Purinergic Signalling

, Volume 14, Issue 2, pp 167–176 | Cite as

P2X7 receptor and klotho expressions in diabetic nephropathy progression

  • A. M. Rodrigues
  • R. S. Serralha
  • C. Farias
  • G. R. Punaro
  • M. J. S. Fernandes
  • Elisa Mieko Suemitsu Higa
Original Article


Diabetes mellitus is characterized by increased levels of reactive oxygen species (ROS), leading to high levels of adenosine triphosphate (ATP) and the activation of purinergic receptors (P2X7), which results in cell death. Klotho was recently described as a modulator of oxidative stress and as having anti-apoptotic properties, among others. However, the roles of P2X7 and klotho in the progression of diabetic nephropathy are still unclear. In this context, the aim of the present study was to characterize P2X7 and klotho in several stages of diabetes in rats. Diabetes was induced in Wistar rats by streptozotocin, while the control group rats received the drug vehicle. From the 1st to 8th weeks after the diabetes induction, the animals were placed in metabolic cages on the 1st day of each week for 24 h to analyze metabolic parameters and for the urine collection. Then, blood samples and the kidneys were collected for biochemical analysis, including Western blotting and qPCR for P2X7 and klotho. Diabetic rats presented a progressive loss of renal function, with reduced nitric oxide and increased lipid peroxidation. The P2X7 and klotho expressions were similar up to the 4th week; then, P2X7 expression increased in diabetes mellitus (DM), but klotho expression presented an opposite behavior, until the 8th week. Our data show an inverse correlation between P2X7 and klotho expressions through the development of DM, which suggests that the management of these molecules could be useful for controlling the progression of this disease and diabetic nephropathy.


Diabetes mellitus Oxidative stress Purinergic receptor Klotho Kidneys 



The authors acknowledge Margaret G Mouro and Deyse Y Lima for their technical assistance and Professor Sergio R R Araujo for the histological analysis. This study was supported by Coordenaçao de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES) and Fundaçao de Apoio a Pesquisa da UNIFESP (FAP).

Compliance with ethical standards

Conflicts of interest

A. M. Rodrigues declares that he has no conflict of interest.

R. S. Serralha declares that he has no conflict of interest.

C. Farias declares that she has no conflict of interest.

G. R. Punaro declares that she has no conflict of interest.

M. J. S. Fernandes declares that she has no conflict of interest.

Elisa Mieko Suemitsu Higa declares that she has no conflict of interest.

Ethical approval

The protocol was approved by the Ethics Committee in Research of Universidade Federal de Sao Paulo under protocol #2056100314.


  1. 1.
    Burnstock G (1978) A basis for distinguishing two types of purinergic receptor. In: Straub RW, Bolis CL (eds) Cell membrane receptors for drugs and hormones: a multidisciplinary approach. Raven Press, New York, pp 107–118Google Scholar
  2. 2.
    Kennedy C, Burnstock G (1985) Evidence for two types of P2-purinoceptor in longitudinal muscle of the rabbit portal vein. Eur J Pharmacol 111:49–56CrossRefPubMedGoogle Scholar
  3. 3.
    Kaczmarek-Hajek K, Lorinczi E, Hausmann R et al (2012) Molecular and functional properties of P2X receptors—recent progress and persisting challenges. Purinergic Signalling 8:375–417CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Surprenant A, Rassendren F, Kawashima E, North RA, Buell G (1996) The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science 272:735–738CrossRefPubMedGoogle Scholar
  5. 5.
    Bjaelde RG, Arnadottir SS, Overgaard MT et al (2013) Renal epithelial cells can release ATP by vesicular fusion. Front Physiol 4:238CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Solini A, Usuelli V, Fiorina P (2014) The dark side of extracellular ATP in kidney diseases. J Am Soc Nephrol 26:1007–1016CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Kwak SH, Park KS, Lee KU, Lee HK (2010) Mitochondrial metabolism and diabetes. J Diabetes Investig 1:161–169CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Rucker B, Abreu-Vieira G, Bischoff LB et al (2010) The nucleotide hydrolysis is altered in blood serum of streptozotocin-induced diabetic rats. Arch Physiol Biochem 116:79–87CrossRefPubMedGoogle Scholar
  9. 9.
    Payne BA, Chinnery PF (2015) Mitochondrial dysfunction in aging: much progress but many unresolved questions. Biochim Biophys Acta 1847:1347–1353CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Gross JL, De Azevedo MJ, Silveiro SP et al (2005) Diabetic nephropathy: diagnosis, prevention, and treatment. Diabetes Care 28:164–176CrossRefPubMedGoogle Scholar
  11. 11.
    Burnstock G, Novak I (2013) Purinergic signalling and diabetes. Purinergic Signalling 9:307–324CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Hansen PB, Schnermann J (2003) Vasoconstrictor and vasodilator effects of adenosine in the kidney. Am J Physiol 285:F590–F599Google Scholar
  13. 13.
    Sallstrom J, Carlsson PO, Fredholm BB et al (2007) Diabetes-induced hyperfiltration in adenosine A(1)-receptor deficient mice lacking the tubuloglomerular feedback mechanism. Acta Physiol (Oxford, England) 190:253–259CrossRefGoogle Scholar
  14. 14.
    Kretschmar C, Oyarzun C, Villablanca C et al (2016) Reduced adenosine uptake and its contribution to signaling that mediates profibrotic activation in renal tubular epithelial cells: implication in diabetic nephropathy. PLoS One 11:e0147430CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Chen K, Zhang J, Zhang W, Zhang J, Yang J, Li K, He Y (2013) ATP-P2X4 signaling mediates NLRP3 inflammasome activation: a novel pathway of diabetic nephropathy. Int J Biochem Cell Biol 45:932–943CrossRefPubMedGoogle Scholar
  16. 16.
    Menzies RI, Booth JWR, Mullins JJ, Bailey MA, Tam FWK, Norman JT, Unwin RJ (2017) Hyperglycemia-induced renal P2X7 receptor activation enhances diabetes-related injury. EBioMedicine 19:73–83CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Kuro-O M (2010) Klotho. Pflugers Arch 459:333–343CrossRefPubMedGoogle Scholar
  18. 18.
    Asai O, Nakatani K, Tanaka T, Sakan H, Imura A, Yoshimoto S, Samejima KI, Yamaguchi Y, Matsui M, Akai Y, Konishi N, Iwano M, Nabeshima Y, Saito Y (2012) Decreased renal alpha-klotho expression in early diabetic nephropathy in humans and mice and its possible role in urinary calcium excretion. Kidney Int 81:539–547CrossRefPubMedGoogle Scholar
  19. 19.
    Maltese G, Fountoulakis N, Siow RC, Gnudi L, Karalliedde J (2017) Perturbations of the anti-ageing hormone klotho in patients with type 1 diabetes and microalbuminuria. Diabetologia 60:911–914CrossRefPubMedGoogle Scholar
  20. 20.
    Zhao Y, Banerjee S, Dey N, LeJeune WS, Sarkar PS, Brobey R, Rosenblatt KP, Tilton RG, Choudhary S (2011) Klotho depletion contributes to increased inflammation in kidney of the db/db mouse model of diabetes via RelA (serine)536 phosphorylation. Diabetes 60:1907–1916CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Huang CL, Moe OW (2011) Klotho: a novel regulator of calcium and phosphorus homeostasis. Pflugers Arch 462:185–193CrossRefPubMedGoogle Scholar
  22. 22.
    Chang Q, Hoefs S, Van Der Kemp AW et al (2005) The beta-glucuronidase klotho hydrolyzes and activates the TRPV5 channel. Science 310:490–493CrossRefPubMedGoogle Scholar
  23. 23.
    Yamamoto M, Clark JD, Pastor JV, Gurnani P, Nandi A, Kurosu H, Miyoshi M, Ogawa Y, Castrillon DH, Rosenblatt KP, Kuro-o M (2005) Regulation of oxidative stress by the anti-aging hormone klotho. J Biol Chem 280:38029–38034CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Saito Y, Yamagishi T, Nakamura T, Ohyama Y, Aizawa H, Suga T, Matsumura Y, Masuda H, Kurabayashi M, Kuro-o M, Nabeshima YI, Nagai R (1998) Klotho protein protects against endothelial dysfunction. Biochem Biophys Res Commun 248:324–329CrossRefPubMedGoogle Scholar
  25. 25.
    Vergani A, Fotino C, D'addio F et al (2013) Effect of the purinergic inhibitor oxidized ATP in a model of islet allograft rejection. Diabetes 62:1665–1675CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Vergani A, Tezza S, Fotino C, Visner G, Pileggi A, Chandraker A, Fiorina P (2014) The purinergic system in allotransplantation. Am J Transplant 14:507–514CrossRefPubMedGoogle Scholar
  27. 27.
    Rodrigues AM, Bergamaschi CT, Fernandes MJ et al (2014) P2x(7) receptor in the kidneys of diabetic rats submitted to aerobic training or to N-acetylcysteine supplementation. PLoS One 9:e97452CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Ochodnicky P, De Zeeuw D, Henning RH et al (2006) Endothelial function predicts the development of renal damage after combined nephrectomy and myocardial infarction. J Am Soc Nephrol 17:S49–S52CrossRefPubMedGoogle Scholar
  29. 29.
    Hampl V, Walters CL, Archer SL (1996) Determination of nitric oxide by the chemiluminescence reaction with ozone. In: Feelisch M, Stamler JS (eds) Methods in nitric oxide research. Wiley, Chichester, pp 310–318Google Scholar
  30. 30.
    Bernheim F, Bernheim ML, Wilbur KM (1948) The reaction between thiobarbituric acid and the oxidation products of certain lipides. J Biol Chem 174:257–264PubMedGoogle Scholar
  31. 31.
    Shimizu MH, Danilovic A, Andrade L et al (2008) N-acetylcysteine protects against renal injury following bilateral ureteral obstruction. Nephrol Dial Transplant 23:3067–3073CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  33. 33.
    Idf (2012) IDF diabetes atlas. In: The global burden. International Diabetes Federation, BrusselsGoogle Scholar
  34. 34.
    Hu MC, Kuro-O M, Moe OW (2010) Klotho and kidney disease. J Nephrol 23(Suppl 16):S136–S144PubMedPubMedCentralGoogle Scholar
  35. 35.
    Kim JH, Xie J, Hwang KH et al (2016) Klotho may ameliorate proteinuria by targeting TRPC6 channels in podocytes. J Am Soc Nephrol 28:140–151CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Maltese G, Psefteli PM, Rizzo B et al (2016) The anti-ageing hormone klotho induces Nrf2-mediated antioxidant defences in human aortic smooth muscle cells. J Cell Mol Med 21:621–627CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Misra S (2016) Explaining common symptoms. In: Reversing diabetes—the high 5 way. Educreation Publishing, New Delhi, p 41–46Google Scholar
  38. 38.
    Unger Rh Fd (1992) Diabetes mellitus. In: Wilson JD FD (ed) Williams textbook of endocrinology. WB Saunders Company, Philadelphia, p 1255–1355Google Scholar
  39. 39.
    Fakhruddin S, Alanazi W, Jackson KE (2017) Diabetes-induced reactive oxygen species: mechanism of their generation and role in renal injury. J Diabetes Res 2017:8379327CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    De Vriese AS, Verbeuren TJ, Van De Voorde J et al (2000) Endothelial dysfunction in diabetes. Br J Pharmacol 130:963–974CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Walsh ME, Shi Y, Van Remmen H (2014) The effects of dietary restriction on oxidative stress in rodents. Free Radic Biol Med 66:88–99CrossRefPubMedGoogle Scholar
  42. 42.
    Masoro EJ (2000) Caloric restriction and aging: an update. Exp Gerontol 35:299–305CrossRefPubMedGoogle Scholar
  43. 43.
    Guzik TJ, West NE, Pillai R et al (2002) Nitric oxide modulates superoxide release and peroxynitrite formation in human blood vessels. Hypertension 39:1088–1094CrossRefPubMedGoogle Scholar
  44. 44.
    Habib S, Ali A (2011) Biochemistry of nitric oxide. Indian J Clin Biochem 26:3–17CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Miwa S, Muller FL, Beckman KB (2008) The basics of oxidative biochemistry. In: Miwa S (ed) Aging medicine: oxidative stress in aging: from model systems to human diseases. Humana Press, New Jersey, pp 11–38Google Scholar
  46. 46.
    Capiotti KM, Siebel AM, Kist LW, Bogo MR, Bonan CD, da Silva RS (2016) Hyperglycemia alters E-NTPDases, ecto-5′-nucleotidase, and ectosolic and cytosolic adenosine deaminase activities and expression from encephala of adult zebrafish (Danio rerio). Purinergic Signalling 12:211–220CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Bian A, Xing C, Hu MC (2014) Alpha klotho and phosphate homeostasis. J Endocrinol Investig 37:1121–112648Google Scholar
  48. 48.
    Lichtman Ma, Miller Dr, Cohen J et al. (1971) Reduced red cell glycolysis, 2, 3-diphosphoglycerate and adenosine triphosphate concentration, and increased hemoglobin-oxygen affinity caused by hypophosphatemia. Ann of intern med 74:562–568Google Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • A. M. Rodrigues
    • 1
    • 2
  • R. S. Serralha
    • 2
    • 3
  • C. Farias
    • 2
    • 3
  • G. R. Punaro
    • 2
    • 3
  • M. J. S. Fernandes
    • 4
  • Elisa Mieko Suemitsu Higa
    • 1
    • 2
    • 3
    • 5
  1. 1.Translational MedicineUniversidade Federal de Sao PauloSao PauloBrazil
  2. 2.Laboratory of Nitric Oxide and Oxidative StressUniversidade Federal de Sao PauloSao PauloBrazil
  3. 3.NephrologyUniversidade Federal de Sao PauloSao PauloBrazil
  4. 4.Neurology and NeurosurgeryUniversidade Federal de Sao PauloSao PauloBrazil
  5. 5.Department of MedicineUniversidade Federal de Sao PauloSao PauloBrazil

Personalised recommendations