Abstract
CDK inhibitors have been used to induce protection in various experimental models. Kidney ischemia–reperfusion (I/R) is a form of acute kidney injury resulting in a cascade of cellular events prompting rapid cellular damage and suppression of kidney function. I/R injury, an inevitable impairment during renal transplant surgery, remains one of the major causes of acute kidney injury and represents the most prominent factor leading to delayed graft function after transplantation. Understanding the molecular events responsible for tubule damage and recovery would help to develop new strategies for organ preservation. This chapter describes procedures to study the effect of CDK inhibitors in the cellular I/R model developed from an epithelial cell line deriving from pig kidney proximal tubule cells (LLC–PK1). We briefly describe methods for determining the protective effect of CDK inhibitors such as activation of caspase 3/7, western blot analysis, gene silencing, and immunoprecipitation.
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References
Orzaez M, Guevara T, Sancho M, Perez-Paya E (2012) Intrinsic caspase-8 activation mediates sensitization of erlotinib-resistant tumor cells to erlotinib/cell-cycle inhibitors combination treatment. Cell Death Dis 3:e415
Iyirhiaro GO, Brust TB, Rashidian J, Galehdar Z, Osman A, Phillips M, Slack RS, Macvicar BA, Park DS (2008) Delayed combinatorial treatment with flavopiridol and minocycline provides longer term protection for neuronal soma but not dendrites following global ischemia. J Neurochem 105(3):703–713
Hilton GD, Stoica BA, Byrnes KR, Faden AI (2008) Roscovitine reduces neuronal loss, glial activation, and neurologic deficits after brain trauma. J Cereb Blood Flow Metab 28(11):1845–1859
Menn B, Bach S, Blevins TL, Campbell M, Meijer L, Timsit S (2010) Delayed treatment with systemic (S)-roscovitine provides neuroprotection and inhibits in vivo CDK5 activity increase in animal stroke models. PLoS One 5(8):e12117
Guevara T, Sancho M, Perez-Paya E, Orzaez M (2014) Role of CDK5/cyclin complexes in ischemia-induced death and survival of renal tubular cells. Cell Cycle 13(10):1617–1626
Bagshaw SM, Mortis G, Godinez-Luna T, Doig CJ, Laupland KB (2006) Renal recovery after severe acute renal failure. Int J Artif Organs 29(11):1023–1030
Kosieradzki M, Rowinski W (2008) Ischemia/reperfusion injury in kidney transplantation: mechanisms and prevention. Transplant Proc 40(10):3279–3288
Singh D, Chander V, Chopra K (2005) Cyclosporine protects against ischemia/reperfusion injury in rat kidneys. Toxicology 207(3):339–347
Delbridge MS, Shrestha BM, Raftery AT, El Nahas AM, Haylor JL (2007) Reduction of ischemia-reperfusion injury in the rat kidney by FTY720, a synthetic derivative of sphingosine. Transplantation 84(2):187–195
Aydemir A, Abbasoglu O, Topaloglu S, Ertoy D, Ayhan A, Kilinc K, Karabulut E, Sayek I (2002) Protective effect of roscovitine on renal ischemia-reperfusion injury. Transplant Proc 34(6):2027–2028
Osuga H, Osuga S, Wang F, Fetni R, Hogan MJ, Slack RS, Hakim AM, Ikeda JE, Park DS (2000) Cyclin-dependent kinases as a therapeutic target for stroke. Proc Natl Acad Sci U S A 97(18):10254–10259
Canela N, Orzaez M, Fucho R, Mateo F, Gutierrez R, Pineda-Lucena A, Bachs O, Perez-Paya E (2006) Identification of an hexapeptide that binds to a surface pocket in cyclin A and inhibits the catalytic activity of the complex cyclin-dependent kinase 2-cyclin A. J Biol Chem 281(47):35942–35953
Hotter G, Palacios L, Sola A (2004) Low O2 and high CO2 in LLC-PK1 cells culture mimics renal ischemia-induced apoptosis. Lab Invest 84(2):213–220
Taniguchi Y, Pippin JW, Hagmann H, Krofft RD, Chang AM, Zhang J, Terada Y, Brinkkoetter P, Shankland SJ (2012) Both cyclin I and p35 are required for maximal survival benefit of cyclin-dependent kinase 5 in kidney podocytes. Am J Physiol Renal Physiol 302(9):F1161–F1171
Brinkkoetter PT, Olivier P, Wu JS, Henderson S, Krofft RD, Pippin JW, Hockenbery D, Roberts JM, Shankland SJ (2009) Cyclin I activates Cdk5 and regulates expression of Bcl-2 and Bcl-XL in postmitotic mouse cells. J Clin Invest 119:3089
Brinkkoetter PT, Pippin JW, Shankland SJ (2010) Cyclin I-Cdk5 governs survival in post-mitotic cells. Cell Cycle 9(9):1729–1731
Fearnhead HO (2001) Cell-free systems to study apoptosis. Methods Cell Biol 66:167–185
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Guevara, T. (2016). Evaluating the Effects of CDK Inhibitors in Ischemia–Reperfusion Injury Models. In: Orzáez, M., Sancho Medina, M., Pérez-Payá, E. (eds) Cyclin-Dependent Kinase (CDK) Inhibitors. Methods in Molecular Biology, vol 1336. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2926-9_10
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DOI: https://doi.org/10.1007/978-1-4939-2926-9_10
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2925-2
Online ISBN: 978-1-4939-2926-9
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