the Bowman’s capsule. Thereafter,a cellular lesion consisting of hypertrophic and hyperplasticglomerular epithelial cells developed. Although hypertrophicpodocytes contributed to these lesions, most cells were proliferatingPEC. Finally, the cellular lesions turned into the adhesivescar that is the hallmark of FSGS. We have evaluated the compositionof the scar using antibodies against heparan sulfates and collagen(IV) 2 and 4, and the data indicated that the ECM that is presentin the scar is derived from the parietal epithelium.
Podocyte changes in our model consisted of podocyte hypertrophyand podocyte foot process effacement. Of note, we never observeddetachment of the podocytes or areas of denuded GBM. Our findingsare in contrast with the observations of Kriz et al., who havepostulated a final common pathway for the development of FSGSon the basis of their studies in various rat models. Their proposedpathway toward FSGS emphasizes the podocyte. Detailed histologicanalyses have pointed to the following sequence of events: (1)podocyte injury; (2) detachment of podocytes and/or loss ofpodocytes (podocytopenia) with denudation of the GBM; (3) adherenceof the naked GBM to PEC, which then lose intercellular contacts;and (4) the formation of an adhesion between the glomerulartuft and Bowman’s capsule, which allows "misdirected"filtration of plasma proteins to the periglomerular and peritubularspace, ultimately leading to global glomerulosclerosis and tubulointerstitialfibrosis (13–17).
In our model, development of FSGS clearly follows other pathways,thus suggesting that the model proposed by Kriz et al. may notbe applicable to FSGS in general. The latter conclusion maynot come as a surprise in view of the large clinical variabilityof patients with FSGS. Thus far, most studies have been donein rat models with evidence of glomerular or systemic hypertension.Most notable is the observation that in the reported studiesthere was always evidence of capillary ballooning and glomerularhypertrophy. It is tempting to speculate that the proposed pathwaytoward FSGS based on the rat models is relevant for the processof secondary FSGS that is observed in patients with reducedrenal mass or longstanding hypertension.
In our mouse model, we did not observe dilation of the glomerularcapillaries or glomerular hypertrophy. Rather, we observed segmentalcollapse of the tuft with prominent extracapillary proliferationand frequent formation of adhesions. Therefore, from a morphologicpoint of view, the lesions in our mouse model closely resemblethe collapsing variant of human FSGS (2,27), although the segmentalrather than global nature of the process and the frequent formationof adhesions are more typical for other variants of human FSGS.Collapsing FSGS is a recently recognized entity, predominantlyseen in African Americans, also associated with HIV infection,and with a poor prognosis and rapid progression toward end-stagerenal disease (ESRD). Collapsing FSGS in humans is associatedwith higher initial proteinuria and by some authors consideredto be early and/or active lesions (29–33). To what extentis our model representative of human collapsing FSGS? Kriz etal. consider collapsing FSGS to be distinct from the classicalform, with a different underlying pathogenetic mechanism (14,15).It has been proposed that the cellular lesions that are prominentlypresent in collapsing FSGS consist of podocytes that are dysregulatedand are dedifferentiated with loss of all podocyte-specificmarkers (34–37) and increased expression of desmin (28).These podocytes are apparently no longer growth restricted andhave regained the ability to proliferate. Normal podocytes areconsidered post-mitotic cells, unable to proliferate due tohigh-level expression of cyclin-dependent kinase inhibitors(CKI) p27 and p57 (34). In our mouse model, we also observedpodocyte hypertrophy and loss of podocyte-specific markers,synaptopodin and ASD33, and an increased expression of desmin,consistent with dedifferentiation. However, because of the continuedexpression of podocyte-specific markers, WT-1 and Thy-1.1, wewere able to show that podocytes in our model do not proliferate.Conversely, the orientation of the proliferating epithelialcells relative to Bowman’s capsule and Bowman’sspace, the strong expression of CD10 and the composition ofthe ECM that is produced by these cells strongly argue thatthe proliferating cells are PEC. PEC have low-level expressionof CKI and are known to proliferate in response to differentstimuli (38). A typical example of PEC proliferation is seenin the formation of crescents (39,40). Taken together, our dataargue against a dedifferentiated and dysregulated podocyte beingresponsible for the formation of FSGS lesions in our model.
How should earlier data be interpreted? In the studies thathave assessed podocyte dedifferentiation, it is noteworthy thatthese "dedifferentiated podocytes" had lost expression of WT-1,GLEPP, synaptopodin, and podocalyxin. Some cells had detachedfrom the GBM, and these cells did not express podocyte markersbut rather demonstrated characteristic features of macrophages(41,42). Thus, it cannot be excluded that some of the cellspresent in cellular lesions in human collapsing FSGS originatefrom proliferating PEC. Contribution of PEC to cellular lesionsin human collapsing FSGS has been previously proposed by otherauthors on the basis of positive staining for cytokeratin (38).We also need to consider the possibility that FSGS lesions maydevelop differently in mouse as compared with humans. In mouseglomeruli, proximal tubular epithelial cells are present withinthe glomerulus close to the urinary pole. It cannot be excludedthat proximal tubular epithelial cells proliferate and influenceFSGS development. The fact that in our mice FSGS lesions developnot only close to the urinary pole but also in the perihilarregion argues against this possibility. Nevertheless, the existenceof proximal tubular epithelial cells within Bowman’s capsulein mice complicates any analysis of glomerular cell changes.
The finding that cellular lesions in our model are of PEC origindoes not preclude an important role for the podocyte in thedevelopment of FSGS lesions. The initial bridge consists ofa hypertrophic podocyte that attaches to PEC or comes into contactwith Bowman’s capsule. Attachment of a hypertrophic podocyteto PEC or appositioning to denuded Bowman’s capsule maybe the critical event that leads to an FSGS lesion. In thisrespect, our findings partly resemble crescent formation ina mouse model of crescentic glomerulonephritis (39). These authorsobserved that podocyte bridges between the capillary tuft andBowman’s capsule precede the development of crescents.In this model, PEC injury with gaps between the PEC and areasof denuded Bowman’s capsule was not observed. The authorssuggest that the bridging podocyte is critically involved inregulating proliferation of PEC. In our model, a similar effectmay then be responsible for the proliferation of the PEC thatwe observed. Le Hir et al. (39) suggests that the podocyte producedbasement membrane-like material that formed in continuity withthe Bowman’s capsule. However, this conclusion was basedon staining for collagen IV with a polyclonal antibody, whichdoes not allow differentiation between different -chains. Ourfindings clearly prove that the adhesion is made up of Bowman’scapsule like material. In Figure 7, we have proposed a schemefor the sequence of events that ultimately lead to FSGS in ourmodel.
Figure 7. Hypothetical sequence of events leading to cellular FSGS lesions in the Thy1.1 tg mice, illustrated in schematic drawings. (A) Days 1–7. Injury of the glomerular epithelial cells. Podocytes (green) show foot process effacement and microvillous transformation (not illustrated). There is no podocyte detachment. PEC (blue) show vacuolization and detachment, leading to "bare" segments of Bowman’s capsule (Bowman’s capsule is drawn as a gray line). (B) Days 3–7. Podocytes become hypertrophic (with activated nuclei, resorption droplets, and pseudocysts) and form bridges between the glomerular tuft and Bowman’s capsule. (C) Days 6 and 7. Proliferation of PEC adjacent to the bridging podocytes, filling up Bowman’s space, giving the appearance of a cellular FSGS lesion. (D) The proliferating PEC in Bowman’s space produce ECM (gray) that eventually forms tuft adhesions.
How to reconcile both models with human data? As mentioned above,the classical pathway of FSGS as described by Kriz et al. inrat models of hypertrophy and hypertension may best fit withsecondary FSGS. Our model may bear more resemblance to primaryFSGS. This needs to be further investigated using human renalbiopsy material. Notably, PEC damage in biopsies from patientswith FSGS is frequently observed (43–45) and possiblycaused by protein overload.
In conclusion, PEC play an important role in the formation ofcellular FSGS lesions in the Thy-1.1 tg mouse.
Acknowledgments
This work was supported by a grant from the Dutch Kidney Foundation(C 99.1844). We thank Dr. Yoshikazu Sado (Shigei Medical ResearchInstitute, Okayama, Japan) for providing the anti-collagen IV2 and 4 antibodies.
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Received for publication August 15, 2003.
Accepted for publication January 21, 2004.
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Top
Abstract
Introduction
2 and 
). The hyperplasia of epithelial cells in Bowman’s space was also encountered near the vascular pole (vp). Podocytes were markedly hypertrophic showing extensive vacuolization, resorption droplets (rd), and formation of pseudocysts (*). In perfusion-fixed tissue, cell bridges formed by a hypertrophic podocyte attached to a PEC were observed (arrows). At the site of attachment between hypertrophic podocytes and PEC junctional complexes were occasionally observed (arrows in inserts 1 and 2). (F) Glomerular epithelial cells positioned in Bowman’s space showed pericellular accumulation of ECM (m). (A, B, and F: immersion-fixed; C-E: perfusion-fixed) Original magnifications: A, x1500; insert, x3000; B, x7500; C, x600; D, x500; E, x1000; insert 1, x12,000; insert 2, x20 000; F, x4000.
