Segmental and Subcellular Distribution of CFTR in the Kidney
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 wordsCystic 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
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.
- 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
- 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.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
- 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