Deletion of ADP Ribosylation Factor-Like GTPase 13B Leads to Kidney Cysts

Abstract

The gene for ADP ribosylation factor-like GTPase 13B (Arl13b) encodes a small GTPase essential for cilia biogenesis in multiple model organisms. Inactivation of arl13b in zebrafish leads to a number of phenotypes indicative of defective cilia, including cystic kidneys. In mouse, null mutation in Arl13b results in severe patterning defects in the neural tube and defective Hedgehog signaling. Human mutations of ARL13B lead to Joubert syndrome, a ciliopathy. However, patients with mutated ARL13B do not develop kidney cysts. To investigate whether Arl13b has a role in ciliogenesis in mammalian kidney and whether loss of function of Arl13b leads to cystic kidneys in mammals, we generated a mouse model with kidney-specific conditional knockout of Arl13b Deletion of Arl13b in the distal nephron at the perinatal stage led to a cilia biogenesis defect and rapid kidney cyst formation. Additionally, we detected misregulation of multiple pathways in the cystic kidneys of this model. Moreover, valproic acid, a histone deacetylase inhibitor that we previously showed slows cyst progression in a mouse cystic kidney model with neonatal inactivation of Pkd1, inhibited the early rise of Wnt7a expression, ameliorated fibrosis, slowed cyst progression, and improved kidney function in the Arl13b mutant mouse. Finally, in rescue experiments in zebrafish, all ARL13B allele combinations identified in patients with Joubert syndrome provided residual Arl13b function, supporting the idea that the lack of cystic kidney phenotype in human patients with ARL13B mutations is explained by the hypomorphic nature of the mutations.

Keywords: polycystic kidney disease; renal fibrosis; signaling.

Figure 1.

Distal nephron–specific inactivation of Arl13b in Arl13b^flox/flox; Ksp-Cre mice leads to rapid progression of PKD. (A) Diagram of targeting vector. Exons are showed by black boxes. FRT indicates FLPE recombinase sites, and loxP indicates Cre recombinase sites. (B) Gross morphology of kidneys from P14 and P17 Arl13b^flox/flox; Ksp-Cre mice compared with control siblings. (C) Increasing KBW ratio in mutants during the neonatal stage (n=3 for each stage and each genotype). (D) Hematoxylin/eosin-stained sections from control and mutant kidneys on P7, P14, and P17. Scale bar, 2 mm. (E) Significant cyst formation quantified by cystic index and increased BUN levels in knockout mice at P14 (n=3 for each genotype). Control indicates the wild type. KO, knockout. *P<0.05; **P<0.01.

Figure 2.

Arl13b is essential for cilia biogenesis, and its depletion leads to cyst formation in mouse kidney. (A) P14 kidney sections shown in the collecting duct are labeled by DBA (red) and Arl13b signal (green; left panel), and cilia signal labeled by antiacetylated tubulin (A-tub; green; right panel) is lacking in the mutant kidney (knockout [KO]). Nuclei are labeled with DAPI in blue. Although insets in control show cilia in the collecting duct, insets in KO show cilia outside of the collecting duct. Arrows point to cilia. Cy, cyst. (B) P7 kidney sections show basal body labeled with anti–γ-tubulin (γ-tub; green) and cilia labeled with antiacetylated tubulin (A-tub; red) in control and mutant (KO) mice. Arrow points to a cilium in DBA-positive (blue) segment. Arrowheads point to basal bodies without cilium in a dilated DBA–positive region (Di). Inset shows a basal body without cilia. (C) Cyst formation is confined to the distal nephron in Arl13b^flox/flox; Ksp-Cre mice. DBA marks the collecting duct. The proximal tubule is labeled with LTL, the medullary thick ascending limb is labeled by THP, and the distal convoluted tubule is labeled with anti-Parvalbumin (Pvalb). Nuclei are labeled with DAPI in blue. Control indicates the wild type. Scale bars, 20 μm in A and B; 100 μm in C.

Figure 3.

Increased cell proliferation and fibrosis in the kidney of Arl13b^flox/flox; Ksp-Cre mice. (A) Kidney sections stained with anti-BrdU (green) and DBA (red) at P14. (B) Quantitation of BrdU-positive nuclei in DBA-positive cells (n=3 animals of each genotype). (C) Trichrome stain indicates collagen deposition (green) in mutant kidneys at P28. (D) Interstitial cells show increased smooth muscle actin (SMA; green) expression in mutant kidney compared with controls at P28. The collecting duct is labeled with DBA in red. Control indicates the wild type. cy, Cyst; KO, knockout. Scale bars, 50 μm in A; 20 μm in C and D. **P<0.01.

Figure 4.

Multiple pathways are misregulated in cystic kidneys of Arl13b^flox/flox; Ksp-Cre mice. qPCR analysis of genes involved in Wnt (cMyc, Wnt7a, Wnt10a, Lef1, Axin2, and RhoU), Hedgehog (Shh, Ihh, Dhh, Ptch1, Ptch2, Gli1, Gli2, and Gli3), Hippo (InhbA, Ctgf, and Birc3), and innate immunity (C3) pathways in control (Con) and knockout (KO) kidneys at P7, P14, P21, and P28. Gene expression is normalized with that of Gapdh. Unit 1 is defined as the expression level in wild-type samples at P7 (n=4 kidneys from two mice). Error bars show SDs. *P<0.05; **P<0.01.

Figure 5.

The HDACi VPA suppresses disease progression in Arl13b^flox/flox; Ksp-Cre mice. (A) qPCR on Hdac1 expression in wild-type (WT; Con) and mutant (KO) kidney. Unit 1 is defined as the expression in WT sample at P7 (n=3 mice). (B) Kidney morphology of vehicle and VPA-injected mutant (KO) mice at P25. (C) Representative hematoxylin/eosin–stained sections of kidneys from vehicle and VPA-treated mutant mice at P25. (D) KBW ratio, cystic index, and BUN in VPA and vehicle-treated mutant (KO) mice at P25 (n=3 mice). (E) Kidney morphology in vehicle and VPA–injected WT mice at P25. (F) KBW ratio in vehicle and VPA–injected WT mice at P25. (G) Trichrome staining of P25 kidney section of vehicle (Con) and VPA–treated Arl13b^flox/flox; Ksp-Cre mice. (H) P25 kidney section of vehicle (Con) and VPA–treated Arl13b^flox/flox; Ksp-Cre mice stained with anti-smooth muscle actin (green) and DBA (red). (I) Wnt7a expression is upregulated in mutant (KO) kidney at P10 as shown by qPCR analysis. Gene expression is normalized with that of Gapdh. Unit 1 is defined as the expression level in WT samples (n=3 kidneys from three mice). (J) qPCR analysis of Wnt7a and Wnt10a expression in mutant (KO) and WT kidney treated with vehicle or VPA. Unit 1 is defined as the expression level in vehicle–treated WT samples (n=3 kidneys from three mice). Error bars show SDs. cy, Cyst. Scale bars, 20 μm in G and H. *P<0.05; **P<0.01.

Figure 6.

Function and localization of four mutant Arl13b identified in patients with JS assayed in zebrafish. (A) Y86C-GFP could rescue the body curvature (left panel) and kidney cyst (enlarged in the right panel; white arrows) of scorpion^hi459 mutants. The truncated form W82X could not rescue either phenotype. (B) PCR genotyping results. Although the wild-type (WT) band identifies the WT allele, the mutant (Mu) band identifies the mutant allele. Each lane is for a single embryo with WT morphology. (C) Western blot showing the protein levels of eGFP-tagged WT and mutant Arl13b at 12-hour postfertilization. Anti–β-tubulin (tub) is used as a loading control. (D) Side views of the trunk region of day 2 scorpion^hi459 mutant embryos subjected to mRNA injection. Arrows point to cilia bundles in the kidney duct. (E) Side views of the pronephric duct in embryos injected with mRNA encoding GFP-tagged WT and mutant Arl13b. Cilia are labeled with antiacetylated tubulin in red. WT inj, Y86C inj, and W82X inj indicate injected with WT, Y86C, and W82X Arl13b-GFP mRNA, respectively. A-tub, antiacetylated tubulin; uninj, uninjected scorpion^hi459 mutant. Scale bar, 10 μm.