Abstract
Autosomal dominant (ADPKD) and autosomal recessive (ARPKD) polycystic kidney disease are caused by mutations in Pkd1/Pkd2 and Pkhd1, which encode polycystins (PCs) and fibrocystin/polyductin (FPC). Our recent study reported that a deficiency in FPC increases the severity of cystic disease in Pkd2 mutants and down-regulates PC2 in vivo, but the precise molecular mechanism of these effects is unknown (Kim, I., Fu, Y., Hui, K., Moeckel, G., Mai, W., Li, C., Liang, D., Zhao, P., Ma, J., Chen, X.-Z., George, A. L., Jr., Coffey, R. J., Feng, Z. P., and Wu, G. (2008) J. Am. Soc. Nephrol. 19, 455-468). In this study, through the use of deletion and mutagenesis strategies, we identified a PC2-binding domain in the intracellular C terminus of FPC and an FPC-binding domain in the intracellular N terminus of PC2. These binding domains provide a molecular basis for the physical interaction between PC2 and FPC. In addition, we also found that physical interaction between the binding domains of PC2 and FPC is able to prevent down-regulation of PC2 induced by loss of FPC. In vivo, we generated a mouse model of ADPKD with hypomorphic Pkd2 alleles (Pkd2nf3/nf3) and show that PC2 down-regulation is accompanied by a phenotype similar to that of Pkhd1(-/-) mice. These findings demonstrate a common mechanism underlying cystogenesis in ADPKD and ARPKD and provide insight into the molecular relationship between PC2 and FPC.
Highlights
Autosomal dominant polycystic kidney disease (ADPKD)2 is characterized by numerous fluid-filled, spherical renal cysts, and autosomal recessive polycystic kidney disease (ARPKD) is characterized by massive, spindle-shaped renal cysts [1, 2]
We used deletion and mutagenesis strategies to identify a PC2-binding domain (PC2BD) in the intracellular C terminus of FPC and an FPC-binding domain (FBD) in the intracellular N terminus of PC2
ADPKD is characterized by focal, spherical renal cysts, whereas ARPKD is characterized by numerous spindle-shaped ectasias of renal tubules [1, 2]
Summary
Mouse Strains—We have previously reported the generation of the gene-targeted mouse model for Pkhd (Pkhd1Ϫ/Ϫ) [22]. We found 206 embryonic stem cell colonies resistant to G418; one (W2A4) was selected after PCR screening using a pair of outside-construct and cassette-based primers. This cell line was confirmed by Southern blot analysis and injected into C57BL/6 blastocysts at the University of Connecticut Health Center Gene Targeting and Transgenic Facility. Western Blotting and Immunoprecipitation—Western analyses and immunoprecipitation experiments were performed using protocols similar to those described in our previous study [23]. The isolation and culture of primary renal epithelial cells from 2-month-old Pkhd1Ϫ/Ϫ and wild-type littermates were described in our previous study [22]. Differences with p values Ͻ0.05 were considered statistically significant
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