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Purinergic Signalling

, Volume 14, Issue 4, pp 485–497 | Cite as

Characterization of purinergic receptor expression in ARPKD cystic epithelia

  • Oleg Palygin
  • Daria V. Ilatovskaya
  • Vladislav Levchenko
  • Christine A. Klemens
  • Lashodya Dissanayake
  • Anna Marie Williams
  • Tengis S. PavlovEmail author
  • Alexander StaruschenkoEmail author
Original Article

Abstract

Polycystic kidney diseases (PKDs) are a group of inherited nephropathies marked by formation of fluid-filled cysts along the nephron. Growing evidence suggests that in the kidney formation of cysts and alteration of cystic electrolyte transport are associated with purinergic signaling. PCK/CrljCrl-Pkhd1pck/CRL (PCK) rat, an established model of autosomal recessive polycystic kidney disease (ARPKD), was used here to test this hypothesis. Cystic fluid of PCK rats and their cortical tissues exhibited significantly higher levels of ATP compared to Sprague Dawley rat kidney cortical interstitium as assessed by highly sensitive ATP enzymatic biosensors. Confocal calcium imaging of the freshly isolated cystic monolayers revealed a stronger response to ATP in a higher range of concentrations (above 100 μM). The removal of extracellular calcium results in the profound reduction of the ATP evoked transient, which suggests calcium entry into the cyst-lining cells is occurring via the extracellular (ionotropic) P2X channels. Further use of pharmacological agents (α,β-methylene-ATP, 5-BDBD, NF449, isoPPADS, AZ10606120) and immunofluorescent labeling of isolated cystic epithelia allowed us to narrow down potential candidate receptors. In conclusion, our ex vivo study provides direct evidence that the profile of P2 receptors is shifted in ARPKD cystic epithelia in an age-related manner towards prevalence of P2X4 and/or P2X7 receptors, which opens new avenues for the treatment of this disease.

Keywords

ATP PCK rat Intracellular calcium flux P2X receptors Kidney Polycystic kidney disease ARPKD P2rx7 P2rx4 P2X7 P2X4 Purinergic receptor 

Notes

Acknowledgements

Christine Duris (Children’s Hospital of Wisconsin) and Elena Sorokina (Department of Pediatrics, Medical College of Wisconsin) are recognized for excellent technical assistance with immunostaining and RNA isolation, respectively.

Funding information

This research was supported by the National Institute of Health grants: R35 HL135749 (to A.S.); R00 HL116603 and P30 DK090868 via Baltimore PKD Center P&F Grant (to TSP); R00 DK105160 and PKD Foundation (221G18a) award (to DVI); T32 HL134643 and CVC A.O. Smith Fellowship (to C.A.K); and American Heart Association grants: 16EIA26720006 (to A.S.) and 17SDG33660149 (to OP); and Department of Veteran Affairs I01 BX004024 (AS).

Compliance with ethical standards

Conflict of interest

Oleg Palygin declares that he/she has no conflict of interest.

Daria V. Ilatovskaya declares that he/she has no conflict of interest.

Vladislav Levchenko declares that he/she has no conflict of interest.

Christine A. Klemens declares that he/she has no conflict of interest.

Lashodya Dissanayake declares that he/she has no conflict of interest.

Anna Marie Williams declares that he/she has no conflict of interest.

Tengis S. Pavlov declares that he/she has no conflict of interest.

Alexander Staruschenko declares that he/she has no conflict of interest.

Ethical approval

Animal use and welfare adhered to the NIH Guide for the Care and Use of Laboratory Animals following a protocol reviewed and approved by the IACUC at the Medical College of Wisconsin.

References

  1. 1.
    Torres VE, Harris PC (2006) Mechanisms of disease: autosomal dominant and recessive polycystic kidney diseases. Nat Clin Pract Nephrol 2(1):40–55PubMedGoogle Scholar
  2. 2.
    Bergmann C, Senderek J, Kupper F, Schneider F, Dornia C, Windelen E, Eggermann T, Rudnik-Schoneborn S, Kirfel J, Furu L, Onuchic LF, Rossetti S, Harris PC, Somlo S, Guay-Woodford L, Germino GG, Moser M, Buttner R, Zerres K (2004) PKHD1 mutations in autosomal recessive polycystic kidney disease (ARPKD). Hum Mutat 23(5):453–463PubMedGoogle Scholar
  3. 3.
    Antignac C, Calvet JP, Germino GG, Grantham JJ, Guay-Woodford LM, Harris PC, Hildebrandt F, Peters DJM, Somlo S, Torres VE, Walz G, Zhou J, Yu ASL (2015) The future of polycystic kidney disease research—as seen by the 12 Kaplan awardees. J Am Soc Nephrol 26(9):2081–2095PubMedPubMedCentralGoogle Scholar
  4. 4.
    Craigie E, Birch RE, Unwin RJ, Wildman SS (2013) The relationship between P2X4 and P2X7: a physiologically important interaction? Front Physiol 4:216PubMedPubMedCentralGoogle Scholar
  5. 5.
    Kennedy C, Chootip K, Mitchell C, Syed NI, Tengah A (2013) P2X and P2Y nucleotide receptors as targets in cardiovascular disease. Future Med Chem 5(4):431–449PubMedGoogle Scholar
  6. 6.
    Menzies RI, Tam FW, Unwin RJ, Bailey MA (2017) Purinergic signaling in kidney disease. Kidney Int 91(2):315–323PubMedGoogle Scholar
  7. 7.
    Gidlöf O, Smith JG, Melander O, Lövkvist H, Hedblad B, Engström G, Nilsson P, Carlson J, Berglund G, Olsson S, Jood K, Jern C, Norrving B, Lindgren A, Erlinge D (2012) A common missense variant in the ATP receptor P2X7 is associated with reduced risk of cardiovascular events. PLoS One 7(5):e37491PubMedPubMedCentralGoogle Scholar
  8. 8.
    Palomino-Doza J, Rahman TJ, Avery PJ, Mayosi BM, Farrall M, Watkins H, Edwards CRW, Keavney B (2008) Ambulatory blood pressure is associated with polymorphic variation in P2X receptor genes. Hypertension 52(5):980–985PubMedGoogle Scholar
  9. 9.
    Wilson PD (2011) Apico-basal polarity in polycystic kidney disease epithelia. Biochim Biophys Acta 1812(10):1239–1248PubMedGoogle Scholar
  10. 10.
    Ilatovskaya DV, Palygin O, Levchenko V, Staruschenko A (2013) Pharmacological characterization of the P2 receptors profile in the podocytes of the freshly isolated rat glomeruli. Am J Phys Cell Phys 305(10):C1050–C1059Google Scholar
  11. 11.
    Rangan G (2013) Role of extracellular ATP and P2 receptor signaling in regulating renal cyst growth and interstitial inflammation in polycystic kidney disease. Front Physiol 4:218PubMedPubMedCentralGoogle Scholar
  12. 12.
    Ilatovskaya DV, Palygin O, Staruschenko A (2016) Functional and therapeutic importance of purinergic signaling in polycystic kidney disease. Am J Physiol Ren Physiol 311(6):F1135–F1139Google Scholar
  13. 13.
    Wilson PD, Hovater JS, Casey CC, Fortenberry JA, Schwiebert EM (1999) ATP release mechanisms in primary cultures of epithelia derived from the cysts of polycystic kidneys. J Am Soc Nephrol 10(2):218–229PubMedGoogle Scholar
  14. 14.
    Schwiebert EM, Wallace DP, Braunstein GM, King SR, Peti-Peterdi J, Hanaoka K, Guggino WB, Guay-Woodford LM, Bell PD, Sullivan LP, Grantham JJ, Taylor AL (2002) Autocrine extracellular purinergic signaling in epithelial cells derived from polycystic kidneys. Am J Physiol Ren Physiol 282(4):F763–F775Google Scholar
  15. 15.
    Vekaria RM, Unwin RJ, Shirley DG (2006) Intraluminal ATP concentrations in rat renal tubules. J Am Soc Nephrol 17(7):1841–1847PubMedGoogle Scholar
  16. 16.
    de Bruijn PIA, Bleich M, Praetorius HA, Leipziger J (2015) P2X receptors trigger intracellular alkalization in isolated perfused mouse medullary thick ascending limb. Acta Physiol 213(1):277–284Google Scholar
  17. 17.
    Guan Z, Fellner RC, Van Beusecum J, Inscho EW (2014) P2 receptors in renal autoregulation. Curr Vasc Pharmacol 12(6):818–828PubMedPubMedCentralGoogle Scholar
  18. 18.
    Birch RE, Schwiebert EM, Peppiatt-Wildman CM, Wildman SS (2013) Emerging key roles for P2X receptors in the kidney. Front Physiol 4:262PubMedPubMedCentralGoogle Scholar
  19. 19.
    Burnstock G (2012) Purinergic signalling: its unpopular beginning, its acceptance and its exciting future. BioEssays 34(3):218–225PubMedGoogle Scholar
  20. 20.
    Vallon V, Rieg T (2011) Regulation of renal NaCl and water transport by the ATP/UTP/P2Y2 receptor system. Am J Physiol Ren Physiol 301(3):F463–F475Google Scholar
  21. 21.
    Vallon V (2008) P2 receptors in the regulation of renal transport mechanisms. Am J Physiol Ren Physiol 294(1):F10–F27Google Scholar
  22. 22.
    Vallon V, Stockand J, Rieg T (2012) P2Y receptors and kidney function. Wiley Interdiscip Rev Membr Transp Signal 1(6):731–742PubMedPubMedCentralGoogle Scholar
  23. 23.
    Geyti CS, Odgaard E, Overgaard MT, Jensen MEJ, Leipziger J, Praetorius HA (2008) Slow spontaneous [Ca2+]i oscillations reflect nucleotide release from renal epithelia. Pflugers Arch 455(6):1105–1117PubMedGoogle Scholar
  24. 24.
    Mori M, Hosomi H, Nishizaki T, Kawahara K, Okada Y (1997) Calcium release from intracellular stores evoked by extracellular ATP in a Xenopus renal epithelial cell line. J Physiol 502:365–373PubMedPubMedCentralGoogle Scholar
  25. 25.
    Turner CM, Ramesh B, Srai SKS, Burnstock G, Unwin RJ (2004) Altered ATP-sensitive P2 receptor subtype expression in the Han:SPRD cy/+ rat, a model of autosomal dominant polycystic kidney disease. Cells Tissues Organs 178(3):168–179PubMedGoogle Scholar
  26. 26.
    Hillman KA, Johnson TM, Winyard PJ, Burnstock G, Unwin RJ, Woolf AS (2002) P2X(7) receptors are expressed during mouse nephrogenesis and in collecting duct cysts of the cpk/cpk mouse. Exp Nephrol 10(1):34–42PubMedGoogle Scholar
  27. 27.
    Chang M-Y, Lu J-K, Tian Y-C, Chen Y-C, Hung C-C, Huang Y-H, Chen Y-H, Wu M-S, Yang C-W, Cheng Y-C (2011) Inhibition of the P2X7 receptor reduces cystogenesis in PKD. J Am Soc Nephrol 22(9):1696–1706PubMedPubMedCentralGoogle Scholar
  28. 28.
    Zaika O, Mamenko M, Berrout J, Boukelmoune N, O'Neil RG, Pochynyuk O (2013) TRPV4 dysfunction promotes renal cystogenesis in autosomal recessive polycystic kidney disease. J Am Soc Nephrol 24(4):604–616PubMedPubMedCentralGoogle Scholar
  29. 29.
    Pavlov TS, Ilatovskaya DV, Palygin O, Levchenko V, Pochynyuk O, Staruschenko A (2015) Implementing patch clamp and live fluorescence microscopy to monitor functional properties of freshly isolated PKD epithelium. J Vis Exp 103:e53035Google Scholar
  30. 30.
    Katsuyama M, Masuyama T, Komura I, Hibino T, Takahashi H (2000) Characterization of a novel polycystic kidney rat model with accompanying polycystic liver. Exp Anim 49(1):51–55PubMedGoogle Scholar
  31. 31.
    Ward CJ, Hogan MC, Rossetti S, Walker D, Sneddon T, Wang X, Kubly V, Cunningham JM, Bacallao R, Ishibashi M, Milliner DS, Torres VE, Harris PC (2002) The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein. Nat Genet 30(3):259–269PubMedGoogle Scholar
  32. 32.
    Palygin O, Levchenko V, Ilatovskaya DV, Pavlov TS, Ryan RP, Cowley AW Jr, Staruschenko A (2013) Real-time electrochemical detection of ATP and H2O2 release in freshly isolated kidneys. Am J Physiol Ren Physiol 305(1):F134–F141Google Scholar
  33. 33.
    Palygin O, Evans LC, Cowley AW Jr, Staruschenko A (2017) Acute in vivo analysis of ATP release in rat kidneys in response to changes of renal perfusion pressure. J Am Heart Assoc 6(9):e006658PubMedPubMedCentralGoogle Scholar
  34. 34.
    Pavlov TS, Levchenko V, O'Connor PM, Ilatovskaya DV, Palygin O, Mori T, Mattson DL, Sorokin A, Lombard JH, Cowley AW Jr, Staruschenko A (2013) Deficiency of renal cortical EGF increases ENaC activity and contributes to salt-sensitive hypertension. J Am Soc Nephrol 24(7):1053–1062PubMedPubMedCentralGoogle Scholar
  35. 35.
    Palygin O, Levchenko V, Evans LC, Blass G, Cowley AW, Staruschenko A (2015) Use of enzymatic biosensors to quantify endogenous ATP or H2O2 in the kidney. J Vis Exp 104:53059Google Scholar
  36. 36.
    Ilatovskaya DV, Palygin O, Levchenko V, Staruschenko A (2015) Single-channel analysis and calcium imaging in the podocytes of the freshly isolated glomeruli. J Vis Exp 100:e52850.  https://doi.org/10.3791/52850 Google Scholar
  37. 37.
    Xing S, Grol MW, Grutter PH, Dixon SJ, Komarova SV (2016) Modeling interactions among individual P2 receptors to explain complex response patterns over a wide range of ATP concentrations. Front Physiol 7:294PubMedPubMedCentralGoogle Scholar
  38. 38.
    North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82(4):1013–1067PubMedGoogle Scholar
  39. 39.
    Coddou C, Yan Z, Obsil T, Huidobro-Toro JP, Stojilkovic SS (2011) Activation and regulation of purinergic P2X receptor channels. Pharmacol Rev 63(3):641–683PubMedPubMedCentralGoogle Scholar
  40. 40.
    Helms N, Kowalski M, Illes P, Riedel T (2013) Agonist antagonist interactions at the rapidly desensitizing P2X3 receptor. PLoS One 8(11):e79213PubMedPubMedCentralGoogle Scholar
  41. 41.
    Khakh BS, Burnstock G, Kennedy C, King BF, North RA, Seguela P, Voigt M, Humphrey PP (2001) International union of pharmacology. XXIV. Current status of the nomenclature and properties of P2X receptors and their subunits. Pharmacol Rev 53(1):107–118PubMedGoogle Scholar
  42. 42.
    Gever JR, Cockayne DA, Dillon MP, Burnstock G, Ford AP (2006) Pharmacology of P2X channels. Pflugers Arch 452(5):513–537PubMedGoogle Scholar
  43. 43.
    Jones CA, Chessell IP, Simon J, Barnard EA, Miller KJ, Michel AD, Humphrey PP (2000) Functional characterization of the P2X(4) receptor orthologues. Br J Pharmacol 129(2):388–394PubMedPubMedCentralGoogle Scholar
  44. 44.
    Allsopp RC, Dayl S, Schmid R, Evans RJ (2017) Unique residues in the ATP gated human P2X7 receptor define a novel allosteric binding pocket for the selective antagonist AZ10606120. Sci Rep 7(1):725PubMedPubMedCentralGoogle Scholar
  45. 45.
    Novak I (2011) Purinergic signalling in epithelial ion transport: regulation of secretion and absorption. Acta Physiol 202(3):501–522Google Scholar
  46. 46.
    Rieg T, Bundey RA, Chen Y, Deschenes G, Junger W, Insel PA, Vallon V (2007) Mice lacking P2Y2 receptors have salt-resistant hypertension and facilitated renal Na+ and water reabsorption. FASEB J 21(13):3717–3726PubMedGoogle Scholar
  47. 47.
    Nanami M, Pech V, Lazo-Fernandez Y, Weinstein AM, Wall SM (2015) ENaC inhibition stimulates HCl secretion in the mouse cortical collecting duct. II. Bafilomycin-sensitive H+ secretion. Am J Physiol Ren Physiol 309(3):F259–F268Google Scholar
  48. 48.
    Pavlov TS, Levchenko V, Ilatovskaya DV, Palygin O, Staruschenko A (2015) Impaired epithelial Na+ channel activity contributes to cystogenesis and development of autosomal recessive polycystic kidney disease in PCK rats. Pediatr Res 77(1–1):64–69PubMedGoogle Scholar
  49. 49.
    Hooper KM, Unwin RJ, Sutters M (2003) The isolated C-terminus of polycystin-1 promotes increased ATP-stimulated chloride secretion in a collecting duct cell line. Clin Sci 104(3):217–221PubMedGoogle Scholar
  50. 50.
    Wildman SS, Hooper KM, Turner CM, Sham JS, Lakatta EG, King BF, Unwin RJ, Sutters M (2003) The isolated polycystin-1 cytoplasmic COOH terminus prolongs ATP-stimulated Cl- conductance through increased Ca2+ entry. Am J Physiol Ren Physiol 285(6):F1168–F1178Google Scholar
  51. 51.
    Buchholz B, Teschemacher B, Schley G, Schillers H, Eckardt KU (2011) Formation of cysts by principal-like MDCK cells depends on the synergy of cAMP- and ATP-mediated fluid secretion. J Mol Med 89(3):251–261PubMedGoogle Scholar
  52. 52.
    Xu C, Shmukler BE, Nishimura K, Kaczmarek E, Rossetti S, Harris PC, Wandinger-Ness A, Bacallao RL, Alper SL (2009) Attenuated, flow-induced ATP release contributes to absence of flow-sensitive, purinergic Cai2+ signaling in human ADPKD cyst epithelial cells. Am J Physiol Ren Physiol 296(6):F1464–F1476Google Scholar
  53. 53.
    Kraus A, Grampp S, Goppelt-Struebe M, Schreiber R, Kunzelmann K, Peters DJ, Leipziger J, Schley G, Schodel J, Eckardt KU, Buchholz B (2016) P2Y2R is a direct target of HIF-1alpha and mediates secretion-dependent cyst growth of renal cyst-forming epithelial cells. Purinergic Signal 12(4):687–695PubMedPubMedCentralGoogle Scholar
  54. 54.
    Odgaard E, Praetorius HA, Leipziger J (2009) AVP-stimulated nucleotide secretion in perfused mouse medullary thick ascending limb and cortical collecting duct. Am J Physiol Ren Physiol 297(2):F341–F349Google Scholar
  55. 55.
    Pochynyuk O, Bugaj V, Rieg T, Insel PA, Mironova E, Vallon V, Stockand JD (2008) Paracrine regulation of the epithelial Na+ channel in the mammalian collecting duct by purinergic P2Y2 receptor tone. J Biol Chem 283(52):36599–36607PubMedPubMedCentralGoogle Scholar
  56. 56.
    Zsembery A, Boyce AT, Liang L, Peti-Peterdi J, Bell PD, Schwiebert EM (2003) Sustained calcium entry through P2X nucleotide receptor channels in human airway epithelial cells. J Biol Chem 278(15):13398–13408PubMedGoogle Scholar
  57. 57.
    Zhang Y, Sanchez D, Gorelik J, Klenerman D, Lab M, Edwards C, Korchev Y (2007) Basolateral P2X4-like receptors regulate the extracellular ATP-stimulated epithelial Na+ channel activity in renal epithelia. Am J Physiol Ren Physiol 292(6):F1734–F1740Google Scholar
  58. 58.
    Hillman KA, Woolf AS, Johnson TM, Wade A, Unwin RJ, Winyard PJD (2004) The P2X7 ATP receptor modulates renal cyst development in vitro. Biochem Biophys Res Commun 322(2):434–439PubMedGoogle Scholar
  59. 59.
    Pandit MM, Inscho EW, Zhang S, Seki T, Rohatgi R, Gusella L, Kishore B, Kohan DE (2015) Flow regulation of endothelin-1 production in the inner medullary collecting duct. Am J Physiol Ren Physiol 308(6):F541–F552Google Scholar
  60. 60.
    Kohan DE, Inscho EW, Wesson D, Pollock DM (2011) Physiology of endothelin and the kidney. Compreh Physiol 2:883–919Google Scholar
  61. 61.
    Pavlov TS, Chahdi A, Ilatovskaya DV, Levchenko V, Vandewalle A, Pochynyuk O, Sorokin A, Staruschenko A (2010) Endothelin-1 inhibits the epithelial Na+ channel through betaPix/14-3-3/Nedd4-2. J Am Soc Nephrol 21(5):833–843PubMedPubMedCentralGoogle Scholar
  62. 62.
    Kim MJ, Turner CM, Hewitt R, Smith J, Bhangal G, Pusey CD, Unwin RJ, Tam FW (2014) Exaggerated renal fibrosis in P2X4 receptor-deficient mice following unilateral ureteric obstruction. Nephrol Dial Transplant 29(7):1350–1361PubMedPubMedCentralGoogle Scholar
  63. 63.
    Guo C, Masin M, Qureshi OS, Murrell-Lagnado RD (2007) Evidence for functional P2X4/P2X7 heteromeric receptors. Mol Pharmacol 72(6):1447–1456PubMedGoogle Scholar

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© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Department of PhysiologyMedical College of WisconsinMilwaukeeUSA
  2. 2.Division of Nephrology, Department of MedicineMedical University of South CarolinaCharlestonUSA
  3. 3.Division of Hypertension and Vascular ResearchHenry Ford HospitalDetroitUSA

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