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. 2007 Jan 30;104(5):1631-6.
doi: 10.1073/pnas.0605266104. Epub 2007 Jan 24.

Glomerulocystic kidney disease in mice with a targeted inactivation of Wwtr1

Zakir Hossain  1 , Safiah Mohamed Ali, Hui Ling Ko, Jianliang Xu, Chee Peng Ng, Ke Guo, Zeng Qi, Sathivel Ponniah, Wanjin Hong, Walter Hunziker
Affiliations

Glomerulocystic kidney disease in mice with a targeted inactivation of Wwtr1

Zakir Hossain et al. Proc Natl Acad Sci U S A. .

Abstract

Wwtr1 is a widely expressed 14-3-3-binding protein that regulates the activity of several transcription factors involved in development and disease. To elucidate the physiological role of Wwtr1, we generated Wwtr1-/- mice by homologous recombination. Surprisingly, although Wwtr1 is known to regulate the activity of Cbfa1, a transcription factor important for bone development, Wwtr1-/- mice show only minor skeletal defects. However, Wwtr1-/- animals present with renal cysts that lead to end-stage renal disease. Cysts predominantly originate from the dilation of Bowman's spaces and atrophy of glomerular tufts, reminiscent of glomerulocystic kidney disease in humans. A smaller fraction of cysts is derived from tubules, in particular the collecting duct (CD). The corticomedullary accumulation of cysts also shows similarities with nephronophthisis. Cells lining the cysts carry fewer and shorter cilia and the expression of several genes associated with glomerulocystic kidney disease (Ofd1 and Tsc1) or encoding proteins involved in cilia structure and/or function (Tg737, Kif3a, and Dctn5) is decreased in Wwtr1-/- kidneys. The loss of cilia integrity and the down-regulation of Dctn5, Kif3a, Pkhd1 and Ofd1 mRNA expression can be recapitulated in a renal CD epithelial cell line, mIMCD3, by reducing Wwtr1 protein levels using siRNA. Thus, Wwtr1 is critical for the integrity of renal cilia and its absence in mice leads to the development of renal cysts, indicating that Wwtr1 may represent a candidate gene for polycystic kidney disease in humans.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Targeting of the Wwtr1 locus and gross anatomy of skeleton and kidneys. (A) Targeting strategy. Schematic representations of the genomic locus of Wwtr1 with exons 1 and 2 (WT allele), the targeting vector and the targeted Wwtr1 locus with exon 2 disrupted by the in-frame insertion of lacZ and a loxP flanked neomycin cassette (Targeted allele). For details, see SI Text. (B) Homologous recombination in ES cells. Southern blot of ScaI digested genomic DNA of selected ES cells hybridized with the labeled DNA probe (sp; see A) for the identification of homologous recombinants. A 7.6- or 12.7-kb ScaI fragment corresponding to the WT or targeted mutant (Mut) Wwtr1 allele, respectively, is detected in targeted (HR) ES cell clones, whereas only the WT allele is present in controls (Con). (C) Western blot of Wwtr1 protein. Kidney lysates from P9 WT, KO, and heterozygous (HZ) littermates were fractionated by SDS/PAGE, transferred to membranes, and blotted with Abs to Wwtr1. Note a cross-reactivity with the highly homologous Yap65. (D) Skeleton anatomy. The skeleton of WT and KO E17.5 embryos was stained with Alizarin red and Alcian blue to stain bone (red) and cartilage (blue), respectively. Note the slightly shorter skeleton of the KO embryo. (E and F) Kidney anatomy. Both kidneys of a 3- to 4-month-old WT and Wwtr1−/− mouse are shown through the abdominal cavity after removal of obstructing organs. Note a larger size and pale appearance of the kidneys and the fluid filled lesions on the renal capsules of the KO animal. (G and H) Renal histology. Hematoxilin and eosin staining of longitudinal kidney sections from an 8-week-old WT and Wwtr1−/− mouse, showing numerous cysts in the corticomedullary region of the KO kidney. (Original magnification, ×20.)
Fig. 2.
Fig. 2.
Embryonic renal expression of Wwtr1 and pathological changes in Wwtr1-null kidneys. (A and B) LacZ expression. β-gal activity was visualized in E14.5 and E15.5 kidneys of Wwtr1-null embryos. Weak expression can first be detected at ≈E14.5 and increases by 15.5. (C–H) Glomerular and tubular dilation. Longitudinal kidney sections of E15.5 and E16.5 embryos and 8-week-old adult mice stained with hematoxylin and eosin (H&E). Glomerular (arrows) and tubular (arrowheads) dilations are first observed around E15.5 and E16.5, respectively, and then gradually progress to cyst formation. (Magnifications, ×400.)
Fig. 3.
Fig. 3.
Renal expression of Wwtr1 and compromised cilia integrity in Wwtr1−/− kidneys. (A–C) Renal and glomerular expression of Wwtr1. Immunofluorescence microscopy of kidney sections from a P9 WT (A) or adult WT (B) or KO (C) mice stained with Abs to Wwtr1 (red). Note the nonuniform expression of Wwtr1 in tubules and glomeruli of control kidney (A and B) and the loss of staining in cystic KO glomerulus (C). T, tuft; cy, cyst. (Original magnifications,×200 in A and ×1,000 in B and C.) (D and E) Cilia integrity. Immunofluorescence microscopy of an adult WT (D) or Wwtr1−/− (E) kidney section labeled with Abs to acetylated α-tubulin (α-Tub, red) to detect primary cilia (arrows) and a labeled CD maker, DBA (green). Intact cilia are abundant and readily detected in control tissue, whereas ciliated cells lining cysts (cy) of Wwtr1−/− kidneys are rare and often appear short when detected. Nuclei were stained with DAPI or nuclear yellow (NY) (blue). (Original magnification, ×1,000.) (F) Quantification. DBA-positive cells lining tubules from control (WT) or Wwtr1 KO littermate kidneys with normal (Tub.), dilated (dil. Tub.), or cystic (cyst) tubules were counted in random sections, and the fraction of ciliated cells was determined (n = 150–200). Error bars indicate standard deviation. Asterisks indicate significant difference from WT (t test, P < 0.005, n = 3). (G and H) Cilia morphology. SEM of a kidney section from a P9 WT (G) or Wwtr1−/− (H) mouse. Abundant intact cilia (arrows) are found on normal tubules, but cilia lining dilated tubules or cysts are frequently short with aberrant morphology, if present, in Wwtr1 KO kidneys. (Scale bar, 1 μm.)
Fig. 4.
Fig. 4.
Down-regulation of genes associated with GCKD and cilia integrity in Wwtr1-null kidneys. (A) Expression of PKD/cilia associated genes. Relative expression levels of the indicated genes in WT and KO kidneys of E17.5 embryos were determined in duplicate by quantitative real-time RT-PCR and normalized with respect to the mRNA level of either Yap or Gapdh, with similar results. Differences for Bicc1, Nphp1, Pkd1, Dnahc11, and D2lic were reproducible but statistically not significant. (B) Expression of control genes is unchanged. Error bars indicate standard deviation (n = 3). Asterisks indicate significant difference from WT (one-tailed t test, P < 0.05, n = 3).
Fig. 5.
Fig. 5.
Compromised cilia integrity and down-regulation of PKD/cilia-associated genes in mIMCD3 cells after Wwtr1 depletion. (A and B) Cilia integrity. Immunofluorescence microscopy of cells treated with control (Con-M) or Wwtr1 (Ri-10, B) siRNA for 72 h stained with Abs to acetylated α-tubulin (red) and ZO-1 (green) to label cilia and tight junctions, respectively. Nuclei were stained with DAPI (blue). Note a reduced number and shorter cilia on cells exposed to Wwtr1 siRNA. (Magnification, ×1,000.) (C and D) Cilia morphology. SEM of a cell treated with control (Con-M) or Wwtr1 (Ri-10) siRNA for 72 h. Cilia (arrows) on Wwtr1 siRNA treated cells were often short with aberrant morphology if present, whereas control cells show intact long cilia. (Scale bar, 1 μm.) (E) Cilia length. Length was measured in random SEM micrographs of untreated (UT) mIMCD3 cells or cells treated with control (Con-M) or Wwtr1 (Ri-10) siRNA. Error bar indicates standard deviation (n = 50). Asterisks, significant difference from UT (t test, P < 0.005). (F) Frequency of ciliated cells. The number of ciliated cells with either intact (≥ 3 μm long) or compromised (<3 μm long) cilia was determined from random SEM micrographs of untreated (UT) mIMCD3 cells or cells treated with control (Con-M) or Wwtr1 (Ri-10) siRNA (Scale bar, 1 μm.). Error bars indicate standard deviation (n = 100). (G) Wwtr1 protein levels. Untreated (UT) mIMCD3 cells or cells treated with two different control (Con-L, Con-M) or Wwtr1 (Ri-10, Ri-11) siRNAs for 72 h were analyzed by Western blot using Abs to Wwtr1. (H) Expression of PKD/cilia associated genes. Relative expression levels of the indicated transcripts in control (Con-M) and Wwtr1 (Ri-10) siRNA treated mIMCD3 cells determined by quantitative real-time RT-PCR. Quantification was performed in duplicate and normalized with respect to the mRNA level of either Yap or Gapdh, with similar results. Error bars indicate standard deviation (n = 3). Asterisks indicate significant differences from controls (t test; P < 0.05, n = 3).
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References

    1. Guay-Woodford LM. Am J Physiol. 2003;285:F1034–F1049. - PubMed
    1. Gretz N, Kranzlin B, Pey R, Schieren G, Bach J, Obermuller N, Ceccherini I, Kloting I, Rohmeiss P, Bachmann S, et al. Nephrol Dial Transplant. 1996;11(Suppl 6):46–51. - PubMed
    1. Calvet JP, Grantham JJ. Semin Nephrol. 2001;21:107–123. - PubMed
    1. Pazour GJ, Witman GB. Curr Opin Cell Biol. 2003;15:105–110. - PubMed
    1. Rosenbaum JL, Witman GB. Nat Rev Mol Cell Biol. 2002;3:813–825. - PubMed

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