Arhgap24 inactivates Rac1 in mouse podocytes, and a mutant form is associated with familial focal segmental glomerulosclerosis

Abstract

The specialized epithelial cell of the kidney, the podocyte, has a complex actin-based cytoskeleton. Dynamic regulation of this cytoskeleton is required for efficient barrier function of the kidney. Podocytes are a useful cell type to study the control of the actin cytoskeleton in vivo, because disruption of components of the cytoskeleton results in podocyte damage, cell loss, and a prototypic injury response called focal segmental glomerulosclerosis (FSGS). Searching for actin regulatory proteins that are expressed in podocytes, we identified a RhoA-activated Rac1 GTPase-activating protein (Rac1-GAP), Arhgap24, that was upregulated in podocytes as they differentiated, both in vitro and in vivo. Increased levels of active Rac1 and Cdc42 were measured in Arhgap24 knockdown experiments, which influenced podocyte cell shape and membrane dynamics. Consistent with a role for Arhgap24 in normal podocyte functioning in vivo, sequencing of the ARHGAP24 gene in patients with FSGS identified a mutation that impaired its Rac1-GAP activity and was associated with disease in a family with FSGS. Thus, Arhgap24 contributes to the careful balancing of RhoA and Rac1 signaling in podocytes, the disruption of which may lead to kidney disease.

Figure 1. Differentiated podocytes show reduced membrane ruffling.

(A) YFP-actin–transduced podocytes were cultured at the permissive temperature (33°C) or differentiated for 7 to 14 days at the nonpermissive temperature (37°C). Kymographs (insets) obtained at the sites of active ruffling (hatched boxes) show prominent ruffling at 33°C that is reduced at 37°C (also see Supplemental Videos 1 and 2). DIC, differential interference contrast. Original magnification, ×400; approximately ×1,000 (insets and time-lapse panels). (B) Quantification of actin spike lengths in kymographs shows that differentiated podocytes (n = 11) have a significant decrease in ruffling activity compared with that of undifferentiated podocytes (n = 15). Individual symbols represent data from individual podocytes. P = 3.29 × 10^–6 by ANOVA with post-test correction.

Figure 2. Arhgap24 transcript and Arhgap24 protein are specifically expressed in podocytes.

(A) MBEI values for microarray data for in vitro–cultured podocytes (33°C, 37°C), ex vivo–isolated podocytes (E13.5, E15.5, and adult), and laser-capture-microdissected glomeruli (E12.5 renal vesicle, E15.5 S-shaped body, and E15.5 renal corpuscle) show an increase in the Arhgap24 transcript with differentiation in vitro and in vivo. (B) By quantitative RT-PCR, differentiated podocytes have higher Arhgap24 mRNA levels compared with those of undifferentiated podocytes, after normalization to 18S rRNA. (C) Immunoblotting shows that differentiated podocytes have higher levels of Arhgap24 protein than undifferentiated podocytes. (D) Confocal imaging of cultured podocytes also shows an increase in levels of Arhgap24 in differentiated podocytes, concentrated in punctate structures at the base of the cell. (E) Arhgap24 colocalizes with the focal adhesion marker vinculin at the tips of actin stress fibers. Original magnification, ×600 (D and E).

Figure 3. Arhgap24 is expressed in kidney podocytes in vivo.

(A) Immunoblotting of tissue lysates shows that Arhgap24 is expressed in the kidney (expected size ~95 kD). Smaller bands likely represent specific degradation products. The positive control (+) is a lysate from HEK293 cells transfected with FLAG-tagged Arhgap24. The negative control (–) is HEK293 cell lysate. Brn, brain; Kid, kidney; Liv, liver; Lng, lung; Spl, spleen. (B) Magnetic separation of glomeruli after beating heart perfusion of mice with magnetic beads results in more than 95% pure isolated glomeruli (top). The flow through consists of tubule fragments (bottom). Original magnification, ×100. (C) Arhgap24 is enriched in the glomerular (Glom) fraction that also contains the podocyte protein, podocin. Tub, tubule. (D) A low-magnification view (original magnification, ×200) of a mouse kidney stained for Arhgap24 shows that the highest signal is detected in the glomeruli. (E) Within mouse glomeruli, the Arhgap24 signal colocalizes with that of the podocyte marker synaptopodin. Original magnification, ×600.

Figure 4. Arhgap24 knockdown in differentiated podocytes increases membrane ruffling.

(A) Two lentivirally transduced cell lines targeting different portions of the Arhgap24 transcript show marked reduction of Arhgap24 protein compared with that of an irrelevant knockdown (Fluc, 100%; line 451, 15%; line 756, 40%). Results are representative of at least 3 independent experiments. (B) Representative images of the 3 knockdown cell lines after differentiation at 37°C for 7 to 10 days. Kymographs (insets) generated from the hatched box area and time-lapse sequences show that the 2 knockdown cell lines have increased membrane ruffling compared with that of the control (also see Supplemental Videos 3–5). Original magnification, ×400; approximately ×1,000 (insets and time-lapse panels). (C) Quantitation of actin spikes in kymographs shows that both the 451 (n = 19) and 756 (n = 11) Arhgap24 cell lines have significantly greater membrane ruffling activity compared with that of the control knockdown Fluc cell line (n = 18) (*P < 0.00001). The ruffling activity is not significantly different between the 2 knockdown cell lines (P = 0.57). Group differences were analyzed by ANOVA with post-test correction.

Figure 5. Arhgap24 knockdown in differentiated podocytes increases active Rac1 and Cdc42 levels and accelerates epithelial monolayer wound closure.

(A) Pull down of active (GTP-bound) Rac1, Cdc42, and RhoA shows that the Arhgap24 knockdown cell lines (lines 451 and 756) have increased levels of active Rac1 and Cdc42 compared with those of the control (Fluc). However, active RhoA levels are similar across all 3 cell lines. Results are representative of 3 independent experiments. (B) Arhgap24 knockdown cells migrate and close a scratch made in a confluent monolayer faster than the control knockdown cell line. Cell nuclei were stained with Hoechst dye. Original magnification ×100.

Figure 6. Individuals with end-stage kidney disease (clinically and/or biopsy proven) are denoted by black symbols.

Deceased family members are represented by diagonal lines. Flanking MSM analysis shows that the unaffected siblings have a distinct haplotype that is different from that of the affected individuals. The columns of numbers and letters under each symbol refer to the alleles that individual carries at the given genetic markers. Where these numbers are within brackets, the haplotype is inferred. The solid black rectangle beneath individuals 1, 101, and 1001 represents the inherited disease haplotype.

Figure 7. Arhgap24 Q158R has defective Rac1-GAP activity and dimerizes with the wild-type protein.

(A) Wild-type FLAG-tagged Arhgap24 (first lane) transfected into HEK293 cells reduces active Rac1 levels compared with Q156R Arhgap24 (last lane). Titration of increasing proportions of Q156R Arhgap24 produces increased levels of active Rac1 (middle lanes). Total Rac1 and FLAG-Arhgap24 protein levels are similar across all lanes. Results are representative of 3 different experiments. (B) FLAG- or GFP-tagged wild-type (W) or Q158R Arhgap24 (Q) constructs were cotransfected into HEK293 cells. Cell lysates were immunoprecipitated with anti-FLAG antibody and then immunoblotted for GFP-tagged Arhgap24 to assess for protein dimerization. Whole cell lysates (WCLs) were immunoblotted for GFP and FLAG to ensure protein expression.