Pathophysiology of Renal Disease

Focal and Segmental Glomerulosclerosis in Mice with Podocyte-Specific Expression of Mutant α-Actinin-4

Jean-Louis Michaud, Lyne I. Lemieux, Manon Dubé, Barbara C. Vanderhyden, Susan J. Robertson and Chris R.J. Kennedy
JASN May 2003, 14 (5) 1200-1211; DOI: https://doi.org/10.1097/01.ASN.0000059864.88610.5E

Abstract

ABSTRACT. Mutations in the gene encoding α-actinin-4 (ACTN4), an actin crosslinking protein, are associated with a form of autosomal dominant focal segmental glomerulosclerosis (FSGS). To better study its progression, a transgenic mouse model was developed by expressing murine α-actinin-4 containing a mutation analogous to that affecting a human FSGS family in a podocyte-specific manner using the murine nephrin promoter. Consistent with human ACTN4-associated FSGS, which shows incomplete penetrance, a proportion of the transgenic mice exhibited significant albuminuria (8 of 18), while the overall average systolic BP was elevated in both proteinuric and non-proteinuric ACTN4-mutant mice. Immunofluorescence confirmed podocyte-specific expression of mutant α-actinin-4, and real-time RT-PCR revealed that HA-ACTN4 mRNA levels were higher in proteinuric versus non-proteinuric ACTN4-mutant mice. Only proteinuric mice exhibited histologic features consistent with human ACTN4-associated FSGS, including segmental sclerosis and tuft adhesion of some glomeruli, tubular dilatation, mesangial matrix expansion, as well as regions of podocyte vacuolization and foot process fusion. Consistent with such podocyte damage, proteinuric ACTN4-mutant kidneys exhibited significantly reduced mRNA and protein levels of the slit diaphragm component, nephrin. This newly developed mouse model of human ACTN4-associated FSGS suggests a cause-and-effect relationship between actin cytoskeleton dysregulation by mutant α-actinin-4 and the deterioration of the nephrin-supported slit diaphragm complex. E-mail: ckennedy@uottawa.ca

Focal segmental glomerulosclerosis (FSGS) is a syndrome of primary glomerular lesions with a population incidence of approximately 2 per million (1). FSGS is a significant cause of end-stage renal disease (ESRD), comprising up to 5% of adults and 20% of children with ESRD (2,3⇓). The clinical hallmarks of FSGS routinely include proteinuria, hypertension, and the progression to ESRD. Renal biopsy often reveals areas of solidification, glomerulosclerosis, and tuft collapse that are both focal (not all glomeruli are affected) and segmental (only a part of each glomerulus is damaged). The accumulating evidence suggests that either damage to or within glomerular visceral epithelial cells (podocytes) plays a key role in the development of these lesions (4–10⇓⇓⇓⇓⇓⇓).

Podocytes are highly differentiated epithelial cells that stabilize the glomerular basement membrane (GBM) and contribute to the formation of the glomerular filtration barrier. Podocytes possess foot processes originating from the cell body that interdigitate over each capillary, forming the filtration barrier and counteracting the distensive forces along the GBM. This unique morphology is supported by a complex network of spatially regulated cytoskeletal proteins dividing podocytes into cell body, major processes, and foot processes. Each foot process is equipped with a microfilament-based contractile apparatus composed of actin, myosin-II, α-actinin, talin, paxillin, and vinculin (11,12⇓). This complex is fixed to focal adhesions at the basal cell membrane of foot processes via an α3β1-integrin complex, which in turn anchors the entire foot process to the underlying GBM (13–16⇓⇓⇓). Key components of the slit diaphragm, including nephrin, which maintains the barrier to protein, are indirectly tethered to this actin cytoskeleton through intermediate molecules such as CD2AP (17,18⇓). This highly ordered architecture is critically related to podocyte function in the glomerulus and the unique location of these cells render them susceptible to damage in many nephropathies, including congenital nephrotic syndrome and FSGS. Indeed, mutations in NPHS1, the gene encoding for nephrin, are associated with Finnish-type nephrotic syndrome (19). Recent studies have linked mutations in a gene encoding for α-actinin-4 (ACTN4), an actin-filament crosslinking protein, with a familial form of autosomal dominant FSGS (20–22⇓⇓). Such mutations increase the affinity of α-actinin-4 for filamentous actin (F-actin). Affected individuals may therefore develop sclerotic lesions as a direct result of dysregulation of the actin cytoskeleton. An elucidation of how increased affinity of α-actinin-4 for actin leads to podocyte damage and subsequent sclerosis would be facilitated by a mouse model for this clinical entity.

We now report the development of mice with podocyte-specific expression of a mutated/high-affinity form of α-actinin-4. These mice develop characteristic FSGS-like features reminiscent of human cases of ACTN4-associated FSGS, including proteinuria in some but not all mice, systolic hypertension that does not correlate with proteinuria, podocyte foot process fusion, glomerular tuft adhesion and collapse, focal and segmental sclerosis, and renal tubular dilatation. Furthermore, we demonstrate that proteinuric ACTN4-mutant mice have reduced nephrin mRNA expression, suggesting a close relationship between the regulation of the actin cytoskeleton and the maintenance of key components of the slit diaphragm complex.

Materials and Methods

Construction of the Targeting Vector

The murine ACTN4 cDNA was cloned into pCDNA3 (Invitrogen, Carlsbad, CA) as follows. Two overlapping I.M.A.G.E. clones (GenBank accession: AI119186 and W57010) with > 95% homology to the human ACTN4 sequence were identified through the I.M.A.G.E. Clone Consortium (http://image.llnl.gov) and purchased from ResGen/Invitrogen. An A766G point mutation (underlined in AI119186 reverse primer) analogous to A763G in the human ACTN4 open reading frame (ORF) was introduced by PCR mutagenesis, resulting in a K256E amino acid substitution. (Note: the original sequence reported for human ACTN4 [gi 4826638] indicating an A682G mutation was recently replaced in the NCBI database by a sequence [gi 12025677] that adds 81 nucleotides to the initial 5′ region, thereby rendering the mutation at position 682 as reported by Kaplan et al. to nt 763 [20]). The untranslated region (UTR) was removed from each I.M.A.G.E. clone by PCR (AI119186 forward primer: 5′-GCG CGA ATT CAT GGT GGA CTA CCA CGC A-3′; reverse primer: 5′-ATA TGT CAT TAT GGC CTC CTC G-3′; W57010 forward primer: 5′-GCT ATG ACG TGG AGA ATG AC-3′; reverse primer: 5′-TGC GGC CGC TCA CAG GTC GCT CTC CCC ATA-3′). A double 5′ hemagglutinin (HA) epitope tag was introduced using overlapping and complementary oligonucleotides (5′-GAT CCA TGT ATC CAT ATG ACG TCC CAG ACT CTG CCT ATC CAT ATG ACG TCC CAG ACT CTG CCG-3′ and 5′-AAT TCG GCA GAG TCT GGG ACG TCA TAT GGA TAG GCA GAG TCT GGG ACG TCA TAT GGA TAC ATG-3′). The HA-ACTN4 mutant ORF construct was generated by ligating 5′-ACTN4 and 3′-ACTN4 I.M.A.G.E. clone sequences digested at a common DraIII site (nt 1485 of the murine ACTN4 ORF). A similar construct was engineered for the wildtype (wt) form of ACTN4 (HA-ACTN4 wt). The ACTN4-pCDNA3 constructs were sequenced to rule out the introduction of mutations other than the desired substitution at nt 766.

Actin-Binding Experiments

The actin-binding assay was performed as described by Kaplan et al. (20). Mutant and wt ACTN4 constructs were in vitro-translated using a TnT T7 Coupled Reticulocyte Lysate kit (Promega, Madison, WI). The in vitro-translated products were incubated with 2.5 μM actin (Cytoskeleton, Inc., Denver, CO) and 10 μM cold α-actinin (Cytoskeleton, Inc.) for 1 h at room temperature. Samples were centrifuged at 105 × g for 1 h to pellet the F-actin-α-actinin complexes. The supernatants containing unbound α-actinin were removed, and the pellet was resuspended in the initial reaction volume (80 μl). Supernatants and resuspended pellets were resolved by 10% SDS-PAGE and transferred to Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech Inc.). The Western blots were probed with a polyclonal anti-HA tag antibody (diluted 1:500; CLONTECH Laboratoires, Inc., Palo Alto, CA) followed by an HRP-conjugated secondary antibody (1:1000; Amersham Pharmacia Biotech Inc.) and developed using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). The membranes were then exposed to film, and images were captured using a Kodak DC290 ZOOM Digital Camera. Blots were then stripped with Pierce IgG elution buffer and reprobed with a rabbit anti-actin antibody (1:1000) (Sigma A-2066) and processed as described above.

Transfection of COS-7 Cells, Cytoskeleton Pulldown, and Western Blotting

COS-7 cells were transfected with either pCDNA3 vector, HA-ACTN4 wt, or HA-ACTN4 mutant using the Effectene Transfection Reagent (QIAGEN Inc., Mississauga, ON, Canada) according to the manufacturer’s instructions. Transfected cells were lysed in buffer (50 mM Tris, 150 mM NaCl, 2 mM EDTA, and 1% Nonidet P40, pH 7.4) containing 1mM PMSF and protease inhibitor cocktail (Sigma-Aldrich, Inc., Saint Louis, MO) by passing them twelve times through 26G1/2 and 30G1/2 needles followed by centrifugation for 1 h at 105 × g and 4°C to pellet the insoluble cytoskeleton. Lysates (10 μg of total protein), supernatants (10 μg of total protein), and resuspended pellets (10 μg of total protein) were resolved by 10% SDS-PAGE and transferred to Hybond ECL Nitrocellulose membranes (Amersham Pharmacia Biotech Inc.). The Western blots were probed with a polyclonal anti-HA tag antibody (diluted 1:1000; CLONTECH Laboratoires, Inc.) followed by an HRP-conjugated secondary antibody (1:1000; Amersham Pharmacia Biotech Inc.) and developed using SuperSignal Chemiluminescent Substrate (Pierce). The membranes were then exposed to film, and images were captured using a Kodak DC290 ZOOM Digital Camera. Densitometry of the 103-kD band was measured using Kodak 1D 3.5 software. Blots were then stripped with Pierce IgG elution buffer and reprobed with a rabbit anti-actin antibody (1:1000; Sigma A-2066) and processed as described above.

Generation and Genotypic Analysis of Transgenic Mice

An 8.3-kb fragment of the murine nephrin promoter (NP) (cloned from a BAC library), which included the 5′ untranslated and 5′ flanking regions of the murine NPHS1 gene was cloned immediately upstream of the HA-ACTN4-mutant ORF. The NP-HA-ACTN4-mutant transgene was linearized with HindIII/RsrII and then microinjected into B6C3F1 (C57Bl6/C3H) mouse embryos at a concentration of 1.758 × 10−13 M and incubated overnight to assure viability. Embryos were then surgically transferred to the oviduct of pseudopregnant CD1 recipient female mice. Both the embryo donor mice and the recipient mice were purchased from Charles River Laboratories, Inc. (Wilmington, MA). Genotyping of resulting pups (founders) was performed by Southern analysis of BamHI-digested tail DNA using an NP probe (an EcoRI/StuI fragment). BamHI-digestion of the endogenous nephrin gene yields a 3.6-kb fragment, and the NP-HA-ACTN4-mutant transgene yields a 1.7-kb fragment. Two founders exhibiting significant proteinuria were backcrossed with purebred C3H mice to generate the N1 mice reported in this study.

Proteinuria, Albuminuria, and Serum Analysis

Individual mice were housed overnight in diuresis cages (Nalge Nunc International), and urine was collected after 24 h. Urine samples (5 μl) were mixed with sample loading buffer, incubated at 95°C for 5 min, and resolved by 10% SDS-PAGE. The gels were stained with Coomassie Brilliant blue for 30 min and destained for 4 h, and images were captured with a Kodak DC290 ZOOM Digital Camera. Urine samples were also assayed for total protein using Bio-Rad Protein Assay reagent (Bio-Rad Laboratories, Hercules, CA). Urinary albumin content was assayed using the Albuwell M Test Kit (Exocell, Inc., Philadelphia, PA) according to the manufacturer’s protocol. At 10 wk of age, mice were anesthetized with halothane and sacrificed, and blood was collected in heparinized syringes by cardiac puncture. Serum samples were analyzed for creatinine, urea, and albumin using a Beckman LX-20 instrument.

BP Studies

Systolic BP was measured daily by tail-cuff plethysmography (BP- 2000; Visitech Systems, Apex, NC). Mice were trained for an initial period of 7 consecutive days, and measurements were subsequently collected for an additional 5 d. Values were compared among wt (n = 11), non-proteinuric ACTN4-mutant (n = 10), and proteinuric ACTN4-mutant mice (n = 8).

Structural Analysis by Light and Electron Microscopy

Kidneys from wt and ACTN4-mutant mice were resected immediately after blood collection. Kidneys to be used for light microscopic analysis were incubated overnight in 4% paraformaldehyde/PBS, subsequently dehydrated, and paraffin-embedded. Sections were cut at 5 μm and stained with either periodic acid-Schiff (PAS), trichrome, or silver stain. For electron microscopy, kidneys were incubated overnight in 2.7% gluteraldehyde. Specimens were then rinsed in sodium cacodylate buffer, followed by OsO4, water, uranyl acetate, and then dehydrated in ethanol and acetone and finally embedded in spurr resin. Sections were then cut and visualized with a Hitachi 7100 transmission electron microscope. Each section and electron micrograph were examined by a renal pathologist in a blinded manner with regard to genotype. Trichrome-stained sections were used to quantify glomerular interstitial and tubular damage for each mouse with respect to % glomeruli sclerosed, % segmental sclerosis, tubular dilatation (none, mild, moderate), and interstitial fibrosis (none, mild, moderate).

Immunofluorescence of Tissue Sections

Tissues for immunohistochemical analysis were embedded in Cryomatrix (Shandon, Pittsburgh, PA) and immediately frozen in a bath of 2-butanol cooled in liquid nitrogen. Embedded tissues were sectioned at 10 μm with a cryostat, and sections were washed three times in PBS and blocked with 5% normal goat serum (Vector Laboratories Inc.) in PBS for 1 h at room temperature. After three washings in PBS, the sections were incubated with the primary antibodies (mouse 12CA5 anti-HA 1:1000, rabbit anti-HA 1:1000, rabbit anti-WT-1 [Zymed, San Francisco, CA] 1:500, or rabbit anti-nephrin 1:1000, a kind gift from Dr. Tomoko Takano, McGill University) for 1 h at room temperature, washed three times, and incubated with the secondary antibodies (anti-mouse-FITC or anti-rabbit-Cy3) for 1 h at room temperature. Sections were washed three times in PBS and mounted with fluorescence mounting media (Vector Laboratories). Sections were visualized using a Zeiss Axioskop 2 fluorescence microscope (Zeiss Axioskop 2 MOT, Zeiss Germany), and images were captured with a Zeiss AxioCam.

RNA Extraction and Real-Time RT-PCR Analyses

Kidneys were snap-frozen in liquid nitrogen, and total RNA was extracted using an RNeasy Kit (QIAGEN Inc., Valencia, CA). NPHS1, HA-ACTN4, endogenous ACTN4, and WT-1 mRNA levels were determined by real-time RT-PCR using TaqMan One-Step RT-PCR Master Mix Reagents (Applied Biosystems, Branchburg, NJ) and an ABI Prism 7000 Sequence Detection System. Reactions were carried out using 50 ng of total kidney RNA under the following conditions: 48°C for 30 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Primers and TaqMan probe for NPHS1: forward primer 5′-AAG CTG GAC GTG CAT TAT GCT-3′, reverse primer 5′-CGG TGC AGA CTA TAT CCA CAG AAC-3′, and probe 6FAM-TGC CCT GAA GGA CCC TAC TGA GGT GAA-TAMRA. Primers and TaqMan probe for WT-1: forward primer 5′-TCT TCC GAG GCA TTC AGG AT-3′, reverse primer 5′-TGC TGA CCG GAC AAG AGT TG-3′, and probe 6FAM-TGC GGC GTG TAT CTG GAG TGG C-TAMRA. Primers and MGB probe for HA-ACTN4 were designed to overlap the hemagglutinin tag and the 5′ terminal ACTN4 sequence to exclusively recognize the transgene: forward primer 5′-CAT ATG ACG TCC CAG ACT CTG C-3′, reverse primer 5′-TGG TTC GCT GCG TGG TAG T-3′, and probe 6FAM-ACT CTG CCG AAT TCA TGG T-MGBNFQ. Primers and TaqMan probe for endogenous ACTN4: forward primer 5′-TGA TCA GGT CAT CGC CTC CTT-3′, reverse primer 5′-GGC AGC TCT CTC CGC AGT TC-3′, and probe 6FAM-AGG TCC TGG CAG GAG ACA AGA ACT TCA TCA C-TAMRA. Values were normalized to GAPDH mRNA levels in each sample as determined by a TaqMan Rodent GAPDH Control Reagent kit (Applied Biosystems). To account for potential differences in podocyte number between wt, proteinuric ACTN4-mutant, and non-proteinuric ACTN4-mutant animals, we normalized relative nephrin mRNA values to the total number of podocytes counted per tissue section as assessed by counting the number of WT-1–positive cells per glomerulus (wt, 11.2 ± 0.2 podocytes/glomerulus; non-proteinuric, 11.1 ± 0.2 podocytes/glomerulus; proteinuric, 11.5 ± 0.1 podocytes/glomerulus).

Statistical Analyses

Values reported are the means ± SEM. Appropriate statistical comparisons were made using either the t test or ANOVA followed by the Newman-Keuls multiple comparison test.

Results

Cloning and Mutagenesis of Murine ACTN4 cDNA

The murine ACTN4 cDNA was cloned by homology to the human form using the mouse EST database. Two overlapping I.M.A.G.E. clones (GenBank accession: AI119186 and W57010) demonstrating >95% homology to the human ACTN4 sequence were obtained. The authenticity of these sequences was later confirmed by alignment with the recently submitted mouse ACTN4 sequence (GenBank accession: NM021895 and AJ289242). In the Kaplan & Mathis studies of familial FSGS, an A→G substitution at nt 763 in the ACTN4 ORF resulted in a K255E substitution in a highly conserved region immediately distal to the actin-binding domain of α-actinin-4 (20–22⇓⇓). Alignment to the human sequence revealed significant homology at both nucleotide and amino acid levels in this affected region (Figure 1). Using PCR-based mutagenesis, we introduced an analogous A766G substitution to obtain a K256E mutation in the translated product. Additionally, an N-terminal double-HA epitope tag was introduced to facilitate analysis of transgene expression in vivo.

Figure1

Figure 1. Homology between human and mouse α-actinin-4. Amino acid sequence comparison reveals the highly conserved region containing one of three point mutations identified by Kaplan et al. (20). An A→G mutation at nucleotide 763 of the human open reading frame (ORF) results in a K255E substitution immediately distal to the actin binding site. An analogous mutation was engineered into the mouse ORF at position 766 by PCR-based mutagenesis.

The α-Actinin-4 Mutant Protein Exhibits Increased Affinity for F-Actin In Vitro

Kaplan et al. (20) observed enhanced in vitro binding to actin for the three mutant human α-actinin-4 proteins. To verify that the K256E murine α-actinin-4 protein exhibits similar properties, we performed an actin pull-down assay. In vitro-translated wt or mutant α-actinin-4 (with or without HA tag) were incubated in the presence of 10 μM cold actinin and 2.5 μM actin under polymerizing conditions, followed by high-speed centrifugation to pellet F-actin/actinin complexes. As shown in Figure 2a, the inclusion of 10 μM cold α-actinin, while able to displace wt α-actinin-4, was unable to displace the mutant α-actinin-4 from F-actin complexes. Similar results were obtained when using [35S]-methionine-labeled in vitro-translated wt and mutant α-actinin-4, thereby confirming that the N-terminal double-HA tag did not alter the co-sedimentation of either wt or mutant α-actinin-4 with F-actin (data not shown). We next confirmed the increased affinity of mutant α-actinin-4 for F-actin in a cell culture model. COS-7 cells were transiently transfected with pCDNA3 vector, HA-ACTN4 wt, or HA-ACTN4 mutant. The actin cytoskeleton was pelleted from cell lysates at 105 × g (23) and the amount of HA-α-actinin-4 assessed by Western blot as analyzed with a polyclonal anti-HA tag antibody (1:1000). As shown in Figure 2b, compared to the wt form, a significantly greater proportion of mutant murine α-actinin-4 associated with the insoluble cytoskeletal fraction containing an equal amount of pelleted actin. These results confirm that, like the mutant variant of human α-actinin-4, the mutant form of murine α-actinin-4 exhibits abnormally high affinity for filamentous actin, thereby suggesting that even low expression levels of the mutant transgene may be sufficient to dysregulate the actin cytoskeleton.

Figure2

Figure 2. Mutant murine α-actinin-4 exhibits increased affinity for actin. (a) Similar quantities of wildtype (wt) and mutant in vitro-translated α-actinin-4 were incubated with 2.5 μM actin and 10 μM cold α-actinin for 1 h. Samples were centrifuged to pellet α-actinin-actin complexes. In vitro-translated products (input, I), supernatants (S) containing unbound α-actinin, and pellets (P) were resolved by SDS-PAGE. Western blotting with an anti-HA tag antibody (a, upper panel) revealed that mutant-HA α-actinin-4 was not competed from actin by cold α-actinin. The blot was reprobed with an anti-actin antibody (a, lower panel). (b) COS-7 cell lysates transfected with vector alone, HA-ACTN4 wt, or HA-ACTN4 mutant were centrifuged at 105 × g and the lysates (L), supernatants (S), and pellets (P) resolved by SDS-PAGE. Western blots were probed with an anti-HA tag antibody (b, upper panel). Blots were then reprobed with an anti-rabbit actin antibody (b, lower panel) to verify that comparable amounts of actin were pelleted from both wt and mutant lysates. Each experiment was performed three times.

Development of ACTN4-Mutant Transgenic Mice

The lesions associated with many forms of FSGS are thought to occur after damage to the podocytes. To address whether the A763G ACTN4 point mutation associated with a familial form of FSGS exerts direct influence in podocytes, we developed mice with podocyte-specific expression of the α-actinin-4 K256E mutant. An 8.3-kb fragment of the murine nephrin promoter (NP), which included the 5′ untranslated and 5′ flanking regions of the murine NPHS1 gene, was chosen to drive the expression of the HA-ACTN4 mutant ORF in a podocyte-specific manner (Figure 3a). Fragments of both the human and mouse nephrin promoter region were recently used to generate mice with podocyte-specific expression of a LacZ reporter gene (24,25⇓) and Cre-recombinase (26). More recently, a similar mouse promoter fragment was employed to generate mice with podocyte-specific expression of mutant WT-1 (27). In the present study, transgenic mice were obtained by pronuclear injection, and founders were identified by Southern analysis of genomic DNA derived from tail biopsies (Figure 3b). Twelve transgenic founders out of 48 pups (25%) were identified carrying the NP-HA-ACTN4-mutant transgene (designated ACTN4-mutant mice). Urinalysis for two male founders revealed significant albuminuria (#6445, 105 μg/ml per g of body weight; #7180, 72 μg/ml per g of body weight), and these were subsequently backcrossed with C3H mice to establish the experimental lines and provide the N1 generation mice used in the present study.

Figure3

Figure 3. Nephrin promoter-ACTN4-mutant construct and genotypic analysis. (a) An 8.3-kb fragment of the murine nephrin promoter (NP) was cloned upstream of the HA-ACTN4 mutant ORF. The resulting construct (NP-HA-ACTN4-mutant) was used to generate transgenic mice overexpressing mutant α-actinin-4 in a podocyte-specific manner. The A766G mutation is indicated by the arrow (↓). An EcoRI/StuI (E/S) fragment was used as a probe in Southern blots. B:BamHI, X:XhoI and H:HindIII. (b) Genomic DNA was isolated from tail snips of resulting mice, digested with BamHI, and analyzed by Southern blot to identify founders carrying the transgene. The endogenous nephrin gene yields a 3.6-kb fragment, whereas the ACTN4-mutant transgene yields a 1.7-kb fragment. Shown is a representative blot with 18 of the 48 candidates. Four of the twelve mice with transgene incorporation are shown on this blot (lanes 4, 9, 11, 13).

ACTN4-Mutant Mice Exhibit Proteinuria

Moderate proteinuria is a common clinical finding in many but not all ACTN4-associated FSGS patients (20,21⇓). The ACTN4-mutant mice from both founder lines could likewise be divided into proteinuric (> 4 μg albumin/ml per g of body weight, mean 40.8 ± 11.9 μg albumin/ml per g of body weight, n = 8; P < 0.001 versus wt and non-proteinuric) and non-proteinuric (0.5 ± 0.1 μg albumin/ml per g of body weight, n = 10) groups based on urinalysis at 10 wk of age (Figure 4). Of the eight proteinuric animals, four were derived from founder 6445 and four from founder 7180. The onset of albuminuria appeared to precede the age of weaning (data not shown) and was not gender-specific. This phenotype continues to be observed for both founder lines as backcrossed onto pure C57Bl/6 and C3H lines (each currently at generation N5). Urine of wt mice exhibited negligible albumin levels (0.3 ± 0.1 μg albumin/ml per g of body weight, n = 11). Three of the eight proteinuric mice (one derived from the 6445 founder line; two from the 7180 founder line) had microscopic hematuria and exhibited elevated serum creatinine (36.7 ± 4.2 μmol/L) and urea levels (28.9 ± 3.7 mmol/L) compared with wt mice (14.8 ± 0.9 μmol/L and 9.6 ± 0.9 mmol/L, respectively; n = 11). Creatinine and urea levels for the remaining proteinuric (12.8 ± 1.9 μmol/L and 8.4 ± 1.5 mmol/L, respectively; n = 5) and non-proteinuric (12.4 ± 0.7 μmol/L and 7.8 ± 0.6 mmol/L, respectively; n = 10) ACTN4-mutant mice were not significantly different from wt.

Figure4

Figure 4. Urinalysis of wildtype and ACTN4-mutant mice. (a) Urine samples (5 μL) of N1 generation mice were resolved by SDS-PAGE, and the gels stained with Coomassie Blue. A significant proportion of the ACTN4-mutant mice exhibit proteinuria. (b) Albumin levels determined by ELISA revealed significant albuminuria in 8 of 18 ACTN4-mutant animals (>4 μg/d per g of body weight). In the other 10 ACTN4-mutant mice, urinary albumin levels did not differ from wt littermates (a and b).

Podocyte-Specific Transgene Expression

Podocyte-specific expression of the mutant HA-α-actinin-4 protein was verified by immunofluoresence of cryostat sections. A representative panel of tissues from two N1 mice (from both founder lines) was probed with a monoclonal anti-HA tag antibody (12CA5, 1:500). As shown in Figure 5 (left panels), the HA-tagged α-actinin-4 protein was detected in the glomeruli of transgenic animals exhibiting an expression pattern that is consistent with podocyte foot process localization. Expression in the podocytes was confirmed by immunofluorescence with nephrin using an anti-nephrin polyclonal antibody (1:1000) (Figure 5, middle panels). Transgene expression was not detected in liver, spleen, lung, skeletal muscle, or heart (data not shown).

Figure5

Figure 5. Podocyte-specific expression of mutant HA-α-actinin-4. Left panels: immunofluorescence using a monoclonal anti-HA tag antibody (12CA5) and FITC-secondary antibody reveals glomerular-specific expression of mutant HA-α-actinin-4 in ACTN4-mutant mice but not in wt littermates. Middle and right panels: the pattern of expression overlapped with that of the podocyte-specific protein - nephrin (Cy3-conjugated secondary antibody).

Elevated Systolic BP in ACTN4-Mutant Mice

The incidence of hypertension in human carriers of ACTN4 mutations is significantly higher than for the general population, yet it seems independent of proteinuria (21). We therefore assessed the systolic BP of the ACTN4-mutant mice by tail cuff plethysmography. As shown in Figure 6, at 10 wk of age the average systolic BP of both proteinuric (110 ± 3 mmHg, n = 8) and non-proteinuric ACTN4-mutant mice (112 ± 3 mmHg, n = 10) was significantly elevated compared with that of the wt animals (101 ± 2 mmHg, n = 11; P < 0.05).

Figure6

Figure 6. Elevated systolic BP in ACTN4-mutant mice. Systolic BP was monitored in wt and ACTN4-mutant mice daily for 5 d by tail cuff plethysmography. Both proteinuric and non-proteinuric ACTN4-mutant mice exhibited elevated mean systolic BP. * P < 0.05.

Proteinuric ACTN4-Mutant Mice Exhibit Histologic Features of FSGS

Renal biopsies from patients with autosomal dominant familial FSGS are characterized by areas of glomerulosclerosis with tuft collapse and solidification that are both focal and segmental (20–22,28⇓⇓⇓). PAS staining and light microscopy of kidney sections from proteinuric mice (n = 6) revealed glomeruli that contained adhesions of the tuft (Figure 7a). Trichrome staining revealed many sections with segmental fibrosis, mainly in glomeruli of the outermost cortex (Figure 7b). Occasional formations reminiscent of “tip lesions” as seen in humans were also noted. Several mice had kidneys exhibiting extensive cortical scarring and prominent tubular dilatations containing proteinaceous material (Figure 7c). Silver stain frequently revealed recognizable increased mesangial matrix (Figure 7d). Proteinuric ACTN4-mutant mice exhibited modest yet significant values of renal damage. For example, proteinuric ACTN4-mutant mice had 7.3 ± 2.8% of their glomeruli globally sclerosed (wt, 0.4 ± 0.2%; non-proteinuric ACTN4-mutant, 0%), and 10.4 ± 4.3% with segmental sclerosis (wt, 0%; non-proteinuric ACTN4-mutant, 0%). Furthermore, five of the eight proteinuric ACTN4-mutant mice had mild to moderate tubular dilatation accompanied by mild to moderate interstitial fibrosis, whereas this pathology was not observed for either wt or non-proteinuric ACTN4-mutant mice. An average of 129, 122, and 140 glomeruli per section were assessed in the wt, non-proteinuric ACTN4, and proteinuric ACTN4 mice, respectively.

Figure7

Figure 7. Proteinuric ACTN4-mutant mice exhibit histologic features of focal segmental glomerulosclerosis (FSGS). Kidney sections from 10 wk-old wt, non-proteinuric ACTN4-mutant, and proteinuric ACTN4-mutant mice were stained with PAS (a), Masson trichrome (b,c), or silver stain (d). (a) A representative glomerulus from a proteinuric ACTN4-mutant mouse showing tuft adhesion by PAS stain (×400); (b) Masson trichrome stain showing segmental sclerosis of a glomerulus (×400); (c) Extensive cortical scarring, tubular dilatation, and accumulation of proteinaceous material in proteinuric ACTN4-mutant kidney (×40); (d) Silver stain showing substantial mesangial matrix (collagen) accumulation in proteinuric ACTN4-mutant glomerulus (×400).

In clinical cases of FSGS, the GBM remains intact while the podocytes exhibit marked structural damage that includes cell hypertrophy, foot process effacement, pseudocyst formation, cytoplasmic overload with reabsorption droplets, and, occasionally, detachment from the GBM. Electron microscopy (EM) analyses of proteinuric ACTN4-mutant mice revealed podocytes with patchy to more extensive foot process fusion, although it was never diffuse (Figure 8, lower left panel). Numerous vacuoles in podocytes with some residual bodies were seen (Figure 8, lower right panel). GBM thickness did not appear to change, nor did we observe any notable differences between the podocytes of non-proteinuric ACTN4-mutant (n = 6) and wt mice (n = 6).

Figure8

Figure 8. Podocyte foot process fusion in ACTN4-mutant mice. Kidney sections from 10-wk-old wt, non-proteinuric ACTN4-mutant and proteinuric ACTN4-mutant mice were assessed by electron microscopy. Representative podocyte foot process fusion in proteinuric ACTN4-mutant mice (lower left panel); Podocyte cell body vacuolization (arrow) in proteinuric ACTN4-mutant mice (lower right panel).

Elevated Expression Levels of Mutant HA-α-actinin-4 in Proteinuric ACTN4-Mutant Mice

To quantitate the relative expression of the HA-ACTN4-mutant gene product in ACTN4-mutant mice, we performed both immunofluorescence of kidney sections and real-time RT-PCR analysis of total kidney RNA derived from either proteinuric or non-proteinuric ACTN4-mutant mice. As shown in Figure 9a, an anti-HA antibody consistently detected more HA-α-actinin-4 in proteinuric versus non-proteinuric mice. This difference was observed for both founder lines. Furthermore, as shown in Figure 9b, relative HA-ACTN4-mutant mRNA expression levels, normalized to GAPDH mRNA, were 3.2-fold greater in proteinuric ACTN4-mutant mice compared with non-proteinuric ACTN4-mutant littermates (non-proteinuric ACTN4-mutant, 0.30 ± 0.11 relative units, n = 10; proteinuric ACTN4-mutant, 0.96 ± 0.23 relative units, n = 8; P < 0.01). The primer/probe combination is specific for the HA-tag; therefore, no signal could be detected for wt mice. We next compared endogenous renal ACTN4 mRNA expression to that of the mutant transgene by real-time RT-PCR using a primer/probe combination that recognizes both forms. The total ACTN4 mRNA expression (i.e., transgenic and endogenous ACTN4) as normalized to GAPDH was not different between groups of mice (wt, 1.16 ± 0.19 relative units; non-proteinuric ACTN4-mutant, 1.16 ± 0.15 relative units; proteinuric ACTN4-mutant, 1.20 ± 0.15 relative units; n = 6), thereby demonstrating low-level expression of the transgene compared with endogenous ACTN4. Such expression is sufficient to generate a proteinuric phenotype in these mice and is consistent with the large gain in affinity for F-actin by the α-actinin-4 mutant protein, as predicted by in vitro results (Figure 2).

Figure9

Figure 9. Expression of mutant HA-α-actinin-4 in proteinuric versus non-proteinuric ACTN4-mutant mice. (a) Immunofluorescence using an anti-HA tag antibody (rabbit polyclonal) and Cy3-conjugated secondary antibody reveals greater mutant HA-α-actinin-4 levels in proteinuric versus non-proteinuric ACTN4-mutant mice. (b) Real-time RT-PCR of total kidney mRNA using a HA-ACTN4-specific MGB probe. Values were standardized to levels of GAPDH mRNA in each sample and have been normalized for the number of podocytes/glomerulus. HA-ACTN4 mRNA levels are significantly higher in proteinuric ACTN4-mutant mice, n = 8 (* P < 0.01 versus non-proteinuric ACTN4-mutant mice, n = 10). No detectable product was amplified using RNA from wt mice (not shown).

Decreased Nephrin mRNA and Protein Levels in Proteinuric ACTN4-Mutant Mice

The maintenance of the filtration barrier at the molecular level is accomplished in part by nephrin, a key component of the slit diaphragm (29). We examined nephrin protein expression by immunofluorescence using an anti-nephrin antibody. As shown in Figure 10a, nephrin expression levels were visibly reduced in proteinuric ACTN4-mutant mice compared with both wt littermate controls and non-proteinuric ACTN4-mutant littermates. We next evaluated the expression of the NPHS1 gene product in ACTN4-mutant mice by real-time RT-PCR analysis of total kidney RNA derived from wt, proteinuric, or non-proteinuric ACTN4-mutant mice. As shown in Figure 10b, relative nephrin mRNA expression levels, corrected for GAPDH mRNA and normalized to the number of podocytes/glomerulus, were reduced by 70% in proteinuric ACTN4-mutant mice compared with both wt littermate controls and non-proteinuric ACTN4-mutant littermates (wt, 1.00 ± 0.16 relative units, n = 6; non-proteinuric ACTN4-mutant, 0.95 ± 0.17 relative units, n = 6; proteinuric ACTN4-mutant, 0.30 ± 0.08 relative units, n = 6; P < 0.01). Furthermore, as shown in Figure 10c, mRNA levels of the podocyte-specific Wilms Tumor-1 (WT-1) transcription factor were slightly lower yet not statistically significant in these transgenic mice as compared with wt controls (wt, 1.00 ± 0.12 relative units, n = 6; non-proteinuric ACTN4-mutant, 0.70 ± 0.13 relative units, n = 6; proteinuric ACTN4-mutant, 0.66 ± 0.15 relative units, n = 6; each NS versus wt).

Figure10

Figure 10. Reduced nephrin expression in proteinuric ACTN4-mutant mice. (a) Immunofluorescence using an anti-nephrin antibody and Cy3-secondary antibody reveals reduced expression of nephrin in proteinuric, but not in wt or non-proteinuric ACTN4-mutant littermates. (b,c) Total kidney RNA isolated from 10-wk-old animals was analyzed by real-time RT-PCR using specific TaqMan probes. Values were standardized to levels of GAPDH mRNA and have been normalized for the number of podocytes/glomerulus. (b) Nephrin mRNA levels were significantly decreased in proteinuric ACTN4-mutant mice, n = 6 (* P < 0.01 versus both wt, n = 6, and non-proteinuric ACTN4-mutant mice, n = 6). (c) Levels of WT-1 mRNA were not significantly different between ACTN4-mutant mice and wt littermates.

Discussion

The renal syndrome known as focal segmental glomerulosclerosis now stands as the second leading cause of renal insufficiency, exceeded only by diabetes (7). The weight of recent evidence suggests that the cellular target in FSGS is the glomerular epithelial cell — or podocyte. Point mutations in ACTN4, a gene encoding for a key component of the podocyte’s complex molecular architecture were recently identified in several families and show strong linkage with a steroid-resistant familial form of autosomal dominant FSGS (20). The sequelae of intermolecular events initiated by aberrant α-actinin-4/actin interactions resulting in the development of sclerosis are unknown. In the present study, we have generated a mouse model for this ACTN4-associated form of FSGS. To our knowledge, this is the first genetically derived animal model based on an inherited form of FSGS.

Clinical cases of ACTN4-associated FSGS are associated with a mild onset of proteinuria in the teenage years with slow but progressive loss of renal function and the development of ESRD in some individuals later in life (20–22⇓⇓). Furthermore, this syndrome is not fully penetrant, because not all carriers of the ACTN4 point mutations are proteinuric (22). Similarly in the present study, not all ACTN4-mutant mice were proteinuric, likely owing to lower expression of mutant α-actinin-4 compared with the proteinuric mice. This phenomenon was observed in both founder lines and could not be correlated with transgene copy number (data not shown). Furthermore, only three of the eight proteinuric mice displayed reduced renal function by 10 wk of age. Variable levels of ACTN4-mutant expression may therefore underlie the incomplete penetrance encountered in human cases of ACTN4-associated FSGS. Hypertension is also a common finding associated with many forms of FSGS, including ACTN4-associated FSGS (21). The incidence of hypertension was reported to be abnormally high in the Oklahoma ACTN4-FSGS family and yet could not be correlated with proteinuria (21). Similarly, we found that the overall systolic BP averages of both proteinuric and non-proteinuric ACTN4-mutant mice were elevated. Such phenotypic heterogeneity observed both in humans and now in mice might be explained by genetic modifier effects.

Our results strongly support the notion that the lesions encountered with this form of familial FSGS find their root cause in the podocyte. We observed a number of renal pathologic changes in mice expressing a high affinity variant of α-actinin-4 in a podocyte-specific manner that are consistent with an FSGS-like phenotype. These include segmental glomerular fibrosis, occasional tip lesions, adhesion of the glomerular tuft, podocyte foot process fusion, podocyte vacuolization, and mesangial matrix accumulation. Additionally, we observed marked tubular dilatation accompanied by the accumulation of a luminal proteinaceous material indicating severe damage due to the proteinuria. This combination of pathologic and clinical data indicates that mice with podocyte-specific expression of mutant α-actinin-4 exhibit a phenotype similar to that seen in humans with this genetic mutation.

The ACTN4-associated form of FSGS was identified by Mathis et al. (21,22⇓), who employed a positional cloning approach to study an FSGS kindred from Oklahoma. Subsequently, Kaplan and co-workers studied three families (including the Oklahoma family) with an autosomal dominant form of FSGS to identify the underlying genetic defect accounting for the observed FSGS (20). They identified an A→G substitution in the ACTN4 open reading frame that resulted in a K→E mutation in a region immediately distal to the actin-binding site (20). Similar mutations in this region were identified in the two other families described in the study. Each of these mutations yield α-actinin-4 proteins that bind to F-actin with greater affinity than the wildtype form, implying that dysregulation of the actin cytoskeleton might underlie the ensuing podocyte damage and thereby render the glomerulus susceptible to sclerosis. Several reports have documented an abnormal distribution and disaggregation of podocyte actin microfilaments during the development of foot process effacement (30–32⇓⇓). Concomitant redistribution of actin and α-actinin in the podocytes of nephrotic rats has also been reported (30,33⇓), resulting in dysregulation of actin filament bundling in foot processes and finally yielding disruption of normal foot process function. Such alterations in cytoskeletal function might explain the characteristic changes to podocytes observed in FSGS.

Accordingly, point mutations in the ACTN4 gene might have significant effects on key components of the podocyte cytoskeleton and slit diaphragm. Nephrin, a key player in the formation of the slit diaphragm, possesses extracellular IgG-like repeats, a single membrane-spanning region, and a cytoplasmic tail that interacts with intracellular molecules including CD2AP, which may anchor it to the actin cytoskeleton (17,18⇓). The importance of this molecule in maintaining the glomerular barrier to protein is illustrated by the fact that humans with mutations in the NPHS1 gene and NPHS1-null mice are nephrotic and fail to develop normal podocyte foot processes (19,34⇓). Many recent studies have associated reduced nephrin mRNA or protein expression with the onset of proteinuria in a number of nephropathies including diabetic nephropathy (35,36⇓), minimal change disease (37), passive Heymann nephritis (38–40⇓⇓), and membranous proliferative glomerulonephritis (37). Our results showing a 70% reduction in nephrin mRNA in proteinuric ACTN4-mutant mice are consistent with the observed foot process fusion and are the first to demonstrate such a correlation in the context of FSGS. The reduced nephrin mRNA and protein levels were not a result of changes in podocyte numbers at this early stage of the disease’s progression, as both the number of podocytes per glomerulus and the number of glomeruli were no different between groups of mice. These changes in nephrin expression may reflect lower transcriptional activity at the NPHS1 locus or perhaps enhanced nephrin mRNA or protein degradation. In either case, these results strongly suggest that a link exists between the regulation of cytoskeletal function and nephrin expression. Expression of the transcription factor WT-1, a podocyte-lineage marker, has been previously shown to be downregulated in some nonfamilial forms of collapsing and noncollapsing FSGS, thereby suggesting a reversion of the podocyte to a more immature/proliferating phenotype (4,41,42⇓⇓). However, we now show that WT-1 mRNA levels and immunofluorescence of synaptopodin (data not shown), another podocyte-specific marker, were not significantly different between the three groups of mice, thereby suggesting that despite the reduced nephrin expression, these cells maintained a somewhat differentiated phenotype.

In summary, we have developed transgenic mice overexpressing a mutated variant of the α-actinin-4 protein in a podocyte-specific manner. As in clinical cases of familial FSGS, a significant proportion of these mice exhibit moderate proteinuria, marked podocyte foot process fusion as well as other classical FSGS hallmarks. Furthermore, nephrin mRNA levels are reduced in proteinuric animals, suggesting a cause and effect relationship between dysregulation of the actin cytoskeleton by mutant α-actinin-4 and the deterioration of the slit diaphragm complex. Future insights gained from such a model uncovering the intracellular and intercellular mechanisms by which mutations in the ACTN4 gene yield an FSGS phenotype may provide novel therapeutic approaches for the prevention and treatment of this disease.

Acknowledgments

We are grateful to Peter Rippstein for the electron microscopy work, to Louise Pelletier for histology, and to Linda Oliveras for the serum analysis. We thank Drs. Kevin D. Burns and David Z. Levine for their thoughtful criticisms of the manuscript. Chris Kennedy is a Kidney Foundation of Canada Biomedical Research Scholar. Jean-Louis Michaud is a recipient of an Ontario Graduate Studies scholarship. This work is supported by an operating grant from the Kidney Foundation of Canada.

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Focal and Segmental Glomerulosclerosis in Mice with Podocyte-Specific Expression of Mutant α-Actinin-4
Jean-Louis Michaud, Lyne I. Lemieux, Manon Dubé, Barbara C. Vanderhyden, Susan J. Robertson, Chris R.J. Kennedy
JASN May 2003, 14 (5) 1200-1211; DOI: 10.1097/01.ASN.0000059864.88610.5E
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