Genetic Variants in ARHGEF6 Cause Congenital Anomalies of the Kidneys and Urinary Tract in Humans, Mice, and Frogs

Abstract

Background: About 40 disease genes have been described to date for isolated CAKUT, the most common cause of childhood CKD. However, these genes account for only 20% of cases. ARHGEF6, a guanine nucleotide exchange factor that is implicated in biologic processes such as cell migration and focal adhesion, acts downstream of integrin-linked kinase (ILK) and parvin proteins. A genetic variant of ILK that causes murine renal agenesis abrogates the interaction of ILK with a murine focal adhesion protein encoded by Parva , leading to CAKUT in mice with this variant.

Methods: To identify novel genes that, when mutated, result in CAKUT, we performed exome sequencing in an international cohort of 1265 families with CAKUT. We also assessed the effects in vitro of wild-type and mutant ARHGEF6 proteins, and the effects of Arhgef6 deficiency in mouse and frog models.

Results: We detected six different hemizygous variants in the gene ARHGEF6 (which is located on the X chromosome in humans) in eight individuals from six families with CAKUT. In kidney cells, overexpression of wild-type ARHGEF6 -but not proband-derived mutant ARHGEF6 -increased active levels of CDC42/RAC1, induced lamellipodia formation, and stimulated PARVA-dependent cell spreading. ARHGEF6-mutant proteins showed loss of interaction with PARVA. Three-dimensional Madin-Darby canine kidney cell cultures expressing ARHGEF6-mutant proteins exhibited reduced lumen formation and polarity defects. Arhgef6 deficiency in mouse and frog models recapitulated features of human CAKUT.

Conclusions: Deleterious variants in ARHGEF6 may cause dysregulation of integrin-parvin-RAC1/CDC42 signaling, thereby leading to X-linked CAKUT.

Graphical abstract

Figure 1

Exome sequencing identifies X-linked recessive variants in the gene ARHGEF6 in six families with CAKUT. (A) Exon structure (upper bar) and protein domains (lower bar) of human ARHGEF6. Positions of start codon (ATG) and stop codon (TAA) are indicated. Exon numbers are marked on an alternating black or white background. Protein domain lengths are shown by the colored boxes in respect to encoding exons. Positions of variants are indicated by black arrows in relation to the exon and the protein domain (see also Table 1). Pedigrees of families with genetic variants in ARHGEF6 are depicted below each family number. (B) CLUSTAL-generated amino acid sequence alignments of ARHGEF6 orthologous proteins are shown for the regions surrounding sites of missense variants. (C) Representative clinical images of individuals with ARHGEF6 variants. Upper row (from left to right): Voiding cysto-urethrogram showing vesico-uretral reflux grade IV or V on the right (B3089-21), renal ultrasound image of proband A5124-33 showing renal hypoplasia, and bladder ultrasound images of B1185-21 indicating bladder exstrophy. Lower row (from left to right): Renal ultrasound of right kidney (B3089-21) showing renal hypoplasia on the right. Voiding cysto-urethrogram showing vesico-uretral reflux grade V on the right (GM2) and renal ultrasound of left kidney of GM2 indicating hypoplastic kidney.

Figure 2

ARHGEF6 increases levels of active CDC42 and RAC1, lamellipodia formation, and FN-induced cell spreading in renal cells. (A) HEK293T cells were transfected with ARHGEF6 WT cDNA and cDNA representing proband variants. After 24 hours, transfection active levels of CDC42 and RAC1, respectively, were measured by G-LISA assay and normalized to MOCK. Each color represents an individual experiment. p.Lys712* indicates splice variant of family GM2. **P<0.01; ***P<0.001; ns, not significant, ordinary one-way ANOVA. (B) Human podocytes were transfected with N-MYC ARHGEF6 WT and mutant cDNA representing proband variants. After 24 hours, cells were fixed and stained with anti-MYC (DaM488) and phalloidin (594) representing F-actin. Lamellipodia/membrane ruffles were counted in a blinded way in three independent experiments (approximately 20 cells/group/experiment counted; p.Glu517Lysfs*27 and p.Pro278Ser only two experiments). Lamellipodia/membrane ruffle was defined as convex cell protrusion. P<0.001 ***, n.s., not significant, Kruskal–Wallis test. (C) Representative images for lamellipodia/membrane ruffle formation, quantified in (B). White arrowheads indicate lamellipodia/membrane ruffles. Scale bar 10 µm. (D) Human podocytes were cotransfected with GFP-tagged WT ARHGEF6 cDNA and c-MYC-tagged ILK cDNA (upper panel), c-MYC-tagged PARVA cDNA (middle panel), or c-MYC-tagged PARVB cDNA (lower panel). After 24 hours, cells were fixed and stained with anti-MYC (secondary antibody DaM594). Note that ARHGEF6 colocalizes with ILK, PARVA, and PARVB, especially in the cell periphery (white arrow). Scale bars 25 µm. (E) IMCD3 cells were cotransfected with GFP-tagged ARHGEF6 (WT/proband-derived variants) and c-MYC-tagged PARVA cDNA. Twenty-four hours after transfection, cells were trypsinized, counted, and plated on FN-coated glass slides. Cells were allowed to attach for 5 hours, washed, and then fixed, followed by antibody staining with anti-MYC (secondary antibody DaM594). Cell morphology was assessed in double-positive cells only and in at least 40 cells per condition. Cell spreading was quantified in a dichotomous way (cells spread or not spread). Scale bars 25 µm. (F) Graph shows quantification of (E) as fractions of spread-out cells per conditions. Different colors represent four independent experiments. (G) HEK293T cells were transfected with GFP-tagged ARHGEF6 (WT versus proband mutants) and c-MYC-tagged PARVA cDNA. Note that GFP ARHGEF6 WT coimmunoprecipitates with MYC-tagged PARVA in HEK293T cells, but not with GFP control vector (MOCK), with early truncating variants (p.Arg191* and p.Le387Alafs*58) and only weakly with late truncating variants (p.Glu517Lys*27 and p.Lys712*). Left panel shows input and right panel immunoprecipitate (IP) immunoblots.

Figure 3

Consequences of ARHGEF6 variants on lumen clearance and cell polarity in MDCK 3D cell cultures. (A) Branching morphogenesis was studied in 3D cultures from MDCK cells stably expressing either ARHGEF6 WT or one CAKUT proband variant (GM1, c.571C>T, p.Arg191*; B1185, c.1158_1159del, p.Leu387Alafs*58; A5124, c.1331T>A, p.Ile444Asn). Cysts were then evaluated and classified as normal (>90% cleared), intermediate (50–90% cleared), or abnormal/disorganized (<50% cleared). Clearance and distribution of ARHGEF6 protein are further depicted by immunofluorescence staining using podocalyxin (PODXL) as an apical marker. Scale bars 10 µm. (B) Lumen clearance of fluid-filled MDCK cysts depends on correct apical-basal polarization. To assess polarization and correct basement membrane assembly, cysts were stained for F-actin and laminin. The panel shows representative images for WT ARHGEF6 and each of the following proband-derived variants: GM1, c.571C>T, p.Arg191*; B1185, c.1158_1159del, p.Leu387Alafs*58; A5124, c.1331T>A, p.Ile444Asn. Scale bars 20 µm. (C) Lumen clearance was assessed as described in (A) and then quantified for GFP control, WT ARHGEF6, and the following proband-derived variants: GM1, c.571C>T, p.Arg191*; B1185, c.1158_1159del, p.Leu387Alafs*58; A5124, c.1331T>A, p.Ile444Asn. All cysts from six independent experiments were evaluated in a blinded manner (approximately 500–700 per condition). The majority of cysts expressing WT ARHGEF6 showed predominantly normal (>90% clearance) or intermediate phenotypes (50%–90% clearance) while cysts expressing any of the tested proband-derived ARHGEF6 variants showed a distribution that was shifted toward intermediate or abnormal/disorganized phenotypes (<50% clearance). (D) Laminin distribution in cysts shown in (B) was evaluated as either normal or abnormal for WT ARHGEF6 and the following proband-derived variants: GM1, c.571C>T, p.Arg191*; B1185, c.1158_1159del, p.Leu387Alafs*58; A5124, c.1331T>A, p.Ile444Asn. Abnormal laminin distribution included absent staining, high luminal staining, irregular or patchy staining, or a combination of these patterns. For each ARHGEF6 variant, cysts of all clearance categories (normal, intermediate, and disorganized) were analyzed. Laminin distribution for cysts expressing WT ARHGEF6 was almost completely normal, whereas cysts expressing any of the tested proband ARHGEF6 variants showed largely abnormal laminin patterns (also see Supplemental Figure 8).

Figure 4

Increased rate of duplex kidneys and renal hypodysplasia in Arhgef6-deficient mice. Heterozygous Arhgef6^+/− were mated with hemizygous Arhgef6^−/Y and litters sacrificed at day 4 postnatally. Kidneys and urinary tract have been evaluated macroscopically and dissected on suspected CAKUT phenotype. Specimens have then been processed for Masson's trichrome (MT) staining and subsequent blinded evaluation of multiple sections of each specimen. (A) Two exemplary kidney pairs from Arhgef6^−/− and Arhgef6^−/Y mice, respectively, are shown demonstrating duplex kidneys. Red arrowhead points to fissure: whole organ overview (upper) and MT-stained section (lower). (B) One exemplary kidney pair of an Arhgef6^−/Y mouse is shown demonstrating a left hypoplastic kidney—whole organ overview. (C) One exemplary kidney of an Arhgef6^−/Y mouse is shown demonstrating a dysplastic kidney—MT-stained section. (D) Graph shows relative frequency of three different observed CAKUT phenotypes (duplex kidney, hypodysplasia, and hydronephrosis) for wild-type Arhgef6^+/Y and hemizygous Arhgef6^−/Y animals from macroscopic evaluation. Unpaired t test; *P<0.05. (E) Graph shows relative frequency of total observed CAKUT for wild-type Arhgef6^+/Y and hemizygous Arhgef6^−/Y animals from macroscopic evaluation. Unpaired t test; *, P<0.05. (F) In a second evaluation, whole litters from Arhgef6^+/− × Arhgef6^−/Y matings were evaluated at 4 weeks of age independent of macroscopic CAKUT. Kidneys were processed for histology, and parenchyma was scored for dysplastic features (cystic structures, very heterogeneous parenchyma, loss of zonal compartmentalization). Bar graph shows relative frequency of dysplasia in wild-type Arhgef6^+/Y and hemizygous Arhgef6^−/Y animals. Unpaired t test; *P<0.05. Nephron quantity was estimated by counting the number of glomeruli in longitudinal midline sections of kidneys. Scatter dot plot shows numbers of glomeruli for wild-type Arhgef6^+/Y and hemizygous Arhgef6^−/Y animals. Each animal is represented by two data points, one for each kidney. Mann–Whitney test; ***P<0.001.

Figure 5

Knockdown or knockout of arhgef6 impairs renal development in Xenopus larvae. (A) Three distinct gRNAs targeting arhgef6 were separately and bilaterally injected in both blastomeres of two-cell X. tropicalis embryos. Embryos were then lysed one day postfertilization, and CRISPR/Cas9 editing efficiencies were determined using Sanger sequencing and ICE deconvolution analysis. This demonstrated in vivo arhgef6 gene editing at high efficiency (86%±10%), enriched toward frameshift variants, yielding high knockout score (78%±10%) for all three gRNAs. (B) Bar graph shows relative frequency of effect on survival from global knockout of arhgef6 compared to noninjected embryos or embryos injected with slc45a2 gRNA, leading to albinism in the frog. (C) Fluorophore-conjugated lectin (LEL) was then used to stain proximal nephron parts to qualitatively compare kidney development in Xenopus embryos from different injections. Exemplary images are shown for arhgef6 gRNA, arghef6 morpholino, and control morpholino injections. LEL, Lycopersicon esculentum Lectin. White scale bar, 100 µm; black scale bar, 20 µm. (D) Scatter plot shows quantification of kidney size from two-cell stage bilateral injections, comparing noninjected, arghef6 gRNA and control slc45a2 gRNA-injected animals. Kruskal–Wallis test; NI, noninjected animal; *P<0.05; ***P<0.001. (E) Side comparison of kidney size from arghef6 gRNA unilateral four-cell stage ventral injection is shown. NI, noninjected side of same animal; Mann–Whitney test; ***P<0.001. (F) Side comparison of kidney size from arghef6 gRNA and tyrosinase gRNA in unilateral four-cell stage ventral injection is shown. Mann–Whitney test; ***P<0.001. (G) Scatter plot shows comparison of kidney size from morpholino-mediated knockdown of arghef6 using unilateral four-cell stage ventral injections using the following conditions: noninjected, arghef6 morpholino, control morpholino. Kruskal–Wallis test; NI, noninjected side; MO, morpholino; ***P<0.001.

Figure 6

Variants in ARHGEF6 lead to CAKUT in humans, mice, and frogs. This graphical summary depicts (1) basic aspects of the ARHGEF6/integrin/ILK/parvin/CDC42/RAC1-dependent pathway and (2) different facets of ARHGEF6 loss of function studied in this work. First, we identified six different X-linked recessive variants in ARHGEF6 as a novel cause of monogenic CAKUT in humans (Fig. 1). On the basis of our findings, ARHGEF6 variants reduce active levels of the small GTPases CDC42 and RAC1 (Fig. 2A). Mutants lead to defective lamellipodia/membrane ruffle formation (Fig. 2, B and C) and decreased FN-induced cell spreading (Fig. 2, E and F). Furthermore, mutants show a reduced binding to PARVA (Fig. 2G) and impaired lumen clearance and cell polarity in MDCK cell 3D spheroids (Fig. 3, A–D). Finally, we replicate human CAKUT phenotypes in Arhgef6 deficient mice (Fig. 4) and frogs (Fig. 5). NPNT, nephronectin; FC, Fraser complex (consisting of FRAS1, FREM2, and FREM1). Created with biorender.com.