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

Abstract

Dominantly inherited mutations in ACTN4, which encodes alpha-actinin-4, cause a form of human focal and segmental glomerulosclerosis (FSGS). By homologous recombination in ES cells, we developed a mouse model deficient in Actn4. Mice homozygous for the targeted allele have no detectable alpha-actinin-4 protein expression. The number of homozygous mice observed was lower than expected under mendelian inheritance. Surviving mice homozygous for the targeted allele show progressive proteinuria, glomerular disease, and typically death by several months of age. Light microscopic analysis shows extensive glomerular disease and proteinaceous casts. Electron microscopic examination shows focal areas of podocyte foot-process effacement in young mice, and diffuse effacement and globally disrupted podocyte morphology in older mice. Despite the widespread distribution of alpha-actinin-4, histologic examination of mice showed abnormalities only in the kidneys. In contrast to the dominantly inherited human form of ACTN4-associated FSGS, here we show that the absence of alpha-actinin-4 causes a recessive form of disease in mice. Cell motility, as measured by lymphocyte chemotaxis assays, was increased in the absence of alpha-actinin-4. We conclude that alpha-actinin-4 is required for normal glomerular function. We further conclude that the nonsarcomeric forms of alpha-actinin (alpha-actinin-1 and alpha-actinin-4) are not functionally redundant. In addition, these genetic studies demonstrate that the nonsarcomeric alpha-actinin-4 is involved in the regulation of cell movement.

Figure 1

(a) α-Actinin-4 expression in mouse kidney shown at low power (image obtained at ×10 magnification). (b) Analysis of α-actinin-1, -2, -3, and -4 expression (red) and synaptopodin (synpo; green) in mouse kidney. Merged images are shown below. α-Actinin-1 and -4 show essentially podocyte-limited expression, whereas α-actinin-2 shows a different expression pattern, consistent with a mesangial cell localization. α-Actinin-3 is not expressed in the mouse glomerulus. α-Actinin-1, -2, and -4 are also expressed in the renal vasculature (not shown). Images were taken at ×40 magnification. (c) Northern blot showing expression of α-actinin-4 transcript in various mouse organs (using mouse multiple-tissue Northern blot from CLONTECH Laboratories Inc.). Lane 1, heart; 2, brain; 3, spleen; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, testis. Hybridization to a β-actin control probe is shown below, with a single 2-kb band in most lanes and an expected 1.8-kb band present in heart, skeletal muscle, and testis. (d) Northern blot showing expression of α-actinin-4 at embryonic days 7, 11, 15, and 17. Hybridization to a β-actin control probe is shown below.

Figure 2

(a) Targeting construct. The construct used was originally designed for the development of a knock-in model. A neomycin resistance cassette is 438 bp from the 3′ end of exon (Ex) 8 of Actn4. (b) Genotyping assay. A PCR fragment amplified from wild-type genomic DNA does not digest with EarI. The targeted allele has an EarI site, allowing simple genotyping. The two left-hand lanes show this assay in two wild-type mice; the middle five lanes show amplification and digestion products of heterozygous mice. The final lane is derived from a mouse homozygous for the targeted allele. (c) Northern blot analysis of Actn4 expression in mice homozygous for the wild-type allele (+/+) and for the targeted allele (–/–). Rehybridization of membrane with β-actin as a control for RNA loading is shown below. (d) Western blot of kidney lysates using anti–α-actinin-4 antibody. The same pattern was seen with lung, liver, brain, and spleen. No difference was seen in the expression of α-actinin-1 (not shown).

Figure 3

(a and b) Kidney sections from 4-week-old mice, stained with H&E. Actn4^+/+ kidney (a) and Actn4^–/– kidney (b) photographed at ×10 magnification. (c and d) At higher magnification (×40), glomerular abnormalities are clearly visible in the Actn4^–/– mouse (d), but not in the Actn4^+/+ littermate (c): the Actn4^–/– kidney shows FSGS, with protein in tubules and areas of glomerular capillary collapse. A diseased glomerulus is indicated by an arrow (labeled “G”). A tubule filled with proteinaceous material is also indicated by an arrow (labeled “T”). (e and f) Kidney sections from 10-week-old mice, stained with H&E. (e) Actn4^+/+ kidney; (f) Actn4^–/– kidney. The Actn4^–/– kidney shows extensive disease, with sclerosed glomeruli, dilated tubules with proteinaceous material, and disrupted architecture. (g) Gross appearance of Actn4^+/+ (left) and Actn4^–/– (right) kidneys from 10-week-old mice.

Figure 4

(a and b) Electron microscopic appearance of kidneys from 10-day-old Actn4^+/+ (a) and Actn4^–/– (b) littermates. (c and d) Kidneys from 5-week-old Actn4^+/+ (c) and Actn4^–/– (d) mice. (e and f) Kidneys from 10-week-old Actn4^+/+ (e) and Actn4^–/– (f) mice. In all cases, Actn4^–/– mice show altered glomerular ultrastructure. In the 10-day-old kidney (b), there is mild disruption of the normal podocyte structure with focal areas of podocyte effacement. The disease is more extensive in the older mice (c and e). (g and h) Early changes in Actn4^–/– mice are shown at higher power. As shown in g, some Actn4^–/– mice demonstrated areas with duplications, or “blebs,” in the GBM on the subepithelial aspect (arrow). These abnormalities were not seen in the control mice, nor were they present in all Actn4^–/– mice. In h, focal areas of foot-process effacement in an Actn4^–/– mouse are evident (arrow).

Figure 5

Immunofluorescence studies of kidneys from Actn4^–/– mice and Actn4^+/+ littermates (×40 magnification). Antibodies to nephrin, podocin, and type IV collagen (col IV) have been previously described (11). Mice were 5.5 weeks of age at the time of sacrifice, and Actn4^–/– mice had mild proteinuria.

Figure 6

(a) Two microliters of mouse urine run on a 10% SDS-PAGE gel and stained with Coomassie. Urine from Actn4^–/– mice is run next to urine from sex- and age-matched littermates. (b) Spot microalbumin measurements from mouse urine of different genotypes estimated by Albustix (Bayer Corp.). (c) Average BUN level, SD, and range, for Actn4^+/+, Actn4^+/–, and Actn4^–/– mice.

Figure 7

(a) Results of a representative chemotaxis assay. Lymphocytes were incubated with varying concentrations of SDF-1 as shown. Base-line movement is greater in the Actn4^–/– cells, as well as in the Actn4^–/– cells stimulated with 1 nmol and 10 nmol SDF-1. (b) Summary of all four sets of pairwise comparisons. Shown is the average increase in cell count in Actn4^–/– expressed as a fraction of the cell count of Actn4^+/+ cells. (c) Western blot analysis of lymphocyte lysates from Actn4^+/+, Actn4^+/–, and Actn4^–/– mice using an anti–α-actinin-4 antibody. Antibody to total ERK-1 and -2 served as a protein loading control.