Abstract
Collapsing glomerulopathy (CG) is associated with disorders that markedly perturb the phenotype of podocytes. The kd/kd mouse has been studied for immune and genetic causes of microcystic tubulointerstitial nephritis with little attention to its glomerular lesion. Because histologic examination revealed classic morphologic features of CG, the question arises whether podocytes in kd/kd mice exhibit additional phenotypic criteria for CG. Utilizing Tg26 mice as a positive control, immunohistochemical profiling of the podocyte phenotype was conducted simultaneously on both models. Similar to Tg26 kidneys, podocytes in kd/kd kidneys showed de novo cyclin D1, Ki-67, and desmin expression with loss of synaptopodin and WT-1 expression. Electron micrographs showed collapsed capillaries, extensive foot process effacement, and dysmorphic mitochondria in podocytes. These results indicate that the kd/kd mouse is a model of CG and raise the possibility that human equivalents of the kd susceptibility gene may exist in patients with CG.
Since its first clinicopathologic descriptions in the 1980s, collapsing glomerulopathy (CG) is increasingly recognized as the cause of renal failure in humans and experimental animals (1–7). In addition to the unique glomerular morphology of hyperplastic and hypertrophic podocytes overlying collapsed capillary loops (1,2), a consistent feature of CG is the marked perturbation to the mature phenotype of podocytes in diseased glomeruli (8–13). This dysregulated podocyte phenotype is captured by select immunohistochemical markers and segregates the podocyte injury in CG from other podocytopathies (8–13). Indeed, the application of these morphologic and immunohistochemical criteria has been instrumental in characterizing several new murine models with similarities to human CG over the last two years (3–7), each in turn furthering knowledge that disruption of normal podocyte function, whether from intrinsic or extrinsic insults, is a critical step in the development of CG.
The kd/kd mouse was first described over three decades ago as a distinctive model of spontaneous proliferative disease of renal epithelium in a subline of CBA/CaH mice (14). Since then, the kd/kd mouse has been studied for immune and genetic causes of its prominent microcystic tubulointerstitial nephritis with little attention to the accompanying glomerular lesion (15–19). Recently, the susceptibility gene for renal disease in kd/kd mice was mapped and found to encode a prenyltransferase-like mitochondrial protein (PLMP) with shared homology to human transprenyltransferase, human geranylgeranyl pyrophosphate synthase, and a putative human tumor suppressor protein (16,19). C57BL/6 (B6) mice bred homozygous for this mutant allele manifest a tubulointerstitial disease identical to the founder strain with variable onset no earlier than 8 wk of age that ultimately progresses to end-stage renal disease by 16 to 40 wk of age (18,19). Introduction of a wild-type PLMP transgene into B6 kd/kd mice can rescue this renal disease (19), suggesting that the kd susceptibility gene is required, but perhaps not sufficient alone, for the development of nephropathy in this model. Because histologic examination of glomeruli in diseased B6 kd/kd mice revealed glomerular collapse and extensive glomerulosclerosis with hypertrophy and hyperplasia of overlying podocytes (Figure 1), we asked whether the additional immunohistochemical and ultrastructural criteria that define CG exist in B6 kd/kd mice. Using heterozygous Tg26 mice as a previously characterized positive control for murine CG (20,21), quantitative profiling of the phenotype of podocytes was conducted simultaneously across the two models.
Collapsing glomerulopathy in B6 kd/kd mice. (A) Normal glomerulus in a B6 wild-type mouse. (B) Normal glomerulus in a nontransgenic Tg26 mouse. (C) B6 kd/kd mouse with glomerular collapse and podocyte hypertrophy and hyperplasia; focal injury to the parietal epithelium is also noted. (D) Tg26 heterozygote with glomerular collapsing features and prominent podocyte hypertrophy and hyperplasia with pseudocrescent formation and bridging to parietal epithelial cells. (E) B6 kd/kd mouse showing glomerular collapse and pseudocrescent formation adjacent to severe tubulointerstitial damage with prominent, protein-filled microcysts. (F) Tg26 heterozygote showing glomerular collapse with pseudocrescent formation adjacent to severe tubulointerstitial damage with prominent microcysts. (G) Electron micrograph of a glomerular capillary in a B6 wild-type mouse shows glomerular basement membranes (GBM) that are normal in thickness and contour, as well as podocytes with well-preserved foot processes. (H) Electron micrograph of a glomerular capillary in a diseased B6 kd/kd mouse shows GBM that are wrinkled and folded (indicating collapse) with subocclusion of the capillary lumen. There is also extensive foot process effacement accompanied by condensation of the actin-based cytoskeleton and swelling of primary processes. (I) A healthy podocyte from a B6 wild-type mouse containing few normal mitochondria with regular matrix density (arrows). (J) A diseased podocyte from a B6 kd/kd mouse containing numerous abnormal mitochondria with compressed cristae forming truncated cisternae and granular-appearing matrix (arrows). Magnification, ×400 in A through D, ×100 in E and F, ×25,000 in G through J. The sections in A through F are silver-stained.
Materials and Methods
Mice
All studies on Tg26 and B6 kd/kd tissues complied with Institutional Animal Care and Use Committee regulations of the New York University School of Medicine and the University of Pennsylvania School of Medicine, respectively. Archival formalin-fixed, paraffin-embedded kidneys from six homozygous B6 kd/kd mice ranging in ages from 15 to 43 wk and from two 15-wk-old wild-type B6 controls were studied. Archival formalin-fixed, paraffin-embedded kidneys from three 6-wk-old heterozygous Tg26 mice and from one 6-wk-old nontransgenic littermate were used as positive and negative controls, respectively, for murine CG (20,21).
Histopathology
Three-μm thick serial sections from each specimen were stained with hematoxylin and eosin (H&E), trichrome, periodic-acid schiff (PAS), or silver. Quantitative histopathology for the extent of glomerular sclerosis, capillary tuft collapse with overlying podocyte hypertrophy and hyperplasia, tubular microcysts, acute tubular injury, tubular atrophy, and interstitial inflammation and fibrosis, was singularly evaluated across the entirety of each section. This quantitation was performed as follows: The percent of all glomeruli with sclerosis (defined as segmental or global solidification of the glomerular tuft on silver or trichrome stain); the percent of all glomeruli with collapse (defined as wrinkling and folding of the glomerular basement membranes of any portion of the capillary tuft on silver stain) with overlying podocyte hypertrophy and hyperplasia, scaled as zero (none), +/− (1 to 5%), 1+ (6 to 25%), 2+ (26 to 50%), or 3+ (>51%); the percent area of the total tubulointerstitial compartment with tubular microcysts (defined as tubules dilated at least 4 times the normal diameter), acute tubular injury (defined as flattening of the tubular epithelium, loss of the brush border, or blebbing of the cytoplasm and nuclear hyperchromasia with prominent nucleoli), tubular atrophy (defined as thickened tubular basement membranes with small cuboidal tubular cells), or interstitial inflammation and fibrosis, scaled as zero (none), +/− (1 to 5%), 1+ (6 to 25%), 2+ (26 to 50%), or 3+ (>51%).
Immunohistochemistry on 3-μm thick serial sections from each specimen to detect changes to the phenotype of mature podocytes was performed using primary antibodies to mark podocyte cell-cycle engagement (cyclin D1, clone SP4, Lab Vision, Fremont, CA), podocyte cell-cycle progression (Ki-67, clone SP6, Lab Vision), the state of podocyte differentiation (synaptopodin, mouse monoclonal, gift of Dr. Peter Mundel, Mount Sinai School of Medicine, New York, NY; WT-1, clone 6F-H2, NovoCastra, Newcastle, UK), and podocyte injury (desmin, clone D33, DAKO, Carpinteria, CA) as described previously on Tg26 kidneys (20,21). Sections stained for synaptopodin or desmin were counterstained with hematoxylin, and sections stained with cyclin D1, Ki-67, or WT-1 were counterstained with PAS. Quantitation of the change in podocyte phenotype in each mouse was calculated as the percent of all nonglobally sclerotic glomeruli containing podocytes with cyclin D1 in one or more nuclei, Ki-67 in one or more nuclei, desmin in at least one segmental distribution, loss of WT-1 in at least one segmental distribution, or with trace or less synaptopodin in the cytoplasm. Globally sclerotic glomeruli were excluded in the analysis due to the absence of podocytes.
Ultrastructural Analysis
Small samples of renal cortex from B6 kd/kd mice were fixed in 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, overnight at 4°C. Samples were postfixed with 2.0% osmium tetroxide in 0.1 M cacodylate buffer for 1 h at 4°C. After additional washing in 0.1 M cacodylate buffer and distilled H2O, samples were stained with 2% aqueous uranyl acetate for 30 min at room temperature. Samples were then rinsed in distilled H2O, dehydrated, infiltrated, and embedded in Embed 812 (Electron Microscopy Science, Fort Washington, PA). Sections were examined in a JEOL100CX electron microscope. Digital images recorded on a Hamamatsu camera were analyzed for the presence of folding and wrinkling of the glomerular basement membrane and foot process effacement.
Results and Discussion
The glomerular lesion of collapsing glomerulopathy is defined morphologically by the presence of hyperplastic and hypertrophic podocytes overlying collapsed capillary loops in either a segmental or global distribution within the glomerular tuft (1,2). These diseased podocytes undergo a marked perturbation in their mature, quiescent phenotype, characterized by proliferation and dedifferentiation, which is not observed in other proteinuric lesions (8–13). Concurrent examination and quantitation of the morphologic injury within glomeruli of B6 kd/kd and Tg26 mice, coupled with quantitative profiling of the podocyte phenotype by immunohistochemical markers, demonstrate that the renal disease in B6 kd/kd mice fulfills the criteria for CG (Figures 1 and 2; Table 1). Similar to CG in Tg26 mice (20,21), diseased glomeruli in B6 kd/kd mice show segmental and global sclerosis and collapse of capillary loops with folding and wrinkling of the glomerular basement membrane, extensive foot process effacement with marked condensation of the actin cytoskeleton and focal loss of primary processes of podocytes, and hyperplastic and hypertrophic podocytes with de novo cyclin D1, Ki-67, and desmin expression and reduced synaptopodin and WT-1 expression. In addition to these significant alterations to podocytes, focal injury to the parietal epithelium lining Bowman’s capsule is evident in B6 kd/kd mice. Despite the variable, age-dependent penetrance of CG in B6 kd/kd mice, there appears to be a positive correlation suggesting causality between the extent of glomerular injury and the downstream tubulointerstitial disease in each animal. Together, these data indicate that the B6 kd/kd mouse is a previously unrecognized model of CG. Moreover, similar to prior observations of changes to the morphology of mitochondria in the tubular epithelium of B6 kd/kd mice (19), abnormal mitochondria are also found in diseased podocytes.
Comparison of the podocyte phenotype between B6 wild-type and B6 kd/kd mice. Synaptopodin stains strongly in the cytoplasm of podocytes in B6 wild-type mice indicating the normal, differentiated state, whereas there is a marked loss of synaptopodin expression in diseased podocytes in B6 kd/kd mice. An identical change is observed in the nuclear staining of WT-1, a second marker of podocyte differentiation. Nuclear staining of Ki-67, a marker of cell-cycle progression, is not detected in glomeruli of B6 wild-type mice, but is focally positive in glomerular epithelial cells forming pseudocrescents in B6 kd/kd mice. Likewise, nuclear staining of cyclin D1, a marker of cell-cycle engagement, is diffusely negative in podocytes of B6 wild-type mice (but positive in some intracapillary cells), whereas it is detected in podocytes in B6 kd/kd mice in areas of podocyte hypertrophy and hyperplasia. Glomerular staining of desmin is found only in mesangial cells in B6 wild-type mice, but is markedly upregulated in injured podocytes in B6 kd/kd mice. Magnification, ×400.
Collapsing glomerulopathy in B6 kd/kd and Tg26 mice: Morphologic injury and changes to the podocyte phenotype
The exact pathogenic steps whereby mutant PLMP causes CG in B6 kd/kd mice are not known. Antisera to PLMP localize to dysmorphic mitochondria in renal epithelium of B6 kd/kd mice (19). This suggests that mutant PLMP might directly alter mitochondrial function in podocytes, lowering the threshold to injury from energetic stress. This is an attractive hypothesis as CG and focal segmental glomerulosclerosis can develop in patients with genetically-acquired mitochondrial cytopathies (22,23). Furthermore, CG is associated with a growing list of disease stresses (1). If this is indeed correct, B6 kd/kd mice would represent the first model of CG due to a mitochondrial disorder, providing a ready system to investigate how environmental factors may influence the manifestation of this abnormality within podocytes. Interestingly, bisphosphonate drugs, small molecules linked to podocyte injury and CG in humans (24,25), can perturb mitochondrial function (26), and human mitochondrial transprenyltransferases sharing homology with PLMP contain specificity determining residues for bisphosphonate binding (i.e., the amino acid sequence DDXXD). However, the extent to which bisphosphonates interact with and inhibit human transprenyltransferases is still unclear (Eric Oldfield, University of Illinois at Urbana-Champaign, personal communication).
Alternatively but not mutually exclusively, an aberrant autoimmune-like response to renal parenchymal damage specific to B6 mice may dictate the development of CG in this model, as suggested by prior studies on B6 kd/kd mice (18). Although the phenotypic manifestation of renal disease after transfer of the kd susceptibility gene to B6 mice, a strain biased toward T-helper type 1 immunity (27), appears to be identical to that of the founder strain, we do not know if the degenerate glomeruli, glomerulosclerosis, and albuminuria noted in the original report on CBA/CaH kd/kd mice (14) is a product of the same podocytopathy reported here (i.e., CBA/CaH kd/kd tissues are no longer available). Indeed, although specific genes that may modify the nephropathy in B6 kd/kd have not been identified, the CG in B6 kd/kd mice may ultimately be attributable to background genetic differences between strains of mice. For example, using a mouse genetics approach with Tg26 mice to investigate the racial predilection of HIV-induced CG, Gharavi et al. identified susceptibility loci and strain-specific modifications to specific features of the renal disease in this model (28), including amelioration by BALB/C mice, a strain biased toward T-helper type 2 immunity (27). These intriguing observations regarding what is likely to be a polygenic disease raise the possibility that human equivalents of the kd susceptibility gene may exist and predispose some patients to develop CG. Further studies on B6 kd/kd mice, the first model of CG caused by a spontaneously occurring mutation identified through forward genetics, not a product of reverse genetics (3,4,6,7,20), and of patients with CG will help answer these questions.
Acknowledgments
We thank Ali Gharavi and David B. Thomas for critical reading of the manuscript, Ke Lin for excellent technical support, Ray Meade from the Biomedical Imaging Core of the University of Pennsylvania for the electron microscopy, and David B. Thomas, J. Charles Jennette, and Surya Seshan for reviewing the pathology in a blinded fashion. This work was supported by National Institutes of Health grants DK55852 (D.L.G.) and DK065498 (P.J.N.).
Footnotes
Published online ahead of print. Publication date available at www.jasn.org
- © 2005 American Society of Nephrology
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