Laboratory Investigation

Kidney International (1995) 48, 1837–1845; doi:10.1038/ki.1995.482

Actin and villin compartmentation during ATP depletion and recovery in renal cultured cells

Nikola Golenhofen, R Brian Doctor, Robert Bacallao and Lazaro J Mandel

Division of Physiology, Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, and Division of Nephrology and Hypertension, Department of Molecular, Cellular, and Structural Biology, Northwestern University, Chicago, Ilinois, USA

Correspondence: Dr Lazaro J Mandel, Department of Cell Biology, Box 3709, Duke University Medical Center, Durham, North Carolina 27710, USA.

Received 24 October 1994; Revised 7 June 1995; Accepted 17 July 1995.

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Abstract

Actin and villin compartmentation during ATP depletion and recovery in renal cultured cells. ATP-depletion in renal cultured cells has been used as a model for studying various cytoskeletal and functional alterations induced by renal ischemia. This communication explores the reversibility of these effects utilizing a novel method [1] that depleted ATP (ATP-D) to 2% of control within 30 minutes and caused complete recovery (REC) of ATP in one hour. Under confocal microscopy, ATP-D (30 min) caused thinning of F-actin from the microvilli, cortical region, and basal stress fibers, with the concurrent appearance of intracellular F-actin patches. These changes were more pronounced after 60 minutes of ATP-D. One hour of REC following 30 minutes of ATP-D produced complete recovery of F-actin in each region of the cell. However, after 60 minutes of ATP-D, a heterogeneous F-actin recovery pattern was observed: almost complete recovery of the apical ring and microvilli, thinned cortical actin with occasional breaks along the basolateral membrane, and a dramatic reduction in basal stress fiber density. The time course of cortical actin and actin ring disruption and recovery coincided with a drop and recovery in the transepithelial resistance and the cytoskeletal dissociation and reassociation of the Na,K-ATPase. Additionally, the microvilli retracted into the cells during ATP-D, a process that was reversed during REC. Triton extraction and confocal microscopy demonstrated that villin remained closely associated with microvillar actin during both ATP-D and REC. These distinctive regional differences in the responses of F-actin to ATP depletion and repletion in cultured renal epithelial cells may help to clarify some of the differential tubular responses to ischemia and reperfusion in the kidney.

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References

  1. Doctor RB, Bacallao R, Mandel LJ: Method for recovering ATP-content and mitochondrial function following chemical anoxia in renal cell cultures. Am J Physiol (Cell Physiol) 266:C1803–C1811, 1994
  2. Venkatachalam MA, Bernard DB, Donohoe JF, Levitsky NG: Ischemic damage and repair in the rat proximal tubule: Differences among the S1, S2 and S3 segments. Kidney Int 14:31–49, 1978 | PubMed | ISI | ChemPort |
  3. Venkatachalam MA, Jones DB, Rennke HG, Sandstrom D, Patel Y: Mechanism of proximal tubule brush border loss and regeneration following mild renal ischemia. Lab Invest 45:355, 1981 | PubMed | ISI | ChemPort |
  4. Johnston PA, Rennke H, Levinski NG: Recovery of proximal tubular function from ischemic injury. Am J Physiol 246:F159–F166, 1984 | PubMed |
  5. Kellerman PS, Clark RAF, Hoilien CA, Linas SL, Molitoris BA: The role of microfilaments in the maintenance of proximal tubule structural and functional integrity. Am J Physiol 259:F279–F285, 1990 | PubMed | ISI | ChemPort |
  6. Molitoris BA, Geerdes A, McIntosh JR: Dissociation and redistribution of Na-K-ATPase from its surface membrane actin cytoskeletal complex during cellular ATP-depletion. J Clin Inv 88:462–469, 1991
  7. Doctor RB, Bennett V, Mandel LJ: Degradation of spectrin and ankyrin in the ischemic rat kidney. Am J Physiol (Cell Physiol) 264:C1003–C1013, 1993 | PubMed | ISI | ChemPort |
  8. Molitoris BA, Falk SA, Dahl RH: Ischemic-induced loss of epithelial polarity. Role of the tight junction. J Clin Invest 84:1334–1339, 1989 | PubMed | ISI | ChemPort |
  9. Mandel LJ, Bacallao R, Zamphigi G: Uncoupling of the molecular "fence" and paracellular "gate" functions in epithelial tight junctions. Nature 361:552–555, 1993 | Article | PubMed | ISI | ChemPort |
  10. Molitoris BA, Wilson P, Schrier R, Simon F: Ischemia induces a partial loss of surface membrane polarity and accumulation of putative calcium ionophores. J Clin Invest 76:2097–2105, 1985 | PubMed | ISI | ChemPort |
  11. Molitoris BA: Ischemia-induced loss of epithelial polarity: Potential role of the actin cytoskeleton. Am J Physiol 260:F769–F778, 1991 | PubMed | ISI | ChemPort |
  12. Kellerman PS, Bogusky RT: Microfilament disruption occurs very early in ischemic proximal tubule cell injury. Kidney Int 42:869–902, 1992
  13. Canfield PE, Geerdes AM, Molitoris B: Effect of reversible ATP depletion on tight-junction integrity in LLC-PK1 cells. Am J Physiol 261:F1038–F1045, 1991 | PubMed | ISI | ChemPort |
  14. Bacallao R, Garfinkel S, Monke G, Zampighi, Mandel LJ: ATP-depletion: A novel method to study junctional properties in epithelial tissues. I. Rearrangement of the actin cytoskeleton. J Cell Sci 107:3301–3313, 1994 | PubMed | ChemPort |
  15. Mandel LJ, Doctor RB, Bacallao R: ATP depletion: A novel method to study junctional properties in epithelial tissues. II. Internalization of Na,K-ATPase and E-cadherin. J Cell Sci 107:3315–3324, 1994
  16. Bacallo R, Fine LG: Molecular events in the organization of renal tubular epithelium: From nephrogenesis to regeneration. Am J Physiol 257(26):F913–F924, 1989
  17. Garza-Quintero R, Weinberg JM, Ortega-Lopez J, Davis JA, Venkatachalam MA: Conservation of structure in ATP-depleted proximal tubules: Role of calcium, polyphosphoinositides, and glycine. Am J Physiol 265:F605–F623, 1993
  18. Amsler K, Cook JS: Linear relationship of phlorizin-binding capacity and hexose uptake during differentiation in a clone of LLC-PK1 cells. J Cell Physiol 122:254–258, 1985
  19. Gstraunthaler G, Pfaller W, Kotanko P: Biochemical characterization of renal epithelial cell cultures (LLC-PK1 and MDCK). Am J Physiol 248:F536–F544, 1985 | PubMed | ISI | ChemPort |
  20. Bacallao R, Stelzer EHK: Preservation of biological specimens for observation in a confocal fluorescence microscope and operational principles of confocal fluorescence microscopy. Meth Cell Biol 31:437–452, 1989
  21. Nelson WJ, Shore EM, Wang AZ, Hammerton RW: Identification of a membrane-cytoskeletal complex containing the cell adhesion molecule uvomurolin (E-cadherin), ankyrin and fodrin in Madin Darby canine kidney epithelial cells. J Cell Biol 110:349–357, 1990 | Article | PubMed | ISI | ChemPort |
  22. Chen J, Doctor RB, Mandel LJ: Cytoskeletal dissociation of ezrin during renal anoxia-Role in microvillar injury. Am J Physiol (Cell Physiol) 267:C784–C795, 1994 | PubMed | ISI | ChemPort |
  23. Venkatachalam MA, Patel YJ, Kreisberg JI, Weinberg JM: Energy thresholds that determine membrane integrity and injury in a renal epithelial cell line (LLC-PK1). J Clin Invest 81:745–758, 1988 | PubMed |
  24. Rodman JS, Mooseker M, Farquhar MG: Cytoskeletal proteins of the rat kidney proximal tubule brush border. Eur J Cell Biol 42:313–327, 1986
  25. Mooseker MS: Organization, chemistry and assembly of the cytoskeletal apparatus of the intestinal brush border. Annu Rev Cell Biol 1:269–293, 1985
  26. Glenney JR, Jr, Kaulfus P, Weber K: F-actin assembly modulated by villin: Ca++-dependent nucleation and capping of the barbed end. Cell 24:471–480, 1981 | Article | PubMed | ISI | ChemPort |
  27. Coluccio LM, Bretscher A: Reassociation of microvillar core proteins: Making a microvillar core in vitro. J Cell Biol 108:495–502, 1989
  28. Friederich E, Kreis TE, Louvard D: Villin-induced growth of microvilli is reversibly inhibited by cytochalasin D. J Cell Sci 105:765–775, 1993
  29. Bretscher A, Osborn M, Wehland J, Weber K: Villin associates with specific microfilamentous structures as seen by immunofluorescence microscopy on tissue sections and cells microinjected with villin. Exp Cell Research 135:213–219, 1981
  30. Franck Z, Footer M, Bretscher A: Microinjection of villin into cultured cells induces rapid and long-lasting changes in cell morphology but does not inhibit cytokinesis, cell motility, or membrane ruffling. J Cell Biol 111:2475–2485, 1990
  31. Stevenson BR, Anderson JM, Braun ID, Mooseker MS: Phosphorylation of the tight junction protein ZO-1 in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance. Biochem J 263:597–599, 1989
  32. Ojakian GK: Tumor promoter-induced changes in the permeability of epithelial tight junctions. Cell 23:95–103, 1991
  33. Citi S: Protein kinase inhibitors prevent junction dissociation induced by low extracellular calcium in MDCK epithelial cells. J Cell Biol 117:169–178, 1992 | Article | PubMed | ISI | ChemPort |
  34. Kobryn CE, Mandel LJ: Decreased protein phosphorylation induced by anoxia in proximal renal tubules. Am J Physiol 267:C1073–C1079, 1994

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