Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells

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Abstract

Several proteins implicated in the pathogenesis of polycystic kidney disease (PKD) localize to cilia. Furthermore, cilia are malformed in mice with PKD with mutations in TgN737Rpw (encoding polaris). It is not known, however, whether ciliary dysfunction occurs or is relevant to cyst formation in PKD. Here, we show that polycystin-1 (PC1) and polycystin-2 (PC2), proteins respectively encoded by Pkd1 and Pkd2, mouse orthologs of genes mutated in human autosomal dominant PKD, co-distribute in the primary cilia of kidney epithelium. Cells isolated from transgenic mice that lack functional PC1 formed cilia but did not increase Ca2+ influx in response to physiological fluid flow. Blocking antibodies directed against PC2 similarly abolished the flow response in wild-type cells as did inhibitors of the ryanodine receptor, whereas inhibitors of G-proteins, phospholipase C and InsP3 receptors had no effect. These data suggest that PC1 and PC2 contribute to fluid-flow sensation by the primary cilium in renal epithelium and that they both function in the same mechanotransduction pathway. Loss or dysfunction of PC1 or PC2 may therefore lead to PKD owing to the inability of cells to sense mechanical cues that normally regulate tissue morphogenesis.

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Figure 1: Polycystin-1 was detected in the cilia of wild-type (wt) mouse embryonic kidney and collecting duct–derived epithelial cells.
Figure 2: Polycystin-1 mediated mechanical flow-induced extracellular Ca2+ influx.
Figure 3: Responses to mechanical flow stimulation require fully developed cilia.
Figure 4: Polycystin-2 was detected in the cilia of wild-type (wt) collecting duct–derived epithelial cells.
Figure 5: Polycystin-2 in flow-induced Ca2+ signaling.
Figure 6: Specificity of antibodies against polycystins.
Figure 7: Ca2+-induced Ca2+ release was required for flow-induced Ca2+ responses.
Figure 8: Schematic diagram of mechanisms of fluid shear stress and Ca2+ signaling in mouse embryonic kidney cells.

References

  1. 1

    Torres, V.E. Extrarenal manifestations of autosomal dominant polycystic kidney disease. Am. J. Kidney. Dis. 34, xlv–xlviii (1999).

  2. 2

    Calvet, J.P. & Grantham, J.J. The genetics and physiology of polycystic kidney disease. Semin. Nephrol. 21, 107–123 (2001).

  3. 3

    Stayner, C. & Zhou, J. Polycystin channels and kidney disease. Trends Pharmacol. Sci. 22, 543–546 (2001).

  4. 4

    Parnell, S.C. et al. The polycystic kidney disease-1 protein, polycystin-1, binds and activates heterotrimeric G-proteins in vitro. Biochem. Biophys. Res. Commun. 251, 625–631 (1998).

  5. 5

    Delmas, P. et al. Constitutive activation of G-proteins by polycystin-1 is antagonized by polycystin-2. J. Biol. Chem. 277, 11276–11283 (2002).

  6. 6

    Chen, X.Z. et al. Transport function of the naturally occurring pathogenic polycystin-2 mutant, R742X. Biochem. Biophys. Res. Commun. 282, 1251–1256 (2001).

  7. 7

    Gonzalez-Perret, S. et al. Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective cation channel. Proc. Natl. Acad. Sci. USA 98, 1182–1187 (2001).

  8. 8

    Vassilev, P.M. et al. Polycystin-2 is a novel cation channel implicated in defective intracellular Ca2+ homeostasis in polycystic kidney disease. Biochem. Biophys. Res. Commun. 282, 341–350 (2001).

  9. 9

    Koulen, P. et al. Polycystin-2 is an intracellular calcium release channel. Nat. Cell Biol. 4, 191–197 (2002).

  10. 10

    Hanaoka, K. et al. Co-assembly of polycystin-1 and -2 produces unique cation-permeable currents. Nature 408, 990–994 (2000).

  11. 11

    Moyer, J.H. et al. Candidate gene associated with a mutation causing recessive polycystic kidney disease in mice. Science 264, 1329–1333 (1994).

  12. 12

    Hou, X. et al. Cystin, a novel cilia-associated protein, is disrupted in the cpk mouse model of polycystic kidney disease. J. Clin. Invest. 109, 533–540 (2002).

  13. 13

    Pazour, G.J., San Agustin, J.T., Follit, J.A., Rosenbaum, J.L. & Witman, G.B. Polycystin-2 localizes to kidney cilia and the ciliary level is elevated in orpk mice with polycystic kidney disease. Curr. Biol. 12, R378–R380 (2002).

  14. 14

    Yoder, B.K. et al. Polaris, a protein disrupted in orpk mutant mice, is required for assembly of renal cilium. Am. J. Physiol. Renal Physiol. 282, F541–F552 (2002).

  15. 15

    Sleigh, M.A., Blake, J.R. & Liron, N. The propulsion of mucus by cilia. Am. Rev. Respir. Dis. 137, 726–741 (1988).

  16. 16

    Perkins, L.A., Hedgecock, E.M., Thomson, J.N. & Culotti, J.G. Mutant sensory cilia in the nematode Caenorhabditis elegans. Dev. Biol. 117, 456–487 (1986).

  17. 17

    Brueckner, M. Cilia propel the embryo in the right direction. Am. J. Med. Genet. 101, 339–344 (2001).

  18. 18

    Pazour, G.J. et al. Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella. J. Cell Biol. 151, 709–718 (2000).

  19. 19

    Murcia, N.S. et al. The Oak Ridge Polycystic Kidney (orpk) disease gene is required for left–right axis determination. Development 127, 2347–2355 (2000).

  20. 20

    Pennekamp, P. et al. The ion channel polycystin-2 is required for left–right axis determination in mice. Curr. Biol. 12, 938–943 (2002).

  21. 21

    Praetorius, H.A. & Spring, K.R. Bending the MDCK cell primary cilium increases intracellular calcium. J. Membr. Biol. 184, 71–79 (2001).

  22. 22

    Davies, P.F. Flow-mediated endothelial mechanotransduction. Physiol. Rev. 75, 519–560 (1995).

  23. 23

    Tulis, D.A., Unthank, J.L. & Prewitt, R.L. Flow-induced arterial remodeling in rat mesenteric vasculature. Am. J. Physiol. 274, H874–H882 (1998).

  24. 24

    Lu, W. et al. Perinatal lethality with kidney and pancreas defects in mice with a targeted Pkd1 mutation. Nat. Genet. 17, 179–181 (1997).

  25. 25

    Lu, W. et al. Comparison of Pkd1-targeted mutants reveals that loss of polycystin-1 causes cystogenesis and bone defects. Hum. Mol. Genet. 10, 2385–2396 (2001).

  26. 26

    Watanabe, M., Muramatsu, T., Shirane, H. & Ugai, K. Discrete distribution of binding sites for Dolichos biflorus agglutinin (DBA) and for peanut agglutinin (PNA) in mouse organ tissues. J. Histochem. Cytochem. 29, 779–780 (1981).

  27. 27

    Wu, G. et al. Somatic inactivation of Pkd2 results in polycystic kidney disease. Cell 93, 177–188 (1998).

  28. 28

    Geng, L. et al. Identification and localization of polycystin, the PKD1 gene product. J. Clin. Invest. 98, 2674–2682 (1996).

  29. 29

    Ong, A.C. et al. Coordinate expression of the autosomal dominant polycystic kidney disease proteins, polycystin-2 and polycystin-1, in normal and cystic tissue. Am. J. Pathol. 154, 1721–1729 (1999).

  30. 30

    Chou, C.L. & Marsh, D.J. Measurement of flow rate in rat proximal tubules with a non-obstructing optical method. Am. J. Physiol. 253, F366–F371 (1987).

  31. 31

    Nauli, S.M., Williams, J.M., Akopov, S.E., Zhang, L. & Pearce, W.J. Developmental changes in ryanodine- and IP(3)-sensitive Ca2+ pools in ovine basilar artery. Am. J. Physiol. Cell Physiol. 281, C1785–C1796 (2001).

  32. 32

    Wilson, P.D. Polycystin: new aspects of structure, function, and regulation. J. Am. Soc. Nephrol. 12, 834–845 (2001).

  33. 33

    Cocks, T.M. & Moffatt, J.D. Protease-activated receptors: sentries for inflammation? Trends Pharmacol. Sci. 21, 103–108 (2000).

  34. 34

    Bogatcheva, N.V., Garcia, J.G. & Verin, A.D. Molecular mechanisms of thrombin-induced endothelial cell permeability. Biochemistry Mosc. 67, 75–84 (2002).

  35. 35

    Kim, K., Drummond, I., Ibraghimov-Beskrovnaya, O., Klinger, K. & Arnaout, M.A. Polycystin 1 is required for the structural integrity of blood vessels. Proc. Natl. Acad. Sci. USA 97, 1731–1736 (2000).

  36. 36

    Boulter, C. et al. Cardiovascular, skeletal, and renal defects in mice with a targeted disruption of the Pkd1 gene. Proc. Natl. Acad. Sci. USA 98, 12174–12179 (2001).

  37. 37

    Muto, S. et al. Pioglitazone improves the phenotype and molecular defects of a targeted Pkd1 mutant. Hum. Mol. Genet. 11, 1731–1742 (2002).

  38. 38

    Lu, W. et al. Late onset of renal and hepatic cysts in Pkd1-targeted heterozygotes. Nat. Genet. 21, 160–161 (1999).

  39. 39

    Weston, B.S. et al. Polycystin expression during embryonic development of human kidney in adult tissues and ADPKD tissue. Histochem. J. 29, 847–856 (1997).

  40. 40

    Ward, C.J. et al. Polycystin, the polycystic kidney disease 1 protein, is expressed by epithelial cells in fetal, adult, and polycystic kidney. Proc. Natl. Acad. Sci. USA 93, 1524–1528 (1996).

  41. 41

    Rossetti, S. et al. The position of the polycystic kidney disease 1 (PKD1) gene mutation correlates with the severity of renal disease. J. Am. Soc. Nephrol. 13, 1230–1237 (2002).

  42. 42

    DeMaria, M., Johnson, R.P. & Rosenzweig, M. Four color immunofluorescence detection using two 488-nm lasers on a Becton Dickinson FACS Vantage flow cytometer. Cytometry 29, 178–181 (1997).

  43. 43

    Lohning, C., Nowicka, U. & Frischauf, A.M. The mouse homolog of PKD1: sequence analysis and alternative splicing. Mamm. Genome 8, 307–311 (1997).

  44. 44

    Wu, G. et al. Molecular cloning, cDNA sequence analysis, and chromosomal localization of mouse Pkd2. Genomics 45, 220–223 (1997).

  45. 45

    Pennekamp, P. et al. Characterization of the murine polycystic kidney disease (Pkd2) gene. Mamm. Genome 9, 749–752 (1998).

  46. 46

    Ong, A.C. et al. Polycystin-1 expression in PKD1, early-onset PKD1, and TSC2/PKD1 cystic tissue. Kidney Int. 56, 1324–1333 (1999).

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Acknowledgements

We thank K.R. Spring, G.J. Pazour, M. Logman and G.B. Witman for discussions and Q. Xi and A.P. Wiebe for technical assistance. This work was supported by grants from the US National Institutes of Health (J.Z.), the US National Aeronautics and Space Administration (D.E.I.), a Howard Hughes Predoctoral Fellowship (F.J.A.) and a Polycystic Kidney Disease Foundation Postdoctoral Fellowship (S.M.N.).

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Correspondence to Jing Zhou.

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