Immunosuppressive calcineurin inhibitor cyclosporine A induces proapoptotic endoplasmic reticulum stress in renal tubular cells

Abstract

Current immunosuppressive strategies in organ transplantation rely on calcineurin inhibitors cyclosporine A (CsA) or tacrolimus (Tac). Both drugs are nephrotoxic, but CsA has been associated with greater renal damage than Tac. CsA inhibits calcineurin by forming complexes with cyclophilins, whose chaperone function is essential for proteostasis. We hypothesized that stronger toxicity of CsA may be related to suppression of cyclophilins with ensuing endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in kidney epithelia. Effects of CsA and Tac (10 µM for 6 h each) were compared in cultured human embryonic kidney 293 (HEK 293) cells, primary human renal proximal tubule (PT) cells, freshly isolated rat PTs, and knockout HEK 293 cell lines lacking the critical ER stress sensors, protein kinase RNA–like ER kinase or activating transcription factor 6 (ATF6). UPR was evaluated by detection of its key components. Compared with Tac treatment, CsA induced significantly stronger UPR in native cultured cells and isolated PTs. Evaluation of proapoptotic and antiapoptotic markers suggested an enhanced apoptotic rate in CsA-treated cells compared with Tac-treated cells as well. Similar to CsA treatment, knockdown of cyclophilin A or B by siRNA caused proapoptotic UPR, whereas application of the chemical chaperones tauroursodeoxycholic acid or 4-phenylbutyric acid alleviated CsA-induced UPR. Deletion of protein kinase RNA–like ER kinase or ATF6 blunted CsA-induced UPR as well. In summary, inhibition of cyclophilin chaperone function with ensuing ER stress and proapoptotic UPR aggravates CsA toxicity, whereas pharmacological modulation of UPR bears potential to alleviate renal side effects of CsA. Current immunosuppressive strategies in organ transplantation rely on calcineurin inhibitors cyclosporine A (CsA) or tacrolimus (Tac). Both drugs are nephrotoxic, but CsA has been associated with greater renal damage than Tac. CsA inhibits calcineurin by forming complexes with cyclophilins, whose chaperone function is essential for proteostasis. We hypothesized that stronger toxicity of CsA may be related to suppression of cyclophilins with ensuing endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in kidney epithelia. Effects of CsA and Tac (10 µM for 6 h each) were compared in cultured human embryonic kidney 293 (HEK 293) cells, primary human renal proximal tubule (PT) cells, freshly isolated rat PTs, and knockout HEK 293 cell lines lacking the critical ER stress sensors, protein kinase RNA–like ER kinase or activating transcription factor 6 (ATF6). UPR was evaluated by detection of its key components. Compared with Tac treatment, CsA induced significantly stronger UPR in native cultured cells and isolated PTs. Evaluation of proapoptotic and antiapoptotic markers suggested an enhanced apoptotic rate in CsA-treated cells compared with Tac-treated cells as well. Similar to CsA treatment, knockdown of cyclophilin A or B by siRNA caused proapoptotic UPR, whereas application of the chemical chaperones tauroursodeoxycholic acid or 4-phenylbutyric acid alleviated CsA-induced UPR. Deletion of protein kinase RNA–like ER kinase or ATF6 blunted CsA-induced UPR as well. In summary, inhibition of cyclophilin chaperone function with ensuing ER stress and proapoptotic UPR aggravates CsA toxicity, whereas pharmacological modulation of UPR bears potential to alleviate renal side effects of CsA. Calcineurin (Cn) inhibitors (CNIs) are considered as the first-line immunosuppressive therapy in patients undergoing organ transplantation (1Williams C.R. Gooch J.L. Calcineurin inhibitors and immunosuppression - a tale of two isoforms.Expert Rev. Mol. Med. 2012; 14e14Google Scholar, 2Casey M.J. Meier-Kriesche H.-U. Calcineurin inhibitors in kidney transplantation: Friend or foe?.Curr. Opin. Nephrol. Hypertens. 2011; 20: 610-615Google Scholar). Two drugs of this class, cyclosporine A (CsA) and tacrolimus (FK506, Tac), have been widely integrated into clinical practice. Cn is a holoenzyme consisting of a catalytic subunit (CnA), a regulatory subunit (CnB), and calmodulin, which functions as a Ca2+-dependent protein serine/threonine phosphatase. Alpha and beta isoforms of the catalytic subunit (CnAα and CnAβ) fulfill distinct and nonredundant tasks in various tissues (1Williams C.R. Gooch J.L. Calcineurin inhibitors and immunosuppression - a tale of two isoforms.Expert Rev. Mol. Med. 2012; 14e14Google Scholar, 3Rusnak F. Mertz P. Calcineurin: Form and function.Physiol. Rev. 2000; 80: 1483-1521Google Scholar). CnAβ-dependent dephosphorylation of nuclear factor of activated T-cell (NFAT) transcription factors is critical to T-lymphocyte activation and underlies the therapeutic action of CNI (4Bueno O.F. Brandt E.B. Rothenberg M.E. Molkentin J.D. Defective T cell development and function in calcineurin A beta -deficient mice.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9398-9403Google Scholar). Off-target effects of CNI include renal complications because of suppression of CnAα activity in kidney tissue (5Gooch J.L. Roberts B.R. Cobbs S.L. Tumlin J.A. Loss of the alpha-isoform of calcineurin is sufficient to induce nephrotoxicity and altered expression of transforming growth factor-beta.Transplantation. 2007; 83: 439-447Google Scholar, 6Naesens M. Kuypers D.R.J. Sarwal M. Calcineurin inhibitor nephrotoxicity.Clin. J. Am. Soc. Nephrol. 2009; 4: 481-508Google Scholar). A majority of organ-transplanted patients receiving CNI exhibit signs of kidney damage after several years of treatment (6Naesens M. Kuypers D.R.J. Sarwal M. Calcineurin inhibitor nephrotoxicity.Clin. J. Am. Soc. Nephrol. 2009; 4: 481-508Google Scholar). CNI-induced renal vasoconstriction, hyperactivity of the renin–angiotensin system (RAS), and dysregulation of major electrolyte transport systems cause hypertension and homeostasis disorders (6Naesens M. Kuypers D.R.J. Sarwal M. Calcineurin inhibitor nephrotoxicity.Clin. J. Am. Soc. Nephrol. 2009; 4: 481-508Google Scholar, 7Hoorn E.J. Walsh S.B. McCormick J.A. Zietse R. Unwin R.J. Ellison D.H. Pathogenesis of calcineurin inhibitor-induced hypertension.J. Nephrol. 2012; 25: 269-275Google Scholar). At the cellular level, CNIs have been shown to induce endoplasmic reticulum (ER) stress and trigger unfolded protein response (UPR), which in turn may promote apoptosis of kidney epithelia (8Kitamura M. Induction of the unfolded protein response by calcineurin inhibitors: A double-edged sword in renal transplantation.Nephrol. Dial. Transplant. 2010; 25: 6-9Google Scholar, 9Cybulsky A.V. Endoplasmic reticulum stress, the unfolded protein response and autophagy in kidney diseases.Nat. Rev. Nephrol. 2017; 13: 681-696Google Scholar). Several retrospective studies suggested that CsA may be associated with stronger nephrotoxicity than Tac (10Ekberg H. Tedesco-Silva H. Demirbas A. Vítko S. Nashan B. Gürkan A. Margreiter R. Hugo C. Grinyó J.M. Frei U. Vanrenterghem Y. Daloze P. Halloran P.F. ELITE-Symphony StudyReduced exposure to calcineurin inhibitors in renal transplantation.N. Engl. J. Med. 2007; 357: 2562-2575Google Scholar, 11Ekberg H. Bernasconi C. Tedesco-Silva H. Vítko S. Hugo C. Demirbas A. Acevedo R.R. Grinyó J. Frei U. Vanrenterghem Y. Daloze P. Halloran P. Calcineurin inhibitor minimization in the symphony study: Observational results 3 years after transplantation.Am. J. Transplant. 2009; 9: 1876-1885Google Scholar, 12Lucey M.R. Abdelmalek M.F. Gagliardi R. Granger D. Holt C. Kam I. Klintmalm G. Langnas A. Shetty K. Tzakis A. Woodle E.S. A comparison of tacrolimus and cyclosporine in liver transplantation: Effects on renal function and cardiovascular risk status.Am. J. Transplant. 2005; 5: 1111-1119Google Scholar, 13Jurewicz W.A. Tacrolimus versus ciclosporin immunosuppression: Long-term outcome in renal transplantation.Nephrol. Dial. Transplant. 2003; 18 Suppl 1: 7i-i11Google Scholar, 14Krämer B.K. Montagnino G. del Castillo D. Margreiter R. Sperschneider H. Olbricht C.J. Krüger B. Ortuño J. Köhler H. Kunzendorf U. Stummvoll H.-K. Tabernero J.M. Mühlbacher F. Rivero M. Arias M. Efficacy and safety of tacrolimus compared with cyclosporin A microemulsion in renal transplantation: 2 year follow-up results.Nephrol. Dial. Transplant. 2005; 20: 968-973Google Scholar). Along the same line, a recent experimental study in cultured human cells demonstrated higher toxicity of CsA compared with Tac (15Fedele A.O. Carraro V. Xie J. Averous J. Proud C.G. Cyclosporin A but not FK506 activates the integrated stress response in human cells.J. Biol. Chem. 2020; 295: 15134-15143Google Scholar). The two CNIs inhibit Cn activity by forming complexes with distinct members of the immunophilin family: CsA binds to cyclophilins, whereas Tac interacts with 12 kDa FK506-binding protein (FKBP12). These individual interaction patterns may provide a reasonable explanation for distinct toxicity profiles of CsA versus Tac. Since the chaperone activity of cyclophilins is required for protein maturation, CsA may impair proteostasis (Fig. 1) (16Ram B.M. Ramakrishna G. Endoplasmic reticulum vacuolation and unfolded protein response leading to paraptosis like cell death in cyclosporine A treated cancer cervix cells is mediated by cyclophilin B inhibition.Biochim. Mol. Scholar, S. M. G. of protein by cyclophilins Biol. Chem. Scholar). studies of CsA versus Tac in cell stronger of CsA with ER stress and UPR, but the of cyclophilins to be (15Fedele A.O. Carraro V. Xie J. Averous J. Proud C.G. Cyclosporin A but not FK506 activates the integrated stress response in human cells.J. Biol. Chem. 2020; 295: 15134-15143Google Scholar, F. M. P. of toxicity in a human kidney cell and comparison with 2011; Scholar). UPR is a that is by of proteostasis leading to of protein C. The unfolded protein cell ER stress and Rev. Mol. Biol. 2012; 13: Scholar, P. D. The unfolded protein stress to 2011; Scholar). protein protein protein kinase ER kinase and activating transcription factor 6 complexes with protein by with the and ATF6 as and a of cell and the factor protein activation of and ATF6 autophagy and protein several as transcription and of protein C. The unfolded protein cell ER stress and Rev. Mol. Biol. 2012; 13: Scholar). of the proapoptotic UPR of the protein which is a transcription factor that expression of proapoptotic A.V. Endoplasmic reticulum stress, the unfolded protein response and autophagy in kidney diseases.Nat. Rev. Nephrol. 2017; 13: 681-696Google Scholar, S. P. C. D. stress renal by the unfolded protein 2011; Scholar, H. F. D. C. ER stress a Mol. Scholar). Cn has been in UPR cell ER stress M. of calcineurin in response to endoplasmic reticulum J. 2002; Scholar). Cn inhibition may impair the of kidney epithelia to for of UPR chemical inhibitors of or autophagy has been considered as an to of kidney disorders A.V. Endoplasmic reticulum stress, the unfolded protein response and autophagy in kidney diseases.Nat. Rev. Nephrol. 2017; 13: 681-696Google Scholar). In this of by CNI may for The study distinct toxicity profiles of CsA versus Tac with a on ER stress and UPR. on of CsA and Tac in cultured human embryonic kidney 293 (HEK 293) cells, primary human renal proximal (PT) cells and freshly isolated rat PTs, provide several lines of in of the that the effects of CsA on proteostasis are in the suppression of cyclophilin chaperone and pharmacological that UPR bears therapeutic potential for of CsA to the CNI and treatment to a of effects on ER and UPR in cultured ER stress and UPR in human cancer and human cells after application of CsA for 6 h (15Fedele A.O. Carraro V. Xie J. Averous J. Proud C.G. Cyclosporin A but not FK506 activates the integrated stress response in human cells.J. Biol. Chem. 2020; 295: 15134-15143Google Scholar). on that a by native HEK 293 cells with of CsA versus Tac each) for 6 h to the effects on cell the for cellular growth and to and Scholar). A of cell was with CsA or Tac (Fig. which as of ER stress and UPR because of the critical of cell Since CsA has been to induce as (16Ram B.M. Ramakrishna G. Endoplasmic reticulum vacuolation and unfolded protein response leading to paraptosis like cell death in cyclosporine A treated cancer cervix cells is mediated by cyclophilin B inhibition.Biochim. Mol. Scholar, toxicity on cultured rat compared effects of CsA versus Tac (10 each) on cell by and after application of CsA or Tac for 6 whereas treatment for h in of in CsA-treated but not in Tac-treated cells (Fig. treatment of cells with CsA for h in a of cell compared with Tac or as by (Fig. that the treatment for human cancer cells (10 CsA versus Tac for 6 is for experimental (15Fedele A.O. Carraro V. Xie J. Averous J. Proud C.G. Cyclosporin A but not FK506 activates the integrated stress response in human cells.J. Biol. Chem. 2020; 295: 15134-15143Google whereas CNI or treatment the cell in HEK 293 cells, primary and rat were CsA versus Tac for 6 the of the treatment in of Cn evaluated of the Cn F. Mertz P. Calcineurin: Form and function.Physiol. Rev. 2000; 80: 1483-1521Google Scholar). an to of in CsA-treated or Tac-treated HEK 293 cells that CNIs dephosphorylation to a for 6 h (Fig. compared effects of CsA versus Tac on key UPR an and UPR V. S. M. M. in ER autophagy in J. Scholar, F. ER stress and the unfolded protein response tissue 2011; as for Compared with CsA significantly of and but not as by for for and for expression of the ER and in response to CsA (Fig. Similar were in cells, whereas Tac effects on the UPR and evaluated nuclear of Compared with CsA and induced of in the nuclear whereas Tac not the nuclear (Fig. the effects of evaluated the as as the proapoptotic protein and M. K. M. V. J. J. I. I. J. The of individual in cell death by in cancer Scholar). that CsA and and whereas Tac not the and the A and of of cells after CsA and but not Tac (Fig. results compared with CsA significantly stronger ER stress and proapoptotic UPR in cultured HEK 293 the in HEK 293 cells in with kidney treated and isolated rat with CNI or Similar to the results in HEK 293 cells, CsA induced significantly stronger UPR compared with Tac in as by of and not and and and as as by for and B and Along the same line, expression of and were significantly higher in isolated rat treated with CsA than in the Tac and Since CsA but not Tac induced UPR, that this may suppression of cyclophilins than Cn cyclophilin A or cyclophilin B expression in HEK 293 cells knockdown These to of or protein and Evaluation of by significantly in cells and cells and In with for in cells and cells and effects of and cells on and were to effects of CsA in cells A and B and a critical of cyclophilin activity in the CsA-induced ER stress and UPR. the potential of the chemical tauroursodeoxycholic acid or 4-phenylbutyric acid to alleviate the CsA-induced ER The CsA-induced of protein were significantly blunted by application of to or to the CsA-induced of cells were blunted by and to or as by of treatment of chemical chaperones or on the of in HEK 293 of in HEK 293 cells treated with and CsA or CsA were with the of cells for cell in the are the and 4-phenylbutyric cyclosporine HEK human embryonic kidney 293 cell tauroursodeoxycholic of UPR the proapoptotic effects of CsA. this for of critical and UPR or in HEK 293 in of or cell lines (Fig. to to cell a critical for this protein in cell and effects on and protein for a of in cells These results that of or ATF6 not effects of CsA or Tac. Since the of or ATF6 may the proteostasis evaluated and The was in cells but in cells as compared with cells (Fig. Evaluation of in and cells (Fig. that ATF6 is associated with altered proteostasis in activation of UPR as A.V. Endoplasmic reticulum stress, the unfolded protein response and autophagy in kidney diseases.Nat. Rev. Nephrol. 2017; 13: 681-696Google Scholar). treated and cells with and the experimental for HEK 293 cells significantly of and in response to whereas of and were not by the treatment (Fig. and whereas Tac the in cells cells to CsA with of and whereas and were not (Fig. the of and whereas Tac the as by of the that CsA-induced of and in and cells were than in cells These results that of UPR by CsA is mediated by and of cyclosporine A tacrolimus and on key unfolded protein response in for and in HEK 293 cells treated with Tac (10 CsA (10 or for 6 results of of and or detection was for and are the activating transcription factor HEK human embryonic kidney 293 cell protein not protein of CsA effects on the UPR in and HEK 293 cells versus HEK 293 cells 293 for effects of CsA in cells and or cells as for effects of CsA in cells and or cells as in a is clinical for CsA in patients on this or Tac to CsA for side effects of Tac as D. K. M. Kuypers D.R.J. study of tacrolimus to cyclosporine A to in patients with after renal transplantation.Am. J. Transplant. Scholar, M. V. A. S. R. V. V. tacrolimus to cyclosporine in patients with after renal Scholar). retrospective of CNI nephrotoxicity suggested that Tac has pharmacological compared with CsA (6Naesens M. Kuypers D.R.J. Sarwal M. Calcineurin inhibitor nephrotoxicity.Clin. J. Am. Soc. Nephrol. 2009; 4: 481-508Google Scholar, 13Jurewicz W.A. Tacrolimus versus ciclosporin immunosuppression: Long-term outcome in renal transplantation.Nephrol. Dial. Transplant. 2003; 18 Suppl 1: 7i-i11Google Scholar, 14Krämer B.K. Montagnino G. del Castillo D. Margreiter R. Sperschneider H. Olbricht C.J. Krüger B. Ortuño J. Köhler H. Kunzendorf U. Stummvoll H.-K. Tabernero J.M. Mühlbacher F. Rivero M. Arias M. Efficacy and safety of tacrolimus compared with cyclosporin A microemulsion in renal transplantation: 2 year follow-up results.Nephrol. Dial. Transplant. 2005; 20: 968-973Google Scholar, H. S. Y. Y. K. M. Tacrolimus versus cyclosporin A study on rat renal Scholar). of CsA has been associated with proteostasis and integrated stress response in cultured human cells (15Fedele A.O. Carraro V. Xie J. Averous J. Proud C.G. Cyclosporin A but not FK506 activates the integrated stress response in human cells.J. Biol. Chem. 2020; 295: 15134-15143Google Scholar). In with the results in cultured cells of renal and isolated rat that CsA UPR as enhanced of and (8Kitamura M. Induction of the unfolded protein response by calcineurin inhibitors: A double-edged sword in renal transplantation.Nephrol. Dial. Transplant. 2010; 25: 6-9Google Scholar, 9Cybulsky A.V. Endoplasmic reticulum stress, the unfolded protein response and autophagy in kidney diseases.Nat. Rev. Nephrol. 2017; 13: 681-696Google Scholar, A. C. I. of the and for the unfolded protein J. 2011; Scholar). of and with in CsA-treated cells of the UPR and to the proapoptotic C. The unfolded protein cell ER stress and Rev. Mol. Biol. 2012; 13: Scholar). In to Tac or effects on the UPR in the These results were not in HEK 293 cells but in and native rat PTs, which in the of CNI cellular toxicity of CsA be by of CsA and Tac in of Cn treatment of the that effects of CsA may be by the suppression of in protein by of the of S. M. G. of protein by cyclophilins Biol. Chem. Scholar, K. G. of protein by Scholar). Since is an its by cyclophilins may be considered as a in protein K. G. of protein by Scholar, and its in protein and protein Mol. Biol. Scholar). on and of cyclophilin isoforms in the kidney is and in studies effects of cyclophilin inhibition or of cyclophilin in of kidney or kidney CsA nephrotoxicity J. J. A. B. G. J.M. U. knockout the kidney cyclosporin J. Scholar, J. inhibition experimental kidney and renal J. Mol. Sci. 2020; Scholar, cell damage and the development of in the Scholar). In studies effects of and in of CsA nephrotoxicity or kidney F. J. C. J. cyclophilin A are to cyclosporin nephrotoxicity Scholar, B. H. H. Y. cyclophilin B proximal cell in and in Scholar, R. C. J. M. cyclophilin endoplasmic 2010; Scholar). is to that of CsA with or may impair the proteostasis because of suppression of whereas complexes to the function J. J. A. B. G. J.M. U. knockout the kidney cyclosporin J. Scholar, F. J. C. J. cyclophilin A are to cyclosporin nephrotoxicity Scholar, B. H. H. Y. cyclophilin B proximal cell in and in Scholar, J.D. cyclophilin cells cell death by Biol. Chem. 2002; Scholar). of or to and in the which a that CsA-induced suppression of chaperone function may cause ER stress and proapoptotic UPR. to may be in and of and whereas may to protein and in ER R. C. J. M. cyclophilin endoplasmic 2010; Scholar, J. activity of cyclophilin A in Natl. Acad. Sci. U. S. A. Scholar). The that knockdown of cyclophilin of and in the study to nonredundant in proteostasis. Since Tac effects on UPR in the that chaperone function is for proteostasis. has been in modulation of the cell than in protein and proteostasis B. K. A. A. the FK506-binding is a of the cell Natl. Acad. Sci. U. S. A. Scholar). of Tac in may stress in kidney epithelia for because of hyperactivity or of renal E.J. Walsh S.B. McCormick J.A. Zietse R. Unwin R.J. Ellison D.H. Pathogenesis of calcineurin inhibitor-induced hypertension.J. Nephrol. 2012; 25: 269-275Google Scholar). chaperone systems has been as an for of kidney A.V. Endoplasmic reticulum stress, the unfolded protein response and autophagy in kidney diseases.Nat. Rev. Nephrol. 2017; 13: 681-696Google Scholar). studies in that ER stress to of kidney disorders to renal which be by of chemical chaperones A.V. Endoplasmic reticulum stress, the unfolded protein response and autophagy in kidney diseases.Nat. Rev. Nephrol. 2017; 13: 681-696Google Scholar, the endoplasmic reticulum renal in and in Scholar). The results that of protein or may cells CsA-induced ER stress, as by blunted of and in HEK 293 cells treated with CsA and chemical chemical chaperones protein in a of UPR by CsA may provide to or its cell this in UPR by or ATF6 in the HEK 293 cell and ATF6 ER stress and distinct to protein C. The unfolded protein cell ER stress and Rev. Mol. Biol. 2012; 13: Scholar). of or ATF6 effects on of whereas of was in which may proapoptotic UPR C. The unfolded protein cell ER stress and Rev. Mol. Biol. 2012; 13: Scholar, H. M. B. ATF6 is a critical of the unfolded protein 2020; Scholar). to cell that is critical to cellular proteostasis and A. C. I. of the and for the unfolded protein J. 2011; Scholar). of has been shown to impair protein and function in J. J. A.V. of in proteostasis and 2020; Scholar). In the cells of and in response to CsA or a blunted UPR. cells blunted effects of CsA and on and as whereas of was in this cell Tac or on UPR in the in and in results that suppression of or may with the proapoptotic UPR and cell studies are to the individual of in the CsA In summary, the of CsA versus Tac toxicity profiles that the effects of CsA on proteostasis suppression of cyclophilins than Cn inhibition (Fig. In in of CsA or Tac may cause or ER stress in kidney epithelia because of and as system and of renal and of kidney epithelia E.J. Walsh S.B. McCormick J.A. Zietse R. Unwin R.J. Ellison D.H. Pathogenesis of calcineurin inhibitor-induced hypertension.J. Nephrol. 2012; 25: 269-275Google Scholar, J. Y. S. K. calcineurin inhibitor nephrotoxicity by 2 expression in kidney 2020; Scholar, S.L. and activation with inhibition of T-cell Scholar). of ER stress chemical chaperones or autophagy as an therapeutic to or CNI nephrotoxicity (8Kitamura M. Induction of the unfolded protein response by calcineurin inhibitors: A double-edged sword in renal transplantation.Nephrol. Dial. Transplant. 2010; 25: 6-9Google Scholar). In the clinical that Tac compared with CsA and the of Tac in organ-transplanted patients calcineurin inhibitor is in renal transplantation: Tacrolimus or Opin. Nephrol. Hypertens. Scholar). HEK 293 cells were cultured in essential with and were cultured in with with in a K. H. S. Y. knockout of and the potential of cancer Scholar). and HEK 293 cell lines were J. V. F. the Scholar). In were and into cells were into were and was by of the were treated with CsA or Tac Both drugs were in for CsA and Tac were in native HEK 293 were to a and with cells were treated with CsA or Tac in of and for 6 h was evaluated was in and the was for of the was to the of the ensuing for the was and were in and with for was in a and the in cells were on the results of this CsA and Tac were for the treatment cells were to treated with CsA or Tac for or and evaluated by and The 6 h the results by as as of cell and was for the experimental effects of CNI on UPR, native HEK 293 cells, or cells were to and treated for 6 h with CsA (10 Tac (10 and as Effects of application of the chemical or were in native HEK 293 cells in the of CsA. were and for or cells were for or were by the on the of for were of and were and were by in with and by M. H. H. M. M. S. F. system Biol. Chem. 2010; Scholar). were a was in of to (Fig. which were into or in of with and for 6 h were and for (Fig. were on and with and after with in (Fig. HEK 293 cells were on to and in for of the cells was in for protein was with in for or were in the and in a cells were with for h were with and the was which was to the were on and were by At with treatment were were in with with a cell and for as V. S. D. P. S. P. S. G. C. J. of to inhibition in 13: Scholar). were in and and an were in with a inhibitor and for was by for and the was were protein were or and to a were with in for by primary for are in were by with the for h by with detection and in was of of primary for in in in in in in 3 in in in in in in in in in in in with in a with was isolated to the transcription was with the of for of the expression of are in 3 F. ER stress and the unfolded protein response tissue 2011; Scholar). was and of was to of for in a and siRNA were by was a cell of to to the of siRNA for in a were by for as the experimental two was by of was by was to A of was as are the K. M. and S. B. that was by that have of with the of this We for and for the and K. M. K. and K. M. D. Y. D. Y. D. H. and H. S. B. and K. M. K. M. S. B. D. Y. K. M. K. M. S. B. and K. M. with

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