Mice deficient in α-actinin-4 have severe glomerular disease

Mouse model development.

We isolated a mouse genomic bacterial artificial chromosome (BAC) clone containing Actn4 by screening an arrayed 129/SvJ genomic library by PCR. We subcloned an 11-kb HindIII genomic fragment containing Actn4 exons 4–10 (Figure 2a). A loxP-flanked neomycin resistance cassette was inserted into an XhoI site 400 bp 3′ of exon 8 as shown. We mutagenized the sequence of exon 8 in order to both alter the encoded amino acid sequence (Lys256Glu) and add a silent EarI restriction site to simplify genotyping (using a Stratagene QuickChange kit; Stratagene, La Jolla, California, USA). We electroporated 129/SvJ ES cells and selected for G418-resistant colonies. We screened 122 resistant colonies for an altered HindIII fragment by Southern blot using an external probe as shown in Figure 2a. We found four homologously recombinant colonies. We injected homologously recombinant cells from one colony into the C57BL/6 blastocysts of pseudopregnant mice (Brigham and Women’s Hospital Transgenic Core facility), identified chimeric offspring, and bred these mice to C57BL/6 females. Initial breeding to C57BL/6 mice produced several lines harboring the targeted allele, verified by Southern blot and/or PCR amplification and EarI digestion. These offspring were on a mixed background, with a 50% contribution from 129/SvJ (the ES cell strain) and a 50% contribution from C57BL/6 (the strain to which the chimeras were bred).

Mouse genotyping.

We isolated DNA from mouse tails. We genotyped mice by PCR amplification: mACTN4-Ex8(–122)-F: GGT TTC CAG TCC AAG AGA GGC CAC T; and mACTN4-Ex8(+64)-R: TGT GGG TCC TAA CAA GTG ATC TCA C; then mACTN4-Ex8(–93)-F: TCT GCA AGA GAA ATG AAG TGA GCG T; and mACTN4-Ex8(+64)-R: TGT GGG TCC TAA CAA GTG ATC TCA C; followed by digestion with EarI (Figure 2b) and electrophoresis on an agarose gel.

Northern blot analyses.

RNA was prepared from kidneys of newly sacrificed mice using Trizol reagent (GIBCO; Invitrogen Corp., Carlsbad, California, USA). Electrophoresis, transfer, and hybridization were performed using standard methods. A radiolabeled probe was generated from an RT-PCR amplicon corresponding to Actn4 nucleotides 655–933 using primers F: GAT GAT CCA GTC ACC AAC CTA AAC and R: CCG AAT CCA CTC TAG AAG ATC AC (Figure 2c). Northern blot analyses were also performed using blots obtained from CLONTECH Laboratories Inc. (Palo Alto, California, USA) to examine Actn4 RNA expression in various tissues and developmental stages (Figure 1, c and d).

Western blot analysis.

We prepared protein from mouse kidney, lung, brain, liver, spleen, and cultured fibroblasts by homogenization in lysis buffer: 150 mM NaCl, 50 mM Tris (pH 8.0), 1% Triton X-100, Na orthovanadate, microcystin, and complete protease inhibitor (Roche Applied Science, Indianapolis, Indiana, USA). Western blot analyses were performed with antibodies to the N-terminal domains of α-actinin-1 and α-actinin-4 (described in ref. 2) using standard methods. Western blot of kidney lysates was also performed with a second α-actinin-4 antibody (raised against a peptide corresponding to amino acids 458–480) (11).

Histology.

Freshly harvested kidneys were fixed in Bouin’s solution. H&E staining was performed using standard methodology. Electron microscopy was performed after fixation in Karnovsky’s media using standard diagnostic protocols. For the electron micrographs, all of the glomeruli that were imaged were from as deep into the renal cortex as possible. No incompletely differentiated glomerulus was imaged. We also performed necropsy of the remaining tissues by gross anatomic observation and light microscopy of tissues fixed in Bouin’s solution. For analysis of mouse embryos, we sacrificed pregnant female mice for analysis of embryos at embryonic days 16.5–18.5 (counting the day of appearance of a vaginal plug as day 0.5).

Immunofluorescence.

For these studies, fresh kidneys were embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, California, USA), frozen in liquid nitrogen, and sectioned at 4 μm. Slides were fixed in cold 100% acetone or methanol and then air dried. Sections were then incubated with 1% BSA at room temperature to block nonspecific binding. They were next incubated with the primary antibodies at room temperature for 1 hour, rinsed with PBS, incubated with secondary antibodies at room temperature for 1 hour, and mounted with VECTASHIELD medium (Vector Laboratories Inc., Burlingame, California, USA). Staining was analyzed by fluorescence microscopy.

For the studies of actinin expression, we incubated anti–α-actinin-1, -2, -3, or -4 rabbit antisera (at 1:250 dilution) together with a mouse monoclonal anti-synaptopodin antibody (at 1:5 dilution; a gift of P. Mundel, Albert Einstein College of Medicine, New York, New York, USA) (12) with fixed sections of kidney from newly sacrificed wild-type mice. Isoform-specific anti-actinin antibodies were described previously (13). A secondary goat anti-rabbit Cy3-labeled antibody and a secondary hamster anti-mouse FITC-labeled antibody were used diluted 1:200 in PBS (Sigma-Aldrich, St. Louis, Missouri, USA) (Figure 1). We also performed immunofluorescence studies of kidneys from Actn4^–/– mice after the onset of mild microalbuminuria (age 5.5 weeks) as well as kidneys from Actn4^+/+ sex-matched littermates. We used primary antibodies to nephrin and podocin, described in ref. 11. We used an anti–collagen type IV antibody obtained from ICN Biomedicals Inc. (Costa Mesa, California, USA). FITC-labeled secondary antibodies were purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, Pennsylvania, USA).

Biochemistry.

Analyses of blood urea nitrogen (BUN) concentrations were performed in the Brigham and Women’s Hospital Clinical Laboratory. Urine protein was assessed by dipstick (Albustix; Bayer Corp., Pittsburgh, Pennsylvania, USA) and/or by acrylamide gel electrophoresis and Coomassie blue staining.

Lymphocyte assays.

We isolated lymphocytes from spleens of newly sacrificed mice and performed chemotaxis assays. The mice had no proteinuria or histologic evidence of kidney disease. Lymphocytes were placed in Transwell chemotaxis chambers (Neuro Probe Inc., Gaithersburg, Maryland, USA). Serial tenfold dilutions of chemokine mSDF-1 were added to the bottom wells. After 37°C incubation for 90 minutes, we counted cells migrating across the filter. Comparisons of migrating cells were analyzed by paired t tests using the results of four sets of independent experiments. We performed Western blot analysis of lymphocyte lysate using the anti–α-actinin-4 antibody.

Expression of α-actinin-4.

We performed immunofluorescence studies of α-actinin expression in mouse kidneys (Figure 1, a and b). In human kidney, we have observed significant expression of α-actinin-4 only (2). In mouse kidney, we observed strong expression of α-actinin-1 and α-actinin-4 in a podocyte pattern, colocalizing with the podocyte marker synaptopodin. In addition, α-actinin-2 was expressed in the glomerulus but in a different pattern, consistent with mesangial cell expression. Multiple-tissue Northern blot demonstrated α-actinin-4 expression in a variety of mouse tissues, with very strong expression in kidney and liver and strong expression in lung and heart (Figure 1c). Signal was minimal in skeletal muscle and testis and relatively low in brain and spleen. Northern blot analysis of mouse embryos demonstrated expression of α-actinin-4 as early as embryonic day 7 (Figure 1d).

We performed Northern and Western blot analysis to examine α-actinin-4 expression at the RNA and protein levels. By both Western blot and Northern blot analysis, we detected no expression of α-actinin-4 in the kidneys of mice homozygous for the targeted allele (Figure 2, c and d). Because we saw no detectable Actn4 expression from the targeted allele, we refer to this allele as Actn4^– for simplicity.

Phenotype.

The genetic background of the original targeted mice (and littermates) derived from both the 129/SvJ ES cells and the C57BL/6 mice to which the chimeras were bred. Mice studied were on a mixed background, with approximately equal contributions from the 129/SvJ and C57BL/6 backgrounds.

We weaned and genotyped mice at approximately 3 weeks of age. Mice heterozygous for the targeted allele (Actn4^+/–) showed no obvious ill effect. We mated pairs of Actn4^+/– mice. In contrast to the expected 25% frequency of homozygotes, we observed 1/18 of the offspring of Actn4^+/– × Actn4^+/– crosses to be homozygous for the targeted allele (42 of 756 pups; Table 1). We sacrificed newborn mice at 0–4 days of age. Ten of the 125 pups genotyped at days 0–4 were homozygous for the targeted allele (and, of these, 2 of 48 mice sacrificed at day 0–1 were homozygous). Approximately 10% of 7- to 12-day-old mice (10 of 92) were homozygous for the targeted allele. We also harvested embryos derived from similar heterozygous matings (embryonic days 16.5–18.5) and found 12 of 49 embryos to be homozygous for the targeted allele. We observed that in the initial weeks of life, those mice identified as Actn4^–/– appeared grossly normal in appearance and behavior, but their rate of growth (compared with that of sex-matched littermates) frequently began to slow starting at approximately 6 weeks, and they subsequently began to lose weight.

Histology.

Light microscopy (Figure 3) showed evidence of FSGS in kidneys of most Actn4^–/– mice over 10 weeks of age. Kidneys of some mice showed areas of glomerular capillary collapse. Kidneys from these mice were typically small and pale on gross examination (Figure 3g). Transmission electron microscopy of Actn4^–/– kidneys showed mild abnormalities at 5 weeks, including the development of areas of focal podocyte foot-process effacement and glomerular basement membrane (GBM) duplication (Figure 4). Slit diaphragms appeared normal. At later ages, the glomerular ultrastructure as visualized by transmission electron microscopy showed significant damage. We performed GBM thickness measurements in the Actn4^–/– and control littermates and found no consistent differences between mice of different genotypes. We found no histologic abnormalities outside of the kidney in any of Actn4^–/– animals sacrificed. Furthermore, we saw no renal or nonrenal abnormalities in any of the Actn4^–/– mice sacrificed in late embryonic stages or in the sacrificed Actn4^–/– neonatal mice.

We performed immunofluorescence using antibodies to podocin, nephrin, and type IV collagen and kidneys from Actn4^–/– mice sacrificed shortly after the onset of microalbuminuria together with sex-matched Actn4^+/+ littermates. We found no consistent differences in expression of these proteins between Actn4^–/– and Actn4^+/+ mice (Figure 5).

Biochemistry.

As shown in Figure 6, we observed increasing albuminuria with advancing age in Actn4^–/– mice but no significant albuminuria in Actn4^+/+ (or Actn4^+/–) littermates. The development of proteinuria was observed in most but not all mice by the time of sacrifice. Similarly, we observed increased BUN in many (but not all) of the Actn4^–/– mice from blood obtained at the time of sacrifice (Figure 6).

Chemotaxis.

α-Actinin-4 has been previously implicated in cell motility (10). Because leukocytes are easily isolated and can be studied directly without propagation in culture, we examined motility in these cells. As chemokines are potent dynamic modulators of the integrin-linked actin cytoskeleton, we assessed lymphocyte motility by measurement of cell movement in response to chemokine SDF-1. We isolated murine lymphocytes from spleens of newly sacrificed mice and performed chemotaxis assays. As shown in Figure 7, we observed increased cell movement across filters in the Actn4^–/– compared with the Actn4^+/+ cells in each of four independent sets of experiments. This difference was observed in unstimulated cells and was augmented by addition of chemokine (SDF-1). These findings were statistically significant (analyzed by two-tailed paired t test, P < 0.005). Flow cytometry demonstrated that there was an equal distribution of lymphocyte subsets from the mutant and control mice.