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Segmental and Subcellular Distribution of CFTR in the Kidney

  • François Jouret
  • Pierre J. Courtoy
  • Olivier Devuyst
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 741)

Abstract

Besides its location at the plasma membrane, CFTR is present in intracellular vesicles along both the exocytic and the endocytic pathways. Immunostaining and subcellular fractionation studies of mouse kidney demonstrate that CFTR is located in endosomes of the cells lining the terminal part of the proximal tubule (PT). The PT cells efficiently reabsorb the ultrafiltered low molecular weight (LMW) proteins by apical endocytosis involving the multiligand receptors megalin and cubilin. The progression from early endosomes to lysosomes depends on the integrity of the cytoskeleton, as well as on vesicular acidification. The latter is mediated by the vacuolar H+-ATPase (V-ATPase) and requires an anionic conductance to dissipate the transmembrane potential gradient. CFTR might ensure such chloride conductance, thereby participating to endosomal acidification and protein uptake by PT cells. Immunostaining with well-characterized antibodies shows that CFTR is located in the terminal segment of PT, where it co-distributes with megalin and cubilin. Subcellular fractionation of total mouse kidneys through Percoll gradients demonstrates the co-localization of CFTR with the V-ATPase and early endosome markers including the Cl/H+ exchanger, ClC-5, and the small GTPase, Rab5a. Deglycosylation studies and immunoblotting show a distinct glycosylation pattern for CFTR in mouse kidney and lung. The segmental and subcellular distribution of CFTR in mouse kidney supports a role for CFTR in PT receptor-mediated endocytosis of ultrafiltered LMW proteins.

Key words

Cystic fibrosis CFTR ClC-5 receptor-mediated endocytosis megalin cubilin endosomal acidification kidney proximal tubule cells low molecular weight protein antigen retrieval immunostaining analytical subcellular fractionation Percoll gradients 

Notes

Acknowledgments

The authors thank Y. Cnops, Th. Lac, and P. Van der Smissen for excellent technical assistance. The Cftr mice were kindly provided by H. R. De Jonge (Erasmus University Medical Center, Rotterdam, The Netherlands) and the anti-CFTR antibody MD1314 by C.R. Marino (University of Tennessee, Memphis, TN).

P. Courtoy wishes to thank G. Dom, M. Leruth, B. Marien, and F. N’Kuli for patiently adapting to mouse kidney the analytical subcellular fractionation procedure originally developed for rat liver (17). These studies were supported by the Belgian agencies FNRS and FRSM, the “Fondation Alphonse and Jean Forton,” Concerted Research Actions, an Inter-university Attraction Pole (IUAP P6/05), the DIANE project (Communauté Française de Belgique), and the EUNEFRON (FP7, GA#201590) program of the European Community.

References

  1. 1.
    Birn, H., and Christensen, E. I. (2006) Renal albumin absorption in physiology and pathology. Kidney Int. 69, 440–449.PubMedCrossRefGoogle Scholar
  2. 2.
    Christensen, E. I., and Birn, H. (2002) Megalin and cubilin: multifunctional endocytic receptors. Nat. Rev. Mol. Cell. Biol. 3, 256–266.PubMedGoogle Scholar
  3. 3.
    Conner, S. D., and Schmid, S. L. (2003) Regulated portals of entry into the cell. Nature 422, 37–44.PubMedCrossRefGoogle Scholar
  4. 4.
    Gekle, M. (2005) Renal tubule albumin transport. Annu. Rev. Physiol. 67, 573–594.PubMedCrossRefGoogle Scholar
  5. 5.
    Shi, L. B., Fushimi, K., Bae, H. R., and Verkman, A. S. (1991) Heterogeneity in ATP-dependent acidification in endocytic vesicles from kidney proximal tubule. Measurement of pH in individual endocytic vesicles in a cell-free system. Biophys. J. 59, 1208–1217.PubMedCrossRefGoogle Scholar
  6. 6.
    Faundez, V., and Hartzell, H. C. (2004) Intracellular chloride channels: determinants of function in the endosomal pathway. Sci. STKE 233, re8.CrossRefGoogle Scholar
  7. 7.
    Jentsch, T. J. (2008) CLC chloride channels and transporters: from genes to protein structure, pathology and physiology. Crit. Rev. Biochem. Mol. Biol. 43, 3–36.PubMedCrossRefGoogle Scholar
  8. 8.
    Devuyst, O., and Guggino, W. B. (2002) Chloride channels in the kidney: lessons learned from knockout animals. Am. J. Physiol. Renal Physiol. 283, F1176–F1191.PubMedGoogle Scholar
  9. 9.
    Moriyama, Y., and Nelson, N. (1987) The purified ATPase from chromaffin granule membranes is an anion-dependent proton pump. J. Biol. Chem. 262, 9175–9180.PubMedGoogle Scholar
  10. 10.
    Bradbury, N. A. (1999) Intracellular CFTR: localization and function. Physiol. Rev. 79, S175–S191.PubMedGoogle Scholar
  11. 11.
    Bradbury, N. A., Jilling, T., Berta, G., Sorscher, E. J., Bridges, R. J., and Kirk, K. L. (1992) Regulation of plasma membrane recycling by CFTR. Science 256, 530–532.PubMedCrossRefGoogle Scholar
  12. 12.
    Barasch, J., Kiss, B., Prince, A., Saiman, L., Gruenert, D., and Al-Awqati, Q. (1991) Defective acidification of intracellular organelles in cystic fibrosis. Nature 352, 70–73.PubMedCrossRefGoogle Scholar
  13. 13.
    Poschet, J. F., Skidmore, J., Boucher, J. C., Firoved, A. M., Van Dyke, R. W, and Deretic, V. (2002) Hyperacidification of cellubrevin endocytic compartments and defective endosomal recycling in cystic fibrosis respiratory epithelial cells. J. Biol. Chem. 277, 13959–13965.PubMedCrossRefGoogle Scholar
  14. 14.
    Poschet, J. F., Fazio, J. A., Timmins, G. S., Ornatowski, W., Perkett, E., Delgado, M., et al. (2006) Endosomal hyperacidification in cystic fibrosis is due to defective nitric oxide-cyclic GMP signalling cascade. EMBO Rep. 7, 553–559.PubMedGoogle Scholar
  15. 15.
    Jouret, F., Bernard, A., Hermans, C., Dom, G., Terryn, S., Leal, T., et al. (2007) Cystic fibrosis is associated with a defect in apical receptor-mediated endocytosis in mouse and human kidney. J. Am. Soc. Nephrol. 18, 707–718.PubMedCrossRefGoogle Scholar
  16. 16.
    Beaufay, H., and Amar-Costesec, A. (1976) Cell fractionation techniques. In (Korn, E. D., ed.) Methods in Membrane Biology, vol. 6. Plenum Press, New York, NY, pp. 1–100.Google Scholar
  17. 17.
    Courtoy, P. J. (1993) Analytical subcellular fractionation of endosomal compartments in rat hepatocytes. In (Bergeron, J. J. M., Harris, J. R., eds) Subcellular Biochemistry: Endocytic Components: Identification and Characterization, vol. 19. Plenum Press, New York, NY, pp. 29–68.Google Scholar
  18. 18.
    Devuyst, O., and Pirson, Y. (2007) Genetics of hypercalciuric stone forming diseases. Kidney Int. 72, 1065–1072.PubMedCrossRefGoogle Scholar
  19. 19.
    Draye, J.-P., Courtoy, P. J., Quintart, J., and Baudhuin, P. (1987) Relations between plasma membrane and lysosomal membrane. 2. Quantitative evaluation of plasma membrane marker enzymes in the lysosomes. Eur. J. Biochem. 170, 405–411.PubMedCrossRefGoogle Scholar
  20. 20.
    Christensen, E. I., Devuyst, O., Dom, G., Nielsen, R., Van der Smissen, P., Verroust, P., et al. (2003) Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney proximal tubules. Proc. Natl. Acad. Sci. USA 100, 8472–8477.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • François Jouret
    • 1
  • Pierre J. Courtoy
    • 2
  • Olivier Devuyst
    • 1
  1. 1.Division of NephrologyUniversité Catholique de Louvain Medical SchoolBrusselsBelgium
  2. 2.CELL Unit, de Duve Institute, Université Catholique de Louvain Medical SchoolBrusselsBelgium

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