Urological Research

, Volume 36, Issue 1, pp 1–10 | Cite as

Oxalate exposure provokes HSP 70 response in LLC-PK1 cells, a line of renal epithelial cells: protective role of HSP 70 against oxalate toxicity

  • Sweaty Koul
  • Meiyi Huang
  • Sidarth Bhat
  • Paul Maroni
  • Randall B. Meacham
  • Hari K. KoulEmail author
Original Paper


We investigated the effects of oxalate on immediate early genes (IEGs) and stress protein HSP 70, commonly induced genes in response to a variety of stresses. LLC-PK1 cells were exposed to oxalate. Gene transcription and translation were monitored by Northern and Western blot analysis. RNA and DNA synthesis were assessed by [3H]-uridine and [3H]-thymidine incorporation, respectively. Oxalate exposure selectively increased the levels of mRNA encoding IEGs c-myc and c-jun as well as stress protein HSP 70. While expression of c-myc and c-jun was rapid (within 15 min to 2 h) and transient, HSP 70 expression was delayed (∼8 h) and stable. Furthermore, oxalate exposure resulted in delayed induction of generalized transcription by 18 h and reinitiation of the DNA synthesis by 24 h of oxalate exposure. Moreover, we show that prior induction of HSP 70 by mild hypertonic exposure protected the cells from oxalate toxicity. To the best of our knowledge this is the first study to demonstrate rapid IEG response and delayed heat-shock response to oxalate toxicity and protective role of HSP 70 against oxalate toxicity to renal epithelial cells. Oxalate, a metabolic end product, induces IEGs c-myc and c-jun and a delayed HSP 70 expression; While IEG expression may regulate additional genetic responses to oxalate, increased HSP 70 expression would serve an early protective role during oxalate stress.


LLC-PK1 cells Immediate early genes Heat shock protein Nephrolithiasis Oxalate toxicity Gene expression 



This work was supported in part by research grant to Dr. Koul from National Institutes of Health (NIH-DK-RO1-54084).


  1. 1.
    Ananthan J, Goldberg AL, Voellmy R (1986) Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science 232:522–524PubMedCrossRefGoogle Scholar
  2. 2.
    Ang D, Liberek K, Skowyra D, Zylicz M, Georgopoulos C (1991) Biological role and regulation of the universally conserved heat shock proteins. J Biol Chem 266:24233–24236PubMedGoogle Scholar
  3. 3.
    Baler R, Welch WJ, Voellmy R (1992) Heat shock gene regulation by nascent polypeptides and denatured proteins: hsp70 as a potential autoregulatory factor. J Cell Biol 117:1151–1159PubMedCrossRefGoogle Scholar
  4. 4.
    Beckmann RP, Mizzen LE, Welch WJ (1990) Interaction of Hsp 70 with newly synthesized proteins: implications for protein folding and assembly. Science 248:850–854PubMedCrossRefGoogle Scholar
  5. 5.
    Capasso JM, Rivard JC, Berl T (2001) Long-term adaptation of renal cells to hypertonicity: role of MAP kinases and Na-K-ATPase. Am J Physiol Renal Physiol 280(5):F768–F776PubMedGoogle Scholar
  6. 6.
    Cajone F, Salina M, Bernelli-Zazzera A (1988) C-myc gene expression in heat-adapted and heat-shocked cells. Cell Biol Int Rep 12:549–553PubMedCrossRefGoogle Scholar
  7. 7.
    Chaturvedi L, Koul S, Sekhon A, Bhandari A, Menon M, Koul H (2002) Oxalate selectively activates p38 mitogen-activated protein kinase and c-Jun N-terminal kinase signal transduction pathways in renal epithelial cells. J Biol Chem 277(15):13321–13330PubMedCrossRefGoogle Scholar
  8. 8.
    Chomczynski P, Sacchi N (1994) Guanidine methods for total RNA preparation. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current protocol in molecular biology. Wiley, New York, pp 4.2.1–4.2.2Google Scholar
  9. 9.
    Cohen DM, Wasserman JC, Gullans SR (1991) Immediate early gene and HSP70 expression in hyperosmotic stress in MDCK cells. Am J Physiol 261:C594–C601PubMedGoogle Scholar
  10. 10.
    Cohen DM, Gullans SR (1993) Urea selectively induces DNA synthesis in renal epithelial cells. Am J Physiol 264:F601–F607PubMedGoogle Scholar
  11. 11.
    Hackett RL, Khan SR (1988) Presence of calcium oxalate crystals in the mammalian thyroid gland. Scanning Microsc 2:241–246PubMedGoogle Scholar
  12. 12.
    Hammes MS, Lieske JC, Pawar S, Spargo BH, Toback FG (1995) Calcium oxalate monohydrate crystals stimulate gene expression in renal epithelial cells. Kidney Int 48:501–509PubMedCrossRefGoogle Scholar
  13. 13.
    Hightower LE (1991) Heat shock, stress proteins, chaperones, and proteotoxicity. Cell 66:191–197PubMedCrossRefGoogle Scholar
  14. 14.
    Koul H, Huang M (2003) Oxalate exposure provokes the immediate early genes (c-myc and c-jun) and HSP70 response in LLC-PK1 cells, a line of renal epithelial cells. J Am Soc Nephrol 14:abstract# SO-PO760Google Scholar
  15. 15.
    Huang MY, Chaturvedi LS, Koul S, Koul HK (2005) Oxalate stimulates IL-6 production in HK-2 cells, a line of human renal proximal tubular epithelial cells. Kidney Int 68(2):497–503PubMedCrossRefGoogle Scholar
  16. 16.
    Iida S, Peck AB, Byer KJ, Khan SR (1999) Expression of bikunin mRNA in renal epithelial cells after oxalate exposure. J Urol 162:1480–1486PubMedCrossRefGoogle Scholar
  17. 17.
    Johnson PF, McKnight SL (1989) Eukaryotic transcriptional regulatory proteins. Annu Rev Biochem 58:799–839PubMedCrossRefGoogle Scholar
  18. 18.
    Kao HT, Capasso O, Heintz N, Nevins JR (1985) Cell cycle control of the human HSP70 gene: implications for the role of a cellular E1A-like function. Mol Cell Biol 5:628–633PubMedGoogle Scholar
  19. 19.
    Kelly K, Cochran BH, Stiles CD, Leder P (1983) Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell 35:603–610PubMedCrossRefGoogle Scholar
  20. 20.
    Khan SR (1995) Calcium oxalate crystal interaction with renal tubular epithelium, mechanism of crystal adhesion and its impact on stone development. Urol Res 23:71–79PubMedCrossRefGoogle Scholar
  21. 21.
    Khan SR, Thamilselvan S (2000) Nephrolithiasis: a consequence of renal epithelial cell exposure to oxalate and calcium oxalate crystals. Mol Urol 4:305–312PubMedGoogle Scholar
  22. 22.
    Kingston RE, Baldwin AS Jr, Sharp PA (1984) Regulation of heat shock protein 70 gene expression by c-myc. Nature 312:280–282PubMedCrossRefGoogle Scholar
  23. 23.
    Knight TF, Senekjian HO, Taylor K, Steplock DA, Weinman EJ (1979) Renal transport of oxalate: effects of diuretics, uric acid, and calcium. Kidney Int 16:572–576PubMedCrossRefGoogle Scholar
  24. 24.
    Kohjimoto Y, Honeyman TW, Jonassen J, Gravel K, Kennington L, Scheid CR (2000) Phospholipase A2 mediates immediate early genes in cultured renal epithelial cells: possible role of lysophospholipid. Kidney Int 58:638–646PubMedCrossRefGoogle Scholar
  25. 25.
    Koul H, Menon M, Scheid C (1996) Oxalate and renal tubular cells: a complex interaction. Ital J Miner Electrolyte Metab 10:67–74Google Scholar
  26. 26.
    Koul H (1999) Idiopathic calcium oxalate urolithiasis: Y2K update. In: Proceedings of the international CME in urology, pp 89–97Google Scholar
  27. 27.
    Koul H, Ebisuno S, Renzulli L, Yanagawa M, Menon M, Scheid C (1994) Polarized distribution of oxalate transport systems in LLC-PK1 cells, a line of renal epithelial cells. Am J Physiol 266:F266–F274PubMedGoogle Scholar
  28. 28.
    Koul H, Kennington L, Nair G, Honeyman T, Menon M, Scheid C (1994) Oxalate-induced initiation of DNA synthesis in LLC-PK1 cells, a line of renal epithelial cells. Biochem Biophys Res Commun 205:1632–1637PubMedCrossRefGoogle Scholar
  29. 29.
    Koul H, Menon M, Chaturvedi L, Koul S, Sekhon A, Bhandari A, Haung M (2002) Activation of the p38-MAP kinase signal transduction pathways by COM-crystals. J Biol Chem 277(39):36845–36852PubMedCrossRefGoogle Scholar
  30. 30.
    Koul S, Chaturved LS, Sekhon A, Bhandari A, Menon M, Koul HK (2002) Effect of oxalate on the re-initiation of DNA synthesis in LLC-PK1 cells do not involve p42/44 MAP kinase activation. Kidney Int 61:525–533PubMedCrossRefGoogle Scholar
  31. 31.
    Kuo SM, Aronson PS (1996) Pathways for oxalate transport in rabbit renal microvillus membrane vesicles. J Biol Chem 271:15491–15497PubMedCrossRefGoogle Scholar
  32. 32.
    Lieske JC, Hammes MS, Hoyer JR, Toback FG (1997) Renal cell osteopontin production is stimulated by calcium oxalate monohydrate crystals. Kidney Int 51:679–686PubMedCrossRefGoogle Scholar
  33. 33.
    Lindquist S, Craig EA (1988) The heat-shock proteins. Annu Rev Genet 22:631–677PubMedCrossRefGoogle Scholar
  34. 34.
    Milarski KL, Morimoto RI (1986) Expression of human HSP70 during the synthetic phase of the cell cycle. Proc Natl Acad Sci USA 83:9517–9521PubMedCrossRefGoogle Scholar
  35. 35.
    Misfeldt DS, Sanders MJ (1981) Transepithelial transport in cell culture: d-glucose transport by a pig kidney cell line (LLC-PK1). J Membr Biol 59:13–18PubMedCrossRefGoogle Scholar
  36. 36.
    Ono K, Yasukohchi A, Kikawa K (1987) Pathogenesis of acquired renal cysts in hemodialysis patients. The role of oxalate crystal deposition in renal tubules. ASAIO Trans 33:245–249PubMedGoogle Scholar
  37. 37.
    Radi MJ (1989) Calcium oxalate crystals in breast biopsies. An overlooked form of microcalcification associated with benign breast disease. Arch Pathol Lab Med 113:1367–1369PubMedGoogle Scholar
  38. 38.
    Rangnekar VM, Aplin AC, Sukhatme VP (1990) The serum and TPA responsive promoter and intron–exon structure of EGR2, a human early growth response gene encoding a zinc finger protein. Nucleic Acids Res 18:2749–2757PubMedCrossRefGoogle Scholar
  39. 39.
    Robertson WG, Peacock M, Heyburn PJ, Marshall RW, Rutherford A, Williams RE, Clark PB (1979) The significance of mild hyperoxaluria in calcium stone-formation. In: Rose GA, Robertson WG, Watts REW (eds) Oxalate in human biochemistry and clinical pathology. The Welcome Foundation Ltd, London, p 173Google Scholar
  40. 40.
    Ryseck RP, Hirai SI, Yaniv M, Bravo R (1988) Transcriptional activation of c-jun during the G0/G1 transition in mouse fibroblasts. Nature 334:535–537PubMedCrossRefGoogle Scholar
  41. 41.
    Scheid C, Koul H, Hill WA, Luber-Narod J, Kennington L, Honeyman T, Jonassen J, Menon M (1996) Oxalate toxicity in LLC-PK1 cells: role of free radicals. Kidney Int 49:413–419PubMedCrossRefGoogle Scholar
  42. 42.
    Sheikh-Hamad D, Garcia-Perez A, Ferraris JD, Peters EM, Burg MB (1994) Induction of gene expression by heat shock versus osmotic stress. Am J Physiol 267:F28–F34PubMedGoogle Scholar
  43. 43.
    Sorger PK (1991) Heat shock factor and the heat shock response. Cell 65:363–366PubMedCrossRefGoogle Scholar
  44. 44.
    Truong LD, Cartwright J Jr, Alpert L (1992) Calcium oxalate in breast lesions biopsied for calcification detected in screening mammography: incidence and clinical significance. Mod Pathol 5:146–152PubMedGoogle Scholar
  45. 45.
    Welch WJ (1991) The role of heat-shock proteins as molecular chaperones. Curr Opin Cell Biol 3:1033–1038PubMedCrossRefGoogle Scholar
  46. 46.
    Welch WJ, Kang HS, Beckmann RP, Mizzen LA (1991) Response of mammalian cells to metabolic stress; changes in cell physiology and structure/function of stress proteins. Curr Top Microbiol Immunol 167:31–55PubMedGoogle Scholar
  47. 47.
    Wu BJ, Morimoto RI (1985) Transcription of the human hsp70 gene is induced by serum stimulation. Proc Natl Acad Sci USA 82:6070–6074PubMedCrossRefGoogle Scholar
  48. 48.
    Wu BJ, Kingston RE, Morimoto RI (1986) Human HSP70 promoter contains at least two distinct regulatory domains. Proc Natl Acad Sci USA 83:629–633PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Sweaty Koul
    • 1
  • Meiyi Huang
    • 1
  • Sidarth Bhat
    • 1
  • Paul Maroni
    • 1
  • Randall B. Meacham
    • 1
  • Hari K. Koul
    • 1
    Email author
  1. 1.Signal Transduction and Molecular Urology Laboratory, Program in Urosciences, Division of Urology, Department of SurgeryUniversity of Colorado, School of MedicineDenverUSA

Personalised recommendations