Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1998 May 15;101(10):2140–2150. doi: 10.1172/JCI1923

Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase.

N J Brunskill 1, J Stuart 1, A B Tobin 1, J Walls 1, S Nahorski 1
PMCID: PMC508802  PMID: 9593770

Abstract

Receptor-mediated endocytosis of albumin is an important function of the kidney proximal tubule epithelium. We have measured endocytosis of [125I]-albumin in opossum kidney cells and examined the regulation of this process by phosphatidylinositide 3-kinase (PI 3-kinase). Albumin endocytosis was inhibited by both wortmannin (IC50 6.9 nM) and LY294002 (IC50 6.5 microM) at concentrations that suggested the involvement of PI 3-kinase in its regulation. Recycling rates were unaffected. We transfected OK cells with either a wild-type p85 subunit of PI 3-kinase, or a dominant negative form of the p85 subunit (Deltap85) using the LacSwitch expression system. Transfects were screened by immunoblotting with anti-PI 3-kinase antibodies. Under basal conditions, transfects demonstrated no expression of p85 or Deltap85, but expression was briskly induced by treatment of the cells with IPTG (EC50 13.7 microM). Inhibition of PI 3-kinase activity by Deltap85 was confirmed by in vitro kinase assay of anti-phosphotyrosine immunoprecipitates from transfected cells stimulated with insulin. Expression of Deltap85 resulted in marked inhibition of albumin endocytosis, predominantly as a result of reduction of the Vmax of the transport process. Expression of p85 had no significant effect on albumin uptake. The results demonstrate that PI 3-kinase regulates an early step in the receptor-mediated endocytosis of albumin by kidney proximal tubular cells.

Full Text

The Full Text of this article is available as a PDF (249.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Araki N., Johnson M. T., Swanson J. A. A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages. J Cell Biol. 1996 Dec;135(5):1249–1260. doi: 10.1083/jcb.135.5.1249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brunskill N. J., Cockcroft N., Nahorski S., Walls J. Albumin endocytosis is regulated by heterotrimeric GTP-binding protein G alpha i-3 in opossum kidney cells. Am J Physiol. 1996 Aug;271(2 Pt 2):F356–F364. doi: 10.1152/ajprenal.1996.271.2.F356. [DOI] [PubMed] [Google Scholar]
  3. Brunskill N. J., Nahorski S., Walls J. Characteristics of albumin binding to opossum kidney cells and identification of potential receptors. Pflugers Arch. 1997 Feb;433(4):497–504. doi: 10.1007/s004240050305. [DOI] [PubMed] [Google Scholar]
  4. Burton C. J., Bevington A., Harris K. P., Walls J. Growth of proximal tubular cells in the presence of albumin and proteinuric urine. Exp Nephrol. 1994 Nov-Dec;2(6):345–350. [PubMed] [Google Scholar]
  5. Christensen E. I., Nielsen S. Structural and functional features of protein handling in the kidney proximal tubule. Semin Nephrol. 1991 Jul;11(4):414–439. [PubMed] [Google Scholar]
  6. Clague M. J., Thorpe C., Jones A. T. Phosphatidylinositol 3-kinase regulation of fluid phase endocytosis. FEBS Lett. 1995 Jul 3;367(3):272–274. doi: 10.1016/0014-5793(95)00576-u. [DOI] [PubMed] [Google Scholar]
  7. Clapp W. L., Park C. H., Madsen K. M., Tisher C. C. Axial heterogeneity in the handling of albumin by the rabbit proximal tubule. Lab Invest. 1988 May;58(5):549–558. [PubMed] [Google Scholar]
  8. Cross M. J., Stewart A., Hodgkin M. N., Kerr D. J., Wakelam M. J. Wortmannin and its structural analogue demethoxyviridin inhibit stimulated phospholipase A2 activity in Swiss 3T3 cells. Wortmannin is not a specific inhibitor of phosphatidylinositol 3-kinase. J Biol Chem. 1995 Oct 27;270(43):25352–25355. doi: 10.1074/jbc.270.43.25352. [DOI] [PubMed] [Google Scholar]
  9. De Camilli P., Emr S. D., McPherson P. S., Novick P. Phosphoinositides as regulators in membrane traffic. Science. 1996 Mar 15;271(5255):1533–1539. doi: 10.1126/science.271.5255.1533. [DOI] [PubMed] [Google Scholar]
  10. Domin J., Waterfield M. D. Using structure to define the function of phosphoinositide 3-kinase family members. FEBS Lett. 1997 Jun 23;410(1):91–95. doi: 10.1016/s0014-5793(97)00617-0. [DOI] [PubMed] [Google Scholar]
  11. Eker P., Holm P. K., van Deurs B., Sandvig K. Selective regulation of apical endocytosis in polarized Madin-Darby canine kidney cells by mastoparan and cAMP. J Biol Chem. 1994 Jul 15;269(28):18607–18615. [PubMed] [Google Scholar]
  12. Fry M. J., Waterfield M. D. Structure and function of phosphatidylinositol 3-kinase: a potential second messenger system involved in growth control. Philos Trans R Soc Lond B Biol Sci. 1993 Jun 29;340(1293):337–344. doi: 10.1098/rstb.1993.0076. [DOI] [PubMed] [Google Scholar]
  13. Gaidarov I., Chen Q., Falck J. R., Reddy K. K., Keen J. H. A functional phosphatidylinositol 3,4,5-trisphosphate/phosphoinositide binding domain in the clathrin adaptor AP-2 alpha subunit. Implications for the endocytic pathway. J Biol Chem. 1996 Aug 23;271(34):20922–20929. doi: 10.1074/jbc.271.34.20922. [DOI] [PubMed] [Google Scholar]
  14. Galaske R. G., Baldamus C. A., Stolte H. Plasma protein handling in the rat kidney: micropuncture experiments in the acute heterologous phase of anti-GBM-nephritis. Pflugers Arch. 1978 Aug;375(3):269–277. doi: 10.1007/BF00582441. [DOI] [PubMed] [Google Scholar]
  15. Hansen S. H., Olsson A., Casanova J. E. Wortmannin, an inhibitor of phosphoinositide 3-kinase, inhibits transcytosis in polarized epithelial cells. J Biol Chem. 1995 Nov 24;270(47):28425–28432. doi: 10.1074/jbc.270.47.28425. [DOI] [PubMed] [Google Scholar]
  16. Hara K., Yonezawa K., Sakaue H., Ando A., Kotani K., Kitamura T., Kitamura Y., Ueda H., Stephens L., Jackson T. R. 1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7415–7419. doi: 10.1073/pnas.91.16.7415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hartley K. O., Gell D., Smith G. C., Zhang H., Divecha N., Connelly M. A., Admon A., Lees-Miller S. P., Anderson C. W., Jackson S. P. DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell. 1995 Sep 8;82(5):849–856. doi: 10.1016/0092-8674(95)90482-4. [DOI] [PubMed] [Google Scholar]
  18. Hawes B. E., Luttrell L. M., van Biesen T., Lefkowitz R. J. Phosphatidylinositol 3-kinase is an early intermediate in the G beta gamma-mediated mitogen-activated protein kinase signaling pathway. J Biol Chem. 1996 May 24;271(21):12133–12136. doi: 10.1074/jbc.271.21.12133. [DOI] [PubMed] [Google Scholar]
  19. Holgado-Madruga M., Moscatello D. K., Emlet D. R., Dieterich R., Wong A. J. Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor. Proc Natl Acad Sci U S A. 1997 Nov 11;94(23):12419–12424. doi: 10.1073/pnas.94.23.12419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hu Q., Klippel A., Muslin A. J., Fantl W. J., Williams L. T. Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase. Science. 1995 Apr 7;268(5207):100–102. doi: 10.1126/science.7701328. [DOI] [PubMed] [Google Scholar]
  21. Ireton K., Payrastre B., Chap H., Ogawa W., Sakaue H., Kasuga M., Cossart P. A role for phosphoinositide 3-kinase in bacterial invasion. Science. 1996 Nov 1;274(5288):780–782. doi: 10.1126/science.274.5288.780. [DOI] [PubMed] [Google Scholar]
  22. Jhun B. H., Rose D. W., Seely B. L., Rameh L., Cantley L., Saltiel A. R., Olefsky J. M. Microinjection of the SH2 domain of the 85-kilodalton subunit of phosphatidylinositol 3-kinase inhibits insulin-induced DNA synthesis and c-fos expression. Mol Cell Biol. 1994 Nov;14(11):7466–7475. doi: 10.1128/mcb.14.11.7466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Joly M., Kazlauskas A., Corvera S. Phosphatidylinositol 3-kinase activity is required at a postendocytic step in platelet-derived growth factor receptor trafficking. J Biol Chem. 1995 Jun 2;270(22):13225–13230. doi: 10.1074/jbc.270.22.13225. [DOI] [PubMed] [Google Scholar]
  24. Jones A. T., Clague M. J. Phosphatidylinositol 3-kinase activity is required for early endosome fusion. Biochem J. 1995 Oct 1;311(Pt 1):31–34. doi: 10.1042/bj3110031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Katagiri H., Asano T., Ishihara H., Inukai K., Shibasaki Y., Kikuchi M., Yazaki Y., Oka Y. Overexpression of catalytic subunit p110alpha of phosphatidylinositol 3-kinase increases glucose transport activity with translocation of glucose transporters in 3T3-L1 adipocytes. J Biol Chem. 1996 Jul 19;271(29):16987–16990. doi: 10.1074/jbc.271.29.16987. [DOI] [PubMed] [Google Scholar]
  26. Koyama H., Goodpasture C., Miller M. M., Teplitz R. L., Riggs A. D. Establishment and characterization of a cell line from the American opossum (Didelphys virginiana). In Vitro. 1978 Mar;14(3):239–246. doi: 10.1007/BF02616032. [DOI] [PubMed] [Google Scholar]
  27. Landwehr D. M., Carvalho J. S., Oken D. E. Micropuncture studies of the filtration and absorption of albumin by nephrotic rats. Kidney Int. 1977 Jan;11(1):9–17. doi: 10.1038/ki.1977.2. [DOI] [PubMed] [Google Scholar]
  28. Lewy J. E., Pesce A. Micropuncture study of albumin transfer in aminonucleoside nephrosis in the rat. Pediatr Res. 1973 Jun;7(6):553–559. doi: 10.1203/00006450-197306000-00002. [DOI] [PubMed] [Google Scholar]
  29. Li G., D'Souza-Schorey C., Barbieri M. A., Roberts R. L., Klippel A., Williams L. T., Stahl P. D. Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10207–10211. doi: 10.1073/pnas.92.22.10207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Liscovitch M., Cantley L. C. Signal transduction and membrane traffic: the PITP/phosphoinositide connection. Cell. 1995 Jun 2;81(5):659–662. doi: 10.1016/0092-8674(95)90525-1. [DOI] [PubMed] [Google Scholar]
  31. Malström K., Stange G., Murer H. Identification of proximal tubular transport functions in the established kidney cell line, OK. Biochim Biophys Acta. 1987 Aug 20;902(2):269–277. doi: 10.1016/0005-2736(87)90305-1. [DOI] [PubMed] [Google Scholar]
  32. Nakanishi S., Catt K. J., Balla T. A wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5317–5321. doi: 10.1073/pnas.92.12.5317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Oken D. E., Flamenbaum W. Micropuncture studies of proximal tubule albumin concentrations in normal and nephrotic rats. J Clin Invest. 1971 Jul;50(7):1498–1505. doi: 10.1172/JCI106635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Reaves B. J., Bright N. A., Mullock B. M., Luzio J. P. The effect of wortmannin on the localisation of lysosomal type I integral membrane glycoproteins suggests a role for phosphoinositide 3-kinase activity in regulating membrane traffic late in the endocytic pathway. J Cell Sci. 1996 Apr;109(Pt 4):749–762. doi: 10.1242/jcs.109.4.749. [DOI] [PubMed] [Google Scholar]
  35. Rodriguez-Viciana P., Warne P. H., Dhand R., Vanhaesebroeck B., Gout I., Fry M. J., Waterfield M. D., Downward J. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature. 1994 Aug 18;370(6490):527–532. doi: 10.1038/370527a0. [DOI] [PubMed] [Google Scholar]
  36. Schu P. V., Takegawa K., Fry M. J., Stack J. H., Waterfield M. D., Emr S. D. Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science. 1993 Apr 2;260(5104):88–91. doi: 10.1126/science.8385367. [DOI] [PubMed] [Google Scholar]
  37. Schwegler J. S., Heppelmann B., Mildenberger S., Silbernagl S. Receptor-mediated endocytosis of albumin in cultured opossum kidney cells: a model for proximal tubular protein reabsorption. Pflugers Arch. 1991 May;418(4):383–392. doi: 10.1007/BF00550876. [DOI] [PubMed] [Google Scholar]
  38. Shepherd P. R., Reaves B. J., Davidson H. W. Phosphoinositide 3-kinases and membrane traffic. Trends Cell Biol. 1996 Mar;6(3):92–97. doi: 10.1016/0962-8924(96)80998-6. [DOI] [PubMed] [Google Scholar]
  39. Stephens L. R., Jackson T. R., Hawkins P. T. Agonist-stimulated synthesis of phosphatidylinositol(3,4,5)-trisphosphate: a new intracellular signalling system? Biochim Biophys Acta. 1993 Oct 7;1179(1):27–75. doi: 10.1016/0167-4889(93)90072-w. [DOI] [PubMed] [Google Scholar]
  40. Stephens L., Cooke F. T., Walters R., Jackson T., Volinia S., Gout I., Waterfield M. D., Hawkins P. T. Characterization of a phosphatidylinositol-specific phosphoinositide 3-kinase from mammalian cells. Curr Biol. 1994 Mar 1;4(3):203–214. doi: 10.1016/s0960-9822(00)00049-x. [DOI] [PubMed] [Google Scholar]
  41. Stephens L., Smrcka A., Cooke F. T., Jackson T. R., Sternweis P. C., Hawkins P. T. A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein beta gamma subunits. Cell. 1994 Apr 8;77(1):83–93. doi: 10.1016/0092-8674(94)90237-2. [DOI] [PubMed] [Google Scholar]
  42. Stoyanov B., Volinia S., Hanck T., Rubio I., Loubtchenkov M., Malek D., Stoyanova S., Vanhaesebroeck B., Dhand R., Nürnberg B. Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase. Science. 1995 Aug 4;269(5224):690–693. doi: 10.1126/science.7624799. [DOI] [PubMed] [Google Scholar]
  43. Thomas M. E., Schreiner G. F. Contribution of proteinuria to progressive renal injury: consequences of tubular uptake of fatty acid bearing albumin. Am J Nephrol. 1993;13(5):385–398. doi: 10.1159/000168653. [DOI] [PubMed] [Google Scholar]
  44. Tiruppathi C., Song W., Bergenfeldt M., Sass P., Malik A. B. Gp60 activation mediates albumin transcytosis in endothelial cells by tyrosine kinase-dependent pathway. J Biol Chem. 1997 Oct 10;272(41):25968–25975. doi: 10.1074/jbc.272.41.25968. [DOI] [PubMed] [Google Scholar]
  45. Toker A., Cantley L. C. Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature. 1997 Jun 12;387(6634):673–676. doi: 10.1038/42648. [DOI] [PubMed] [Google Scholar]
  46. Tsakiridis T., Taha C., Grinstein S., Klip A. Insulin activates a p21-activated kinase in muscle cells via phosphatidylinositol 3-kinase. J Biol Chem. 1996 Aug 16;271(33):19664–19667. doi: 10.1074/jbc.271.33.19664. [DOI] [PubMed] [Google Scholar]
  47. Valentich J. D. Morphological similarities between the dog kidney cell line MDCK and the mammalian cortical collecting tubule. Ann N Y Acad Sci. 1981;372:384–405. doi: 10.1111/j.1749-6632.1981.tb15490.x. [DOI] [PubMed] [Google Scholar]
  48. Vlahos C. J., Matter W. F., Hui K. Y., Brown R. F. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem. 1994 Feb 18;269(7):5241–5248. [PubMed] [Google Scholar]
  49. Volinia S., Dhand R., Vanhaesebroeck B., MacDougall L. K., Stein R., Zvelebil M. J., Domin J., Panaretou C., Waterfield M. D. A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system. EMBO J. 1995 Jul 17;14(14):3339–3348. doi: 10.1002/j.1460-2075.1995.tb07340.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wall D. A., Maack T. Endocytic uptake, transport, and catabolism of proteins by epithelial cells. Am J Physiol. 1985 Jan;248(1 Pt 1):C12–C20. doi: 10.1152/ajpcell.1985.248.1.C12. [DOI] [PubMed] [Google Scholar]
  51. Yao R., Cooper G. M. Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science. 1995 Mar 31;267(5206):2003–2006. doi: 10.1126/science.7701324. [DOI] [PubMed] [Google Scholar]
  52. Zoellner H., Höfler M., Beckmann R., Hufnagl P., Vanyek E., Bielek E., Wojta J., Fabry A., Lockie S., Binder B. R. Serum albumin is a specific inhibitor of apoptosis in human endothelial cells. J Cell Sci. 1996 Oct;109(Pt 10):2571–2580. doi: 10.1242/jcs.109.10.2571. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES