Skip Navigation



NDT Advance Access published online on October 1, 2009
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfp516
This Article
Right arrow Extract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Kitamura, M.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kitamura, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2009. Published by Oxford University Press [on behalf of ERA-EDTA]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Induction of the unfolded protein response by calcineurin inhibitors: a double-edged sword in renal transplantation

Masanori Kitamura

Department of Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi 409-3898, Japan

Correspondence and offprint requests to: Masanori Kitamura; E-mail: masanori{at}yamanashi.ac.jp

Keywords: calcineurin inhibitor; cyclosporine A; endoplasmic reticulum stress; tacrolimus; transplantation

The endoplasmic reticulum (ER) provides a unique environment for appropriate protein folding and assembly to produce functional, mature proteins. A number of pathophysiological insults cause accumulation of unfolded proteins in the ER, namely, ER stress. In response to ER stress, cells adapt themselves to the stress conditions via the unfolded protein response (UPR), leading to attenuation of translation, induction of ER chaperones and activation of ER-associated degradation (ERAD) to eliminate immature proteins. The UPR is involved in a diverse range of pathophysiological events [1,2], including renal diseases [3].

In response to ER stress, three major branches of the UPR are activated, as summarized in Figure 1A. Those include the RNA-dependent protein kinase-like ER kinase (PERK) pathway, the activating transcription factor 6 (ATF6) pathway and the inositol-requiring ER-to-nucleus signal kinase 1 (IRE1) pathway. Activation of PERK causes phosphorylation of the eukaryotic translation initiation factor 2{alpha} (eIF2{alpha}), which leads to general inhibition of protein synthesis. In response to ER stress, p90ATF6 transits to the Golgi where it is cleaved by the proteases S1P and S2P, yielding an active transcription factor p50ATF6. Similarly, activated IRE1 catalyses removal of a small intron from the mRNA of X-box binding protein 1 (XBP1). This splicing event creates translational frameshift in XBP1 mRNA to produce an active transcription factor. Active p50ATF6 and XBP1 subsequently bind to the ER stress response element (ERSE) and the UPR element (UPRE), leading to expression of target genes including ER chaperones and ERAD factors that degrade unfolded proteins.


Figure 1
View larger version (27K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 (A) Induction of three major branches of the UPR by ER stress. ER stress activates three major branches of the UPR; i.e. the PERK, ATF6 and IRE1 pathways. Activation of PERK causes phosphorylation of eIF2{alpha}, which leads to general translational suppression. In response to ER stress, p90ATF6 transits to the Golgi where it is cleaved by proteases S1P and S2P, yielding an active transcription factor p50ATF6. Similarly, activated IRE1 catalyses splicing of XBP1 mRNA to produce an active transcription factor. Active p50ATF6 and XBP1 bind to the ERSE and the UPRE, leading to expression of target genes including ER chaperones and ERAD factors involved in appropriate folding and degradation of unfolded proteins, respectively. (B) The light and dark sides of the UPR triggered by calcineurin inhibitors. Calcineurin inhibitors cause the UPR and thereby up-regulate C/EBP that interferes with function of p65 NF-{kappa}B subunit. The UPR also induces A20, an inhibitor of the TNF-{alpha} signalling at the step of RIP. Furthermore, the UPR induces selective degradation of TRAF2, an essential component for the TNF-{alpha} signalling. These molecular events suppress activation of NF-{kappa}B and attenuate immune/inflammatory responses in renal transplants. On the other hand, ER stress caused by calcineurin inhibitors also triggers cytotoxic UPR. Proapoptotic CHOP is induced mainly by the PERK–eIF2{alpha} pathway. ER stress activates caspase-12 through an interaction with IRE1 and TRAF2. The IRE1–TRAF2 interaction also allows for recruitment and activation of ASK1 and JNK, both of which are involved in a variety of proapoptotic signalling.

 
Cyclosporine A (CsA) and tacrolimus (FK506) are pivotal immunosuppressive agents to prevent allograft rejection in renal transplantation. Use of these immunosuppressants leads to significant reduction in the incidence of acute graft rejection and improvement in the survival of kidney transplants [4]. CsA binds to the cyclophilin family of molecules that have high affinity for calcineurin, a key protein phosphatase in the activation of T cells. FK506 is another calcineurin inhibitor, the mechanism of which is similar to that of CsA. It forms complexes with its cytosolic partner FK506-binding protein 12, and the complexes bind to calcineurin. By blocking calcineurin, CsA and FK506 inhibit phosphatase-controlled translocation of nuclear factor of activated T-cells (NF-AT) into the nucleus and prevent induction of cytokines and their receptors required for activation and proliferation of lymphocytes and other immune cells [5]. These agents are, therefore, regarded as inhibitors of immune cell function. However, several reports have also demonstrated that CsA and FK506 suppress activation of non-immune cells including keratinocytes, endothelial cells, mesangial cells, synovial fibroblasts and hepatocytes. For example, CsA inhibits mitogenesis of endothelial cells and growth factor-driven proliferation of keratinocytes [6,7]. CsA also attenuates induction of cytokines and their receptors in keratinocytes and expression of nitric oxide synthase in mesangial cells [8,9]. Similarly, other investigators reported that FK506 inhibits secretion of TNF-{alpha} in keratinocytes, production of matrix metalloproteinase 13 by rheumatoid synovial fibroblasts and generation of reactive oxygen species and nitric oxide in hepatocytes [10–12]. Currently, molecular mechanisms underlying the suppressive effects of CsA and FK506 on the non-lymphoid lineages are largely unknown.

Recently, we found that in renal tubular cells, induction of monocyte chemoattractant protein 1 (MCP-1) by inflammatory cytokines is blunted by CsA and FK506 [13]. We identified that this suppressive effect is ascribed, at least in part, to induction of ER stress and consequent UPR. This phenomenon is observed not only in tubular cells but also in other non-immune cells including smooth muscle cells, preadipocytes, mesangial cells and endothelial cells. Indeed, administration with CsA in mice causes rapid induction of the UPR in several organs [13]. In tumour necrosis factor-{alpha} (TNF-{alpha})-exposed cells, suppression of MCP-1 by CsA and FK506 is associated with the blunted activation of nuclear factor-{kappa}B (NF-{kappa}B), and the suppression of NF-{kappa}B is reproduced by other UPR inducers. CsA and FK506, as well as other UPR inducers, cause up-regulation of CCAAT/enhancer-binding proteins (C/EBP), especially C/EBPβ that interacts with the p65 NF-{kappa}B subunit. Overexpression of C/EBPβ significantly attenuates TNF-{alpha}-triggered NF-{kappa}B activation, and down-regulation of C/EBPβ by small interfering RNA substantially reverses the suppressive effect of CsA on TNF-{alpha}-induced MCP-1 expression [13]. In addition to this mechanism, A20, an inhibitor of the TNF-{alpha} signalling at the step of TNF receptor-interacting protein (RIP), is rapidly induced in renal cells under ER stress conditions [14]. Selective degradation of TNF receptor-associated factor 2 (TRAF2), an essential component for the TNF-{alpha} signalling, is also caused in renal cells by ER stress [14,15]. These results indicate that calcineurin inhibitors confer insensitiveness to inflammatory stimuli on renal cells through UPR-mediated induction of C/EBP and A20, degradation of TRAF2 and consequent suppression of NF-{kappa}B and NF-{kappa}B-dependent gene expression (Figure 1B, left).

The anti-inflammatory potential of the UPR has been further confirmed by our recent investigation using cultured podocytes, mesangial cells and macrophages [14–18]. Furthermore, we found that administration with a subtoxic dosage of subtilase cytotoxin, a selective inducer of the UPR, protects mice from lipopolysaccharide (LPS)-induced endotoxic lethality and collagen arthritis [18]. Together with the fact that in vivo administration of CsA causes systemic UPR [13], I suggest a possibility that calcineurin inhibitors, especially CsA, interfere with not only the function of effector cells (i.e., immune cells) but also activation of responder cells (i.e. resident renal cells) through induction of the UPR. It is the ‘light side’ of the UPR that is triggered by calcineurin inhibitors in renal transplantation.

However, the UPR induced by CsA and FK506 has not only the ‘light side’ but also the ‘dark side’ that incites cellular damage. It is well known that CsA and FK506 cause acute and chronic nephrotoxicity [19]. These agents produce identical pathological changes. Acute toxicity is characterized by necrosis and early hyalinosis of smooth muscle cells in the glomerular afferent arterioles and/or isometric vacuolation of the proximal tubules. In the chronic phase, damage of vascular smooth muscle cells, renal tubular atrophy and tubulointerstitial fibrosis are observed [20]. Several mechanisms have been postulated to explain the nephrotoxicity of these agents [21], and induction of ER stress may be involved in their pathogenic effects. In response to ER stress, several proapoptotic UPRs are transduced [3,22], as shown in Figure 1B (right). For example, proapoptotic C/EBP-homologous protein (CHOP) [also called growth arrest and DNA damage-inducible protein 153 (GADD153)] is induced mainly by the PERK–eIF2{alpha} pathway. ER stress activates caspase-12 (or caspase-4 in humans) localized at the ER membrane through an interaction with IRE1 and TRAF2, leading cells to undergo apoptosis. The IRE1–TRAF2 interaction also allows for recruitment and activation of apoptosis signal-regulating kinase 1 (ASK1) and downstream c-Jun N-terminal kinase (JNK), both of which are involved in a variety of proapoptotic signalling. Activation of these pathways may be involved in the nephrotoxicity caused by calcineurin inhibitors. Indeed, Pallet et al. demonstrated that CsA induced ER stress and cellular damage in tubular cells in vitro [23]. The same group also suggested that CsA induced ER stress in tubular cells and endothelial cells, which may contribute to dedifferentiation and death of these cell types [24,25]. In vivo exposure to CsA causes ER stress in normal kidneys and kidney transplants in animals [3,22], suggesting involvement of ER stress in CsA nephrotoxicity. Recently, Han et al. tested effects of CsA-induced ER stress on apoptotic cell death in an experimental model of chronic CsA nephropathy [26]. In this report, CsA was administered into rats for 7 or 28 days, and induction of 78-kDa glucose-regulated protein (GRP78), CHOP and caspase-12 was evaluated. They found that short-term treatment with CsA caused induction of GRP78, expression of CHOP and activation of caspase-12. In contrast, long-term treatment with CsA decreased anti-apoptotic GRP78 whereas it increased proapoptotic CHOP, leading to disruption of the ER structure and apoptotic cell death. These results suggest that calcineurin inhibitor-triggered ER stress causes apoptosis in the kidney by depleting ER chaperones and inducing proapoptotic proteins.

Currently, it is not fully elucidated why the kidney is preferentially targeted by the toxicity of calcineurin inhibitors. One possible answer to this question is preferential induction of CHOP in the kidney after administration with these agents. We found that although induction of ER stress (indicated by GRP78) was observed in several organs after systemic administration with CsA, substantial induction of CHOP was detectable only in the kidney [13]. It may explain why the kidney is selectively damaged by calcineurin inhibitors. If so, blockade of the proapoptotic UPR, especially the UPR responsible for the induction of CHOP, may be a possible way to attenuate the nephrotoxicity of calcineurin inhibitors.

In contrast to CsA, information is limited regarding the potential of FK506 to induce ER stress. There is currently no report showing induction of ER stress by FK506 in vivo. Recently, Bouvier et al. reported that CsA, but not FK506, induced ER stress in endothelial cells [24]. Although FK506 induced ER stress in renal tubular cells, high concentrations, ~20–30 µM, were required to trigger the UPR [13]. Further investigation will be required to determine involvement of ER stress and consequent UPR in the pharmacological actions of FK506 in vivo.

The UPR triggered by calcineurin inhibitors is a double-edged sword. It possesses both ‘light side’ and ‘dark side’. Understanding the roles of individual UPR in renal pathophysiology will lead us to the development of more effective immunosuppressants with less adverse effects.

Conflict of interest statement. None declared.



   References
Top
 References
 

  1. Kaufman RJ. Orchestrating the unfolded protein response in health and disease. J Clin Invest (2002) 110:1389–1398.[CrossRef][Web of Science][Medline]
  2. Wu J, Kaufman RJ. From acute ER stress to physiological roles of the unfolded protein response. Cell Death Differ (2006) 13:374–384.[CrossRef][Web of Science][Medline]
  3. Kitamura M. Endoplasmic reticulum stress and unfolded protein response in renal pathophysiology: Janus faces. Am J Physiol Renal Physiol (2008) 295:F323–F342.[Abstract/Free Full Text]
  4. Halloran PF. Immunosuppressive drugs for kidney transplantation. N Engl J Med (2004) 351:2715–2729.[Free Full Text]
  5. Schreiber SL, Crabtree GR. The mechanism of action of cyclosporine A and FK-506. Immunol Today (1992) 12:136–142.
  6. Storogenko M, Pech-Amsellem MA, Kerdine S, et al. Cyclosporin A inhibits human endothelial cells proliferation through interleukin-6-dependent mechanisms. Life Sci (1997) 60:1487–1496.[CrossRef][Web of Science][Medline]
  7. Sharpe RJ, Arndt KA, Bauer SI, et al. Cyclosporine inhibits basic fibroblast growth factor-driven proliferation of human endothelial cells and keratinocytes. Arch Dermatol (1989) 125:1359–1362.[Abstract/Free Full Text]
  8. Won YH, Sauder DN, McKenzie RC. Cyclosporin A inhibits keratinocyte cytokine gene expression. Br J Dermatol (1994) 130:312–319.[CrossRef][Web of Science][Medline]
  9. Kunz D, Walker G, Eberhardt W, et al. Interleukin 1β-induced expression of nitric oxide synthase in rat renal mesangial cells is suppressed by cyclosporin A. Biochem Biophys Res Commun (1995) 216:438–446.[CrossRef][Web of Science][Medline]
  10. Lan CC, Yu HS, Wu CS, et al. FK506 inhibits TNF-{alpha} secretion in human keratinocytes via regulation of NF-{kappa}B. Br J Dermatol (2005) 153:725–732.[CrossRef][Web of Science][Medline]
  11. Migita K, Miyashita T, Maeda Y, et al. FK506 suppresses the stimulation of matrix metalloproteinase 13 synthesis by interleukin-1beta in rheumatoid synovial fibroblasts. Immunol Lett (2005) 98:194–199.[CrossRef][Web of Science][Medline]
  12. Tuñón MJ, Sánchez-Campos S, Gutiérrez B, et al. Effects of FK506 and rapamycin on generation of reactive oxygen species, nitric oxide production and NF-{kappa}B activation in rat hepatocytes. Biochem Pharmacol (2003) 66:439–445.[CrossRef][Web of Science][Medline]
  13. Du S, Hiramatsu N, Hayakawa K, et al. Suppression of NF-{kappa}B by cyclosporine A and tacrolimus (FK506) via induction of the C/EBP family: implication for unfolded protein response. J Immunol (2009) 182:7201–7211.[Abstract/Free Full Text]
  14. Hayakawa K, Hiramatsu N, Okamura M, et al. Acquisition of anergy to proinflammatory cytokines in non-immune cells through endoplasmic reticulum stress response: a mechanism for subsidence of inflammation. J Immunol (2009) 182:1182–1191.[Abstract/Free Full Text]
  15. Okamura M, Takano Y, Hiramatsu N, et al. Suppression of cytokine responses by indomethacin in podocytes: a mechanism through induction of unfolded protein response. Am J Physiol Renal Physiol (2008) 295:F1495–F1503.[Abstract/Free Full Text]
  16. Hayakawa K, Hiramatsu N, Okamura M, et al. Blunted activation of NF-{kappa}B and NF-{kappa}B-dependent gene expression by geranylgeranylacetone: involvement of unfolded protein response. Biochem Biophys Res Commun (2008) 365:47–53.[CrossRef][Web of Science][Medline]
  17. Takano Y, Hiramatsu N, Okamura M, et al. Suppression of cytokine response by GATA inhibitor K-7174 via unfolded protein response. Biochem Biophys Res Commun (2007) 360:470–475.[CrossRef][Web of Science][Medline]
  18. Harama D, Koyama K, Mukai M, et al. A sub-cytotoxic dose of subtilase cytotoxin prevents LPS-induced inflammatory responses, depending on its capacity to induce the unfolded protein response. J Immunol (2009) 183:1368–1374.[Abstract/Free Full Text]
  19. Liptak P, Ivanyi B. Primer: histopathology of calcineurin-inhibitor toxicity in renal allografts. Nat Clin Pract Nephrol (2006) 2:398–404.[CrossRef][Web of Science][Medline]
  20. Busauschina A, Schnuelle P, van der Woude FJ. Cyclosporine nephrotoxicity. Transplant Proc (2004) 36(Suppl):229S–233S.[CrossRef][Web of Science][Medline]
  21. Naesens M, Kuypers DR, Sarwal M. Calcineurin inhibitor nephrotoxicity. Clin J Am Soc Nephrol (2009) 4:481–508.[Abstract/Free Full Text]
  22. Kim R, Emi M, Tanabe K, et al. Role of the unfolded protein response in cell death. Apoptosis (2006) 11:5–13.[CrossRef][Web of Science][Medline]
  23. Pallet N, Rabant M, Xu-Dubois YC, et al. Response of human renal tubular cells to cyclosporine and sirolimus: a toxicogenomic study. Toxicol Appl Pharmacol (2008) 229:184–196.[CrossRef][Web of Science][Medline]
  24. Bouvier N, Flinois JP, Gilleron J, et al. Cyclosporine triggers endoplasmic reticulum stress in endothelial cells: a role for endothelial phenotypic changes and death. Am J Physiol Renal Physiol (2009) 296:F160–F169.[Abstract/Free Full Text]
  25. Pallet N, Bouvier N, Bendjallabah A, et al. Cyclosporine-induced endoplasmic reticulum stress triggers tubular phenotypic changes and death. Am J Transplant (2008) 8:2283–2296.[CrossRef][Web of Science][Medline]
  26. Han SW, Li C, Ahn KO, et al. Prolonged endoplasmic reticulum stress induces apoptotic cell death in an experimental model of chronic cyclosporine nephropathy. Am J Nephrol (2008) 28:707–714.[CrossRef][Web of Science][Medline]
Received for publication: 9. 7.09
Accepted in revised form: 4. 9.09


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Extract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Kitamura, M.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kitamura, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?