Mechanism of cyclosporine A nephrotoxicity: Oxidative stress, autophagy, and signalings

Food and Chemical Toxicology 118 (2018) 889–907

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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

Mechanism of cyclosporine A nephrotoxicity: Oxidative stress, autophagy, and signalings

T

Qinghua Wua,b,c,∗, Xu Wangd, Eugenie Nepovimovab, Yun Wanga, Hualin Yanga, Kamil Kucab,∗∗ a

College of Life Science, Yangtze University, Jingzhou, 434025, China Department of Chemistry, Faculty of Science, University of Hradec Kralove, Hradec Kralove, Czech Republic c Jingchu Food Research and Development Center, Yangtze University, Jingzhou, 434025, China d National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, 430070, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Cyclosporine Nephrotoxicity Biomarker Oxidative stress Autophagy Therapy

Cyclosporine A (CsA) is a widely used immunosuppressive agent that greatly reduces the rates of kidney-, heart-, and liver-transplant rejection. However, CsA nephrotoxicity is a serious side effect that limits the clinical use of CsA. While the mechanisms underlying CsA nephrotoxicity are still not fully understood, increasing lines of evidence suggest that oxidative stress plays an important role in this phenomenon. Specifically, CsA induces endoplasmic reticulum stress and increases mitochondrial reactive oxygen species production: this modifies the redox balance, which causes lipid peroxidation and thereby induces nephrotoxicity. Recent studies on the pathogenesis of CsA nephrotoxicity suggest that CsA-induced autophagy can alleviate the deleterious effects of CsA-induced endoplasmic reticulum stress, thereby preventing nephrotoxicant-induced renal injury. A variety of signaling pathways participate in the pathogenesis of CsA nephrotoxicity. Specifically, the p38, ERK, and JNK MAPK subfamilies are all involved in CsA nephrotoxicity, while NF-κB is a target molecule of CsA. Moreover, the fibrogenic cytokine TGF-β1 contributes to CsA-induced renal fibrosis, while Nrf2 modulates CsA-induced cellular oxidative stress. In addition, CsA generally inhibits nitric oxide synthesis and impairs endothelium-dependent relaxation in the renal artery. However, some reports also suggest that nitric oxide synthesis is enhanced in the kidney cortex during CsA nephrotoxicity. Notably, the biomarkers of CsA nephrotoxicity associated with CsA have not been reviewed previously. Therefore, in this review, we will first provide an update on CsA nephrotoxicity in humans and describe the potential biomarkers of CsA nephrotoxicity. The molecular and cellular mechanisms that underlie CsA nephrotoxicity and the roles played by oxidative stress, autophagy, and signaling pathways will then be comprehensively summarized and discussed. Finally, the current therapeutical strategies for CsA nephrotoxcixity are summarized. We hope this review will provide a better understanding of CsA nephrotoxicity, thereby improving the management of patients who are treated with CsA.

1. Introduction Cyclosporine

A

(CsA)

(Fig.

1)

is

a

very

important

immunosuppressive drug and greatly improves the survival rates of patients and grafts after solid-organ transplantation (Tafazoli, 2015; Ziaei et al., 2016; Zimmermann et al., 2017). It is also increasingly

Abbreviations: CsA, Cyclosporine; GFR, glomerular filtration rate; EMT, epithelial-mesenchymal transition; RBP, retinol-binding protein; miR-494, microRNA-494; TLR4, Toll-like receptor 4; ER stress, endoplasmic reticulum stress; ROS, reactive oxygen species; LC-3II, light chain-3II; VEGF, vascular endothelial growth factor; CAA, CsA associated arteriolopathy; FSG, focal segmental glomerulosclerosis; IgAN, IgA nephropathy; FRNS, frequently relapsing nephrotic syndrome; SDNS, steroid-dependent nephrotic-syndrome; HO-1, heme oxygenase1; PCT, proximal convoluted tubules; KIM-1, kidney injury molecule-1; NGAL, neutrophil gelatinase-associated lipocalin; SOD, superoxide dismutase; CDH-1, cadherin-1; NO, nitric oxide; MDA, malonaldehyde; GSH, glutathione; GOX, glucose oxidase; TMBIM6, transmembrane BAX inhibitor motif containing 6; KRG, Korean red ginseng; TER, transepithelial electrical resistance; EGF, Epidermal growth factor; NOS, NO synthase; nNOS, neuronal NOS; iNOS, inducible NOS; eNOS, endothelial NOS; VEGF, vascular endothelial growth factor; RAS, reninangiotensin system; TAK1, TGF β-activated kinase1; HPTECs, human proximal tubular epithelial cells; SCCs, quamous cell carcinomas; EGFR-TKIs, epidermal growth factor receptortyrosine kinase inhibitors; NSCLC, non-small cell lung cancer; PKA, protein kinase A; NEO, Sandimmun Neoral; IMM, SDZ IMM 125; VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; HIV, human immunodeficiency virus; SDNS, steroid-dependent nephrotic syndrome; Nox2, nicotinamide adenosine diphosphate oxidase 2; PTEN, phosphatase and tensin homolog deleted on chromosome 10 ∗ Corresponding author. College of Life Science, Yangtze University, Jingzhou, 434025, China. ∗∗ Corresponding author. E-mail addresses: [email protected] (Q. Wu), [email protected] (K. Kuca). https://doi.org/10.1016/j.fct.2018.06.054 Received 8 May 2018; Received in revised form 21 June 2018; Accepted 22 June 2018 Available online 28 June 2018 0278-6915/ © 2018 Elsevier Ltd. All rights reserved.

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Fig. 1. Structure of cyclosporine A (CsA) and its main metabolites.

already been established (Carlos et al., 2014). They include urinary retinol-binding protein (RBP), which accurately predicts progressive CsA nephrotoxicity in heart-transplant patients (Camara et al., 2001). Moreover, microalbuminuria along with elevated urinary levels of kidney injury molecule-1 (KIM-1), tumor necrosis factor (TNF)-α, and fibronectin are indicative of acute CsA nephrotoxicity while high urinary osteopontin and transforming growth factor (TGF)-β1 levels may be potential biomarkers for chronic CsA nephrotoxicity (Carlos et al., 2014). The levels of these urinary kidney injury biomarkers also correlate well with the temporal development of CsA nephrotoxicity (Carlos et al., 2014). Potential biomarkers of CsA nephrotoxicity in the blood include indoxyl sulfate and nicotinamide adenosine diphosphate oxidase 2 (Nox2), both of which are significantly elevated in patients with CsA nephrotoxicity (Umino et al., 2010). Several very recent studies (Gooch et al., 2017; Yuan et al., 2015; González-Guerrero et al., 2017) also suggest that several genetic elements, including microRNA494 (miR-494) and Toll-like receptor 4 (TLR4), could also serve as CsA nephrotoxicity biomarkers. The validation of these molecules as effective CsA nephrotoxicity biomarkers, and the identification of new biomarkers, will greatly help to rapidly diagnose CsA nephrotoxicity, thereby making the treatment much safer. At present, the cellular and molecular biological mechanisms underlying CsA nephropathy are not completely understood. However, a series of early and more recent studies suggest that an important mechanism is oxidative stress (Redondo-Horcajo and Lamas, 2005; ElBassossy and Eid, 2018). This notion is supported by several lines of evidence. First, CsA administration significantly increases the xanthine oxidase activity and the levels of oxidants (Josephine et al., 2007). A recent study also showed that oxidative stress combined with

being used to treat autoimmune diseases such as psoriasis and rheumatoid arthritis (Di Lernia et al., 2016; Kisiel et al., 2015). However, the chronic use of CsA associates with high incidences of nephrotoxicity and the eventual development of chronic renal failure (Caires et al., 2018; Lai et al., 2017). For example, most CsA-treated cardiac-transplant patients present with some renal damage after transplantation (Camara et al., 2001; Tedesco and Haragsim, 2012) while all patients on long-term treatment with CsA experience chronic nephrotoxicity (da Silva et al., 2014). The chronic CsA nephrotoxicity is usually characterized by tubular atrophy, inflammatory cell influx, striped tubulointerstitial fibrosis, arteriolopathy, and increased intrarenal immunogenicity (Yoon and Yang, 2009; Lee, 2010). CsA exerts its nephrotoxic activity by targeting renal tubular epithelial cells (McMorrow et al., 2005). Thus, Liu et al. showed that CsA promotes the epithelial-mesenchymal transition (EMT) in these cells (Liu et al., 2017a). EMT is thought to mediate the inflammation-induced fibrosis and eventual failure of organs such as the kidney (LópezNovoa and Nieto, 2009). Several other studies also found that CsA inhibits the DNA synthesis of tubular epithelial cells and induces their apoptosis (Kim et al., 2000). Indeed, all renal biopsy specimens from patients with CsA nephrotoxicity exhibit tubular cell apoptosis (Rao et al., 2017; de Arriba et al., 2013). A very recent study (Wirestam et al., 2017) suggests that CsA can also damage renal tubular cells by indirect mechanisms, namely, by inducing the production of osteopontin, which promotes inflammation that injures the renal cells. Given the common occurrence of CsA nephrotoxicity in organtransplant patients, many researchers have sought to isolate biomarkers of this disease that will allow it to be identified in a timely manner. Several biomarkers of CsA nephrotoxicity in the urine and blood have 890

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First, Rafiee et al. (2002) found that CsA inhibits NO synthesis, including in the kidney. Later, Shihab et al. (2003) found that in chronic CsA nephrotoxicity, the renal expression of vascular endothelial growth factor (VEGF) and its receptors is upregulated and that this effect is due to CsA-mediated suppression of NO synthesis. However, other studies show that the NO pathway is an important regulatory mechanism that protects against CsA-associated vasoconstriction (Ikesue et al., 2000). Thus, there are many unanswered questions regarding the role of the NO pathway in CsA nephrotoxicity. In 2004, the CsA nephrotoxicity and its potential strategies for the prevention of nephrotoxicity were briefly dicussed by Busauschina et al. (2004). However, to date, a systematic review that comprehensively summarizes the mechanisms that underlie CsA nephrotoxicity, which describes the roles played by oxidative stress, autophagy, and signaling pathways, has not been published. Indeed, most studies on CsA are clinical case reports on CsA nephrotoxicity (Mulic et al., 2017; Pavleska-Kuzmanovska et al., 2014) or reviews on the role of CsA in hypertension and dermatology (Dehesa et al., 2012; El-Gowelli and ElMas, 2015). Only Xiao et al. (2013a,b) collected and analyzed the clinical data of transplant patients who were treated with CsA. However, they mainly discussed the apoptotic profiles of these patients in their meta-analysis. Yoon and Yang (2009) published a review article that discusses concepts in CsA nephropathy and preventive strategies. However, some important information was not discussed, including the current progress in identifying biomarkers of CsA nephrotoxicity. They also did not describe the role of oxidative stress, the recent findings regarding the role of autophagy, or the signaling pathways that are involved in CsA nephrotoxicity. In addition, none of these earlier publications discussed the biomarkers of CsA nephrotoxicity. Therefore, in the present review, we will first provide an update on CsA-induced nephrotoxicity in humans and animals. This section will include a summary of the potential biomarkers of CsA nephrotoxicity that have been found. We will then progress to our main goal, which is to fully summarize and discuss the roles of oxidative stress, autophagy, and various signaling pathways in the pathogenic processes that drive CsA nephrotoxicity. Finally, we will summarize the current therapeutical approaches for CsA nephrotoxicity. We hope that this review will help to further illuminate the cellular and molecular mechanisms that drive CsA nephrotoxicity and that this will spur the development of effective treatments for this disease.

mitochondrial damage plays an important role in CsA hepatotoxicity (Korolczuk et al., 2016). Second, there is evidence that the induction of CsA nephropathy involves endoplasmic reticulum (ER) stress, which is closely connected to oxidative stress (Chong et al., 2017). This notion is supported by the fact that renal-transplant patients who are treated with CsA exhibit upregulation of an ER stress marker, immunoglobulinbinding protein, in kidney-transplant biopsies (Pallet et al., 2008a). Notably, CsA-induced ER stress also triggers phenotypic changes that resemble the EMT and apoptotic death in human tubular cells both in vivo and in vitro (Pallet et al., 2008b); both of these phenomena have been implicated previously to participate in CsA nephrotoxicity (Liu et al., 2017a). Third, CsA-induced apoptosis in renal tubular cells relates to oxidative damage and mitochondrial fission (de Arriba et al., 2013). Notably, reactive oxygen species (ROS) have been implicated in CsA nephrotoxicity (Ciarcia et al., 2015; Lai et al., 2017). Fourth, as well as increasing oxidative stress, CsA treatment also greatly decreases the antioxidant capacity of the kidneys. This could sensitize the kidneys to various stresses, thus making them more susceptible to nephrotoxic agents (Ghaznavi et al., 2007). Another important mechanism that may underlie CsA nephrotoxicity is autophagy (Kim et al., 2014; Fakharnia et al., 2017). Although studies on the role of autophagy in CsA nephrotoxicity started only 10 years ago, the evidence to date suggests that autophagy plays very complicated functions in CsA nephrotoxicity (Pallet et al., 2008c; Høyer-Hansen and Jaattela, 2007; Fakharnia et al., 2017). On one hand, it appears that autophagy protects renal tubular cells from CsA-induced nephrotoxicity (Pallet et al., 2008c; Ciechomska et al., 2013; Kim et al., 2014). Thus, Pallet et al. (2008c) showed that when primary human renal tubular cells are treated with CsA, they undergo autophagy, as indicated by the expression of the autophagy marker microtubule-associated protein light chain-3II (LC3II) and the development of autophagosomes. They also showed that this autophagic response to CsA treatment is driven by CsA-induced ER stress: not only did various ERstress inducers activate autophagy, the administration of salubrinal, an agent that protects cells from ER stress, inhibited the LC3II expression of CsA-treated tubular cells (Pallet et al., 2008c). Later, Yoo and Jeung (2010) showed that CsA-induced autophagy alleviates the deleterious effects of ER stress by eliminating misfolded proteins. Thus, when renal cells encounter CsA, they not only undergo ER stress and oxidative stress, they also undergo autophagy, which attenuates the ER stressinduced nephrotoxicity. On the other hand, CsA-induced autophagy itself enhances the nephrotoxicity caused by CsA treatment (Xiang et al., 2013; Lai et al., 2015). Thus, autophagy plays a Janus-faced role in CsA nephrotoxicity. These findings suggest that fine regulation of autophagy could be an important therapeutic target for preventing the chronic allograft dysfunction that is caused by long-term CsA treatment. Further research that elucidates the complex functions of autophagy in CsA nephrotoxicity is warranted. The molecular mechanisms that underlie CsA nephrotoxicity involve a number of important signaling pathways. In particular, the p38, ERK, and JNK MAPK subfamilies all participate in CsA nephrotoxicity (Kiely et al., 2003; Martin-Martin et al., 2010; Yang et al., 2003) and the crosstalk between MAPK and Smad regulates CsA-induced apoptosis in renal proximal tubular cells (Iwayama et al., 2011). Moreover, NF-κB is a crucial target molecule of CsA (Nakahara et al., 2003). Interestingly, Jin et al. (2017) showed very recently that the PDLIM2/NF-κB p65 signaling pathway mediates the ability of Klotho, an anti-aging protein, to prevent CsA nephropathy. Furthermore, the TGF-β1/interleukin (IL)-2/cyclooxygenase (COX)-2 crosstalk pathway participates in CsA-induced renal tubular atrophy and interstitial fibrosis (El-Gowelli et al., 2014). Moreover, CsA treatment of renal proximal tubular epithelial cells strongly activates the Nrf-2 pathway (Hamon et al., 2014), and this activity modulates the cellular oxidative stress caused by CsA (Lai et al., 2017). Nitric oxide (NO) is a vasoactive factor that plays a key role in maintaining vascular tone in the kidney (Liu et al., 2017b) and several studies suggest that it also participates in CsA nephropathy.

2. CsA nephrotoxicity A number of clinical reports show that CsA induces nephrotoxicity in humans, especially in renal-transplant patients (Benway and Iacomini, 2018; Xiao et al., 2013a; b; Yoon and Yang, 2009). It has also been reported in recipients of solid organs other than the kidney and in patients with autoimmune diseases (Schwaiger et al., 2014; Manito et al., 2011). It manifests as two distinct and well-characterized forms, namely, acute and chronic nephrotoxicity (Burdmann et al., 2003). Multiple biomarkers of CsA neprotoxicity have been identified to date. These biomarkers, which have not been reviewed to date, include urine proteins such as RBP and blood proteins such as indoxyl sulfate. The identification of biomarkers that effectively detect CsA nephrotoxicity is crucial as it permits the quick clinical detection and treatment of this disease (Umino et al., 2010). In this section, the clinical reports of CsA nephrotoxity in humans, and the specific symptoms of this disease, are summarized. Moreover, the potential biomarkers of CsA nephrotoxicity are reviewed and discussed. 2.1. CsA nephrotoxicity in humans CsA is nephrotoxic in humans, especially transplant patients (Damiano et al., 2015). For example, the clinical experience of a Romanian medical center for more than 10 years (1999–2011) showed that when heart-transplant recipients were treated with CsA, 20% 891

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that CsA treatment greatly improved their renal survival and remission rates. However, many of the children required ongoing immunosuppression. There is some controversy regarding whether CsA is nephrotoxic in patients with SDNS and SRNS. Fujinaga et al. (2012) found that two of ten SDNS patients who underwent once-daily CsA therapy developed chronic CsA nephrotoxicity. However, this study lacked a control group. Similarly, however, Fujinaga and Shimizu (2013) also found that long-term (2–3 years) CsA administration in patients with SRNS is an independent risk factor for chronic nephrotoxicity. Moreover, when a patient who developed SRNS at the age of 14 was treated with CsA, her renal function declined and she had to undergo renal transplantation at age 23 (Nakazawa et al., 2015). Notably, her post-transplant course was stable with no symptoms of rejection or CsA nephrotoxicity. It is likely that this patient could not respond properly to CsA because of a congenital absence of the portal vein that impaired drug metabolism (Nakazawa et al., 2015). By contrast, Kranz et al. (2008) showed that even long-term (> 5 years) CsA treatment is safe in SDNS. This position was supported very recently by Kuroyanagi et al. (2018): they retrospectively evaluated the target serum C2 levels in children (average age, 5.2 ± 2.9 years) with SDNS at the time CsA treatment was initiated and 2 years later (when the kidneys were biopsied). Only one very mild case of CsA-associated nephrotoxicity was identified. Moreover, CsA treatment did not affect the estimated GFR. Similar to Kranz et al. that 2 years of treatment with a medium dose of CsA is relatively safe for SDNS in terms of CsA nephrotoxicity (Kuroyanagi et al., 2018). It should be noted, however, that these two studies are limited by their retrospective design and the fact that only 25% of the patients underwent renal biopsy after longterm nephrotoxic treatment (Kranz et al., 2008). While prospective studies on the outcomes of long-term CsA therapy in children with SDNS and SRNS would help resolve this controversy, they may not be reasonable from an ethical perspective. Therefore, the long-term use of CsA in humans, especially in renaltransplant patients, usually causes nephrotoxicity. Based on the data from a meta-analysis (da Silva et al., 2014), a greater than 30% incidence of nephrotoxicity is observed in the patients on long term CsA therapy. CsA direct affects renal tubular epithelial cells: specifically, it promotes EMT by increasing TGF-β1 protein levels, and it induces tubular cell apoptosis, DNA damage, and great increases in intracellular calcium levels. CsA is used to treat SDNS and SRNS and there is controversy about whether it causes nephrotoxity in these patients. This largely reflects the fact that most of these studies involved limited patient numbers and had a retrospective design, or even lacked a control group. The precis of relevant literature of CsA nephrotoxicity in humans are summarized in Table 1.

developed significant chronic decreases in their kidney function (Ispas et al., 2012). Moreover, CsA generates nephrotoxicity by direct effects on renal tubular epithelial cells that include the induction of EMT (Liu et al., 2017a). EMT not only plays an important role in embryonic development and tumorigenesis, it also participates in the organ remodeling that occurs when chronically diseased parenchymal organs such as the kidney undergo fibrosis (Simeoni et al., 2018). A human renal cell line treated with CsA, elevated the expression of both the profibrotic protein TGF-β1 and connective tissue growth factor (CTGF) expression. Since both the EMT and CTGF expression were abrogated by neutralizing TGF-β1 antibody treatment, it appears that CsA directly stimulates EMT in renal tubulular epithelial cells by elevating TGF-β1 expression: this induces the expression of CTGF, which serves as the downstream mediator of the profibrotic activity of TGF-β1 (McMorrow et al., 2005). CsA also induces nephrotoxicity by directly inducing tubular cell apoptosis. Indeed, renal biopsy specimens from patients with CsA nephrotoxicity always exhibit apoptosis (Rao et al., 2017). In addition, CsA is directly toxic to LLC-PK1 renal tubular cells and that this effect associates with DNA synthesis inhibition and the induction of apoptosis that is mediated by the Fas antigen-ligand system (Kim et al., 2000). Notably, Granchi et al. (2018) showed recently that when renal tubular cells are injured by CsA, their cell calcium levels rise and activate the expression of the glucose transporter GLUT1, which is a stress response protein. This is significant because Kim et al. (2000) reported many years previously that GLUT1 mediates the cytotoxicity of CsA in tubular cells. Thus, it appears that CsA increases the calcium levels of renal tubular cells: this activates GLUT1, which in turn upregulates glycolysis. This metabolic response may play a prominent role in the recovery of tubules that were injured by CsA. IgA nephropathy (IgAN) is an autoimmune disease and is the most common glomerulonephritis worldwide (Xie et al., 2017). A study on 18 children (4.2–13.9 years of age) with IgAN who were treated with CsA for 8 or 12 months and who underwent renal biopsies before and after treatment showed that CsA treatment promoted the development of interstitial fibrosis, perhaps because it increased TGF-β1 expression in the kidney. Thus, the long-term use of CsA in IgAN patients may not be indicated. Interestingly, the study also found that the CsA-induced increase in interstitial fibrosis did not associate with increased osteopontin production and macrophage numbers in the kidney (Lim et al., 2009). Although the CsA nephrotoxicity is correlated with the duration of CsA treatment in pediatric patient, Hamasaki et al. (2017) recently found that most of cases of CsA nephrotoxicity involved arteriolar hyalinosis, suggesting that long-term CsA treatment is useful for treatment of frequently relapsing nephrotic syndrome. Osteopontin is produced by various immune cells and acts as a chemoattractant for macrophages. Wirestam et al. (2017) recently reported that patients with systemic lupus erythmatosis, which is an autoimmune disease that often associates with kidney fibrosis, have high circulating levels of osteopontin. This molecule may promote inflammatory damage to the kidney, which in turn induces interstitial fibrosis. These findings could suggest that osteopontin may also participate in renal interstitial fibrosis that is induced by CsA. Indeed, this notion is supported by the fact that osteopontin-null mice are less susceptible to chronic CsA nephropathy than wild-type mice (Mazzali et al., 2002). However, since CsA treatment of the CsA-treated children with IgAN did not increase their kidney levels of osteopontin and macrophages, it may be that osteopontin does not itself increase TGF-β1 expression and may thus be involved in renal fibrosis only indirectly. Notably, CsA is sometimes a useful treatment for nephrosis. In particular, once-daily CsA is the treatment of choice for children with steroid-dependent nephrotic syndrome (SDNS) (Hamasaki et al., 2009). Children with steroid-resistant nephrotic syndrome (SRNS) also benefit from CsA treatment: when Hamasaki et al. (2013) prospectively analyzed the 5-year outcomes of children with SRNS who enrolled in their previous prospective multicenter trial on CsA and steroids, they found

2.2. Biomarkers of CsA nephrotoxicity The recent advances in genomics, proteomics, and molecular pathology have led to the identification of many biomarkers that may be of clinical value (Ludwig and Weinstein, 2005). It is of considerable interest to identify biomarkers of CsA nephrotoxicity (especially those in the urine and blood) because they could greatly aid the early detection of this disease (Camara et al., 2001; Umino et al., 2010; Fernando et al., 2014). This is important because detecting acute and chronic CsA nephrotoxicity at an early stage will help minimize kidney injury, which can become irreversible if the CsA therapy is not halted or reduced in a timely fashion (Carlos et al., 2014; Filler, 2011). A number of biomarkers in the urine that could be used to rapidly detect CsA nephrotoxicity have been identified. Camara et al. (2001) showed that urinary RBP levels accurately predict progressive CsA nephrotoxicity in heart-transplant patients. RBP is a low-molecular-mass protein (21 kDa) that is easily filtered in the renal glomeruli and is very efficiently reabsorbed by the proximal convoluted tubules (Woll et al., 2017). When the proximal convoluted tubules are dysfunctional, the 892

5.4

4.2–13.9

29

48 males, 14 females

13 males; 5 females

1 male

Patients with frequently relapsing nephrotic syndrome Patients with IgAN

893 4–24 h

72 h

1 μg/ml

420 nM-42 μM

Human renal proximal tubular cells

Human with hepatitis B virus and HIV

Human mesangial cells

8 and 12 months

24 months

CsA significantly increased cell apoptosis and induced the DNA damage. Positive staining for both iNOS and p53 proteins was observed. CsA-induced EMT was associated with increased TGFβ1 protein levels; EMT was markedly attenuated in the presence of anti-TGF-β1 antibody.

Interstitial fibrosis developed or was aggravated; TGF-β expression was significantly increased; Osteopontin and macrophages may be indirectly involved in renal fibrosis by prolonging interstitial inflammation. Acute renal dysfuntion was observed.

Renal survival at 5 years was 94.3%. Patient status of complete remission was 88.6%; Partial remission in one; and non-remission in three, including chronic kidney disease and end-stage kidney disease. CsA therapy resulted in a significant reduction in the median relapse rate; Two patients showed evidence of chronic CsA nephrotoxicity. CsA nephrotoxicity was detected in only 8.6% of patients after 2 years of treatment.

12 months

> 24 months

50% of patients with SRNS treated with CsA showed histological evidence of CsA nephrotoxicity.

The prevalence of CsA nephrotoxicity was 64%, with the vast majority of patients having mild arteriolar hyalinosis without significant interstitial fibrosis. Patients with and without nephrotoxic, CsA therapy showed a similar drop in GFR. CsA increased tubular expression of TLR4 and its ligand HSP70.

Only one very mild case of CyA-associated nephrotoxicity was identified

Heart transplant patients with high RBP levels had worse renal prognostic, kidney survival, and renal failure. CsA associated nephrotoxicity was not observed.

Sixteen patients were diagnosed with CsA nephroxicity; The mRNA levels of TGF-β, collagen, fibronec tin, MMP-2, and osteopontin were significantly upregulated. Nephrotoxicity was present even at low trough CsA concentration.

Major results

> 24 months

8–24 months

5 years

> 4 years

2 years

3 days

6 weeks

5 mg/kg/day

80–100 ng/ml; 60–80 ng/ml

2.8 ± 0.6 mg/kg/day

120–150 ng/ml for 3 months, followed by 80–100 ng/ml for 9 months

2–3 mg/kg/day

100–150 mg/m2 per body surface area 5 mg/kg/day

422.2 ± 133.5 ng/ml

4 mg/kg/day

6 months

4–78 months

Time

1.25–3 mg/kg/day

2.2–13.5

5.4 ± 2.2

9.4

12 males; 8 females 14 males; 4 females

5.7 ± 4.0

9 males; 2 females

19 males; 9 females

Patients with SDNS

5.2 ± 2.9

Patients with steroiddependent MCNS

17 males, 11 females

Patients with SDNS

23

7.7

1 female

Renal recipients

150–174.5 ng/ml

44.8 ± 11.1

21 males; 14 females

34 males; 3 females

Heart transplant patients

50 ng/ml

325 mg/day; 189 ng/ml

Dose

9

1.3–13.8

1 male

Kidney transplant patient

41–49

7 males, 5 females

47 males

Transplant patients

Age (years)

Patients with SDNS Patients with IgAN or minimal change nephrotic syndrome (MCNS) Patients with steroidresistant nephrotic syndrome Patients with steroidresistant nephrosis

Sex

Objectives

Table 1 Summary of cyclosporine A-induced nephrotoxicity in humans.

(Jankauskiene et al., 2001)

This is an unusual case of nephrotoxicity and impaired renal function with a very low CsA blood trough concentration on post-transplant treatment. RBP is useful marker of tubulointerstitial injury in transplant patients, mainly caused by CsA toxicity.

CsA is a direct stimulus for EMT in renal tubule epithelial cells and implicate TGF-β1 as mediators of this response.

CsA-induced nephrotoxicity in patients with autoimmune diseases is even greater in patients who are HIV positive. CsA-induces apoptosis is mediated by iNOS via p53.

Microemulsified CsA administered according is safe and effective in children with frequently relapsing nephrotic syndrome Increased levels of TGF-β and the development of interstitial fibrosis limit the long-term use of CsA in IgAN patients.

Once-daily CsA therapy appears to be effective in children with steroid-dependent MCNS.

CsAN prevalence appears to be higher in patients with SRNS than in those with steroid-sensitive nephrotic syndrome. Although SRNS treatment with CsA provides high renal survival and remission rates, many children require ongoing immunosuppression.

CsA seems to be safe even in long-term treatment for more than 5 years TLR4 is influenced by both direct toxicity and impediment of renal microcirculation in human CsA nephrotoxicity.

The post-transplant course is stable with no symptoms of rejection or CsA nephrotoxicity. A 2-year treatment with a medium dose of cyclosporine A is relatively safe with regard to the development of CsA nephrotoxicity. The risk of CsA nephrotoxicity is positively associated with the duration of continuous CsA treatment but not with the total treatment period.

(Khanna et al., 2002)

The expression of TGF-β and profibrogenic molecules in the renal tissues of renal transplant recipients treated with CsA.

(McMorrow et al., 2005)

(Amore et al., 2000)

(Mignogna et al., 2005)

(Lim et al., 2009)

(Ishikura et al., 2010)

(Fujinaga et al., 2012)

(Hamasaki et al., 2013)

(Fujinaga and Shimizu, 2013)

(Lim et al., 2009)

(Kranz et al., 2008)

(Fujinaga and Urushihara, 2017)

(Nakazawa et al., 2015) (Kuroyanagi et al., 2018)

(Camara et al., 2001)

References

Major conclusions

Q. Wu et al.

Food and Chemical Toxicology 118 (2018) 889–907

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important biomarker and pharmacological target in CsA nephrotoxicity (González-Guerrero et al., 2017). Moreover, since Xiao et al. (2013a,b) found that angiotensin II and the NO and ER pathways are novel treatment targets in chronic CsA nephrotoxicity and Cheng et al. (2012) showed that Bip/Grp78 regulates CsA-induced tubular vacuolization, these molecules may also be biomarkers of CsA nephrotoxicity. In summary, the identification of potential biomarkers of CsA nephrotoxity has become an important field of research in the last decade because these biomarkers could aid the early detection of CsA nephrotoxity and facilitate the safer and more rational use of CsA. This field of research also enhances our understanding of the mechanisms that underlie CsA nephrotoxicity. These biomarkers may first regulate the proinflammatory signalings (for example MAPK and JAK/STAT), cytokines, and subsequently induce the renal cell oxidative stress, autophagy, and apoptosis. Therefore, to manipulate the expression of these biomarkers may attenuate the nephrotoxicity. However, up to date, the real function of these biomarkers in the mechanisms of CsA nephrotoxicity is poorly understood. The most promising urinary biomarkers to date are RBP, which may predict CsA nephrotoxicity in heart-transplant patients, and KIM-1, TNF-α, fibronectin, and microalbuminuria, which are markers of acute CsA nephrotoxicity. Blood biomarkers of interest include indoxyl sulfate level, which may predict chronic CsA nephropathy, and Nox2, which modulates CsA-induced hypoxia. Moreover, since inhibiting miR-494 and TLR4 suppresses CsA nephrotoxicity, they may be important genetic biomarkers of this disease. The validity of these potential biomarkers should be tested in larger numbers of patients. The potential biomarkers for the diagnosis of CsA nephrotoxicity are summarized in Table 2.

urine concentrations of RBP rise noticeably (Camara et al., 2001). Urine RBP levels may be a better biomarker than the urine levels of other proteins such as β2-microglobulin and lysozyme because its production is relatively constant (Umino et al., 2010). Moreover, since proteinuria precedes serum creatinine elevation, urinary RBP levels may be a marker of relatively early tubulointerstitial involvement. This is of clinical importance because the degree of tubulointerstitial injury is believed to be the best predictor of GFR and the long-term prognosis of chronic progressive renal disease (Camara et al., 2001). In another study, Carlos et al. (2014) found that microalbuminuria and urinary KIM-1, TNF-α, and fibronectin levels rose in the early phase of CsA nephrotoxicity while the urinary levels of TGF-β, and osteopontin increased in the late phase of CsA nephrotoxicity. These urinary biomarkers exhibited consistent correlations between the kidney expression of cytokines and the temporal changes in kidney function and structure that are caused by CsA. Thus, early microalbuminuria and increases in urinary KIM-1, TNF-α, and fibronectin indicate acute CsA nephrotoxicity while late elevation of urinary osteopontin and TGF-β1 indicate chronic CsA nephrotoxicity. In addition, the proteomic analysis of the urine of mice with CsA nephrotoxicity by O'Connell et al. (2011) showed that cadherin-1, superoxide dismutase, and vinculin are involved in early CsA nephropathy. However, whether these proteins could serve as early biomarkers of CsA nephrotoxicity was not assessed. A number of biomarkers in blood samples that detect CsA-induced renal injury have also been identified. When Umino et al. (2010) measured the cystatin C, indoxyl sulfate, creatinine, and β2-microglobulin levels in the blood from CsA-treated patients with frequently relapsing SDNS, they found that only indoxyl sulfate was significantly elevated in the patients with CsA nephrotoxicity. They speculated that when evaluating chronic CsA nephropathy in pediatric patients with frequently relapsing SDNS, repeated serum indoxyl sulfate measurements could be used instead of repeated renal biopsies. In addition, the prospective study of live related renal-allograft recipients by Naqvi et al. (2005) showed that fractional excretion of serum magnesium is a useful marker of CsA toxicity that is independent of CsA blood levels. Recently, Djamali et al. (2016) provided several lines of evidence that suggested that Nox2 plays an important role in CsA-induced chronic hypoxia. First, chemical and genetic inhibition of Nox2 in rats abrogated CsA-induced hypoxia independently of regional perfusion. Second, Nox2 knockout associated with less oxidative stress and fibrogenesis. Third, liver-transplant recipients with chronic CsA nephrotoxicity exhibited significantly greater Nox2 expression. Thus, Nox2 appears to be a modulator of CsA-induced hypoxia and may be a potential biomarker of CsA nephrotoxicity. Several studies show that microRNAs also participate in CsA nephrotoxicity (Gooch et al., 2017; Yuan et al., 2015). In particular, Yuan et al. (2015) found that mice with CsA nephrotoxicity exhibited an early increase in the miR-494 expression in the kidney. They also found that when tubular epithelial cells underwent EMT due to CsA treatment in vitro, they exhibited upregulated miR-494 expression and decreased levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). They then showed that miR-494 directly targets PTEN and negatively regulates its expression, and that blocking this activity of miR-494 abrogated CsA-induced EMT. Thus, miR-494 plays a key role in CsA-induced EMT and nephrotoxicity by downregulating PTEN. This study suggests miR-494 in the kidney may be a useful biomarker of CsA nephrotoxicity. Moreover, strategies that block miR-494 expression may be a novel way to prevent this disease. Notably, González-Guerrero et al. (2017) showed very recently that TLR4 is a potential pharmacological target in CsA nephrotoxicity. They found that CsA treatment induced renal TLR4 expression in wild-type mice and that targeting TLR4 pharmacologically or genetically inhibited the CsA-induced activation of the proinflammatory JNK/c-jun, JAK2/STAT3, IRE1α, and NF-κB signaling pathways. Inhibiting TLR4 also reduced CsA-induced tubular damage and markedly suppressed the development of kidney fibrosis. Thus, the TLR4 gene may be an

3. Mechanism of oxidative stress in CsA nephrotoxicity Oxidative stress has various adverse effects on many biological systems. It is also a major effect of CsA nephrotoxicity (RedondoHorcajo and Lamas, 2005; El-Bassossy and Eid, 2018).When rats are administered 25 mg/kg/day CsA for 21 days, their renal tissues exhibit increased levels of oxidants, xanthine oxidase activity, and inducible NO synthase (NOS) mRNA (Josephine et al., 2007). There is also a concomitant increase in the plasma NO levels. Thus, CsA clearly induces nitrosative stress. This stress is likely to induce apoptosis since CsA increases the cysteine protease, caspase-3, and caspase-6 production by rat hepatocytes (Grub et al., 2000). However, since enzyme leakage is not observed, it appears that CsA does not damage the integrity of the outer cell membrane. The genome-wide expression analysis of rat tubular renal cells by Pallet et al. (2008a) showed that CsA induced ER stress activity. They also observed that CsA treatment in renal-transplant patients associates with the upregulation of immunoglobulin-binding protein, an ER stress marker, in kidney-transplant biopsies. A large-scale study that tests the ability of immunoglobulin-binding protein to serve as an early CsA nephrotoxicity biomarker in transplant patients is warranted. Pallet et al. (2008b) then showed that CsA-induced ER stress triggers EMT-like phenotypic changes in human tubular cells and their death, and that these effects can be prevented by salubrinal, which protects against ER stress. Notably, the ability of CsA to suppress T-cell activation is also mediated by its ability to induce ER stress and oxidative stress; these effects lead to the apoptosis of T cells (Hama et al., 2013; O'Connell et al., 2011; Schmeits et al., 2015). These observations suggest that CsA induces nephrotoxicity by activating an ER-specific apoptotic pathway, and that ER stress may be a factor that predicts nephrotoxicity and its clinical outcomes (Xiao et al., 2013a; b). ROS production in mitochondrion and oxidative stress have been implicated in the pathogenesis of a variety of renal diseases, including IgAN and chronic kidney disease (Damiano et al., 2015). Multiple studies show that ROS are also involved in CsA nephrotoxicity (Ciarcia et al., 2015; Lai et al., 2017; Satyanarayana and Chopra, 2002). In particular, CsA treatment significantly increased the production of ROS 894

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(González-Guerrero et al., 2017)

(Djamali et al., 2016)

(Yuan et al., 2015)

TLR4 inhibition reduced tubular damage and drastically prevented the development of kidney fibrosis. Kidney TLR4

Mice

Nox2 is a modulator of CsA-induced hypoxia. Liver transplant recipients Blood Nox2

Mice Kidney miR-494

Salt-depleted rats Urine

by human renal mesangial cells (O'Connell et al., 2011). CsA may also promote its neurological side effects in transplantation patients by augmenting the generation of ROS; Mun and Ha (2010) showed that CsA treatment of glioma cells induced ROS production and reduced their antioxidant status, although it did not alter their levels of the oxidative stress marker malondialdehyde (MDA). Moreover, when rats were treated with CsA, their renal function dropped and their kidneys exhibited tissue peroxidation (Satyanarayana and Chopra, 2002). These findings together show clearly that ROS plays a pivotal role in CsAinduced renal dysfunction. Co-treatment of rats with CsA, which can induce tissues to produce ROS (Ay et al., 2007; Francis and Baynosa, 2017), significantly reduced CsA-induced oxidative stress while having no effect on CsA-induced nephrotoxicity. Hyperbaric oxygen by itself did not increase renal oxidative stress. This suggests that ROS are involved in CsA nephrotoxicity but are not the direct cause of this toxic activity (Ay et al., 2007). Djamali et al. (2016) reported recently that NADPH-oxidase 2 is responsible for the CsA-induced increases in oxidative stress. It was thought that Rho-associated kinase may mediate this role of NADPHoxidase 2 because Park et al. (2011) found that fasudil, a Rho/Rhokinase inhibitor, ameliorates CsA nephrotoxicity. However, this possibility was challenged recently by El-Yazbi et al. (2017, 2018): they found that while the hemodynamic, autonomic, left ventricular and histopathological disturbances of CsA-treated rats are abrogated by inhibiting NADPH-oxidase 2, fasudil had no effect. Fasudil also did not reduce NADPH-oxidase 2 levels. This suggests that CsA exerts its cardiotoxic effects via Rho-associated kinase-independent upregulation of NADPH-oxidase 2 (Xiao et al., 2013a; b; El-Yazbi et al., 2017; El-Yazbi et al., 2018). Notably, Redondo-Horcajo and Lamas (2005) showed that when bovine aortic endothelial cells are treated with CsA, they produce ROS and nitrogen intermediates that may lead to the intracellular formation of peroxynitrite. The latter agent may be a crucial mediator by which CsA induces nitration of tyrosine (antioxidant-sensitive), a marker for nitrosative stress-mediated endothelial damage (RedondoHorcajo and Lamas, 2005). Several studies show that CsA-induced apoptosis in renal tubular cells relates to mitochondrial fission (de Arriba et al., 2013). Since one of the main sources of intracellular ROS is the mitochondria, de Arriba et al. (2013) studied the effects of CsA on the mitochondrial functions in LLC-PK1 tubular cells. First, they showed that CsA induces ROS synthesis and decreases the levels of the antioxidant glutathione. Subsequently, they found that CsA reduces the mitochondrial membrane potential (ΔΨm), generates mitochondrial permeability transition pores, and causes cytochrome c to be released into the intermembrane space. It is noteworthy that as well as increasing oxidative stress, CsA treatment also decreases the antioxidant capacity of the kidney. This is exemplified by the study of Ghaznavi et al. (2007): when they treated rats with CsA, the total antioxidant capacity in the kidney tissues dropped significantly. A low antioxidant status is likely to sensitize the kidney to various stresses and thereby lead to nephrotoxicity. It is also possible that the CsA-induced dietary effect or nutritional status promote nephrotoxicity. Jeon et al. (2012) also found that when the BEAS2B human bronchial epithelial cell line was treated with CsA, ROS production increased and the biological antioxidant potential dropped. The resulting radical-induced damage increased the levels of the oxidative stress marker MDA levels. How CsA reduces antioxidant capacity is not clear but it is possible that CsA increases the production of oxygen-free radicals or the consumption of antioxidant molecules. Interestingly, when CsA is administered to asphyxiated newborn piglets after reoxygenation, it has a neuroprotective effect (Gill et al., 2012): it significantly attenuates the production of hydrogen peroxide by the cerebral cortex and the cerebral oxidized glutathione levels. Notably, when Wang et al. (2018) treated intracerebral hemorrhage model rats with the antioxidant melatonin, it reduced the DNA damage, inflammation, oxidative stress apoptosis, and mitochondrial damage in

Manipulating miR-494 expression may represent a novel approach to preventing CsA nephrotoxicity. Nox2 could be used for the diagnosis and monitoring of chronic CsA nephrotoxicity. TLR4 is a potential pharmacological target in CsA nephrotoxicity.

(O'Connell et al., 2011) (Carlos et al., 2014) A number of novel potential markers of CsA nephropathy are identified. These biomarkers correlated well with the temporal development of CsA nephrotoxicity. Cadherin 1, superoxide dismutase, vinculin KIM-1, TNF-α, fibronectin, osteopontin, TGF-β

Serum indoxyl sulfate

Urine

Patients with steroiddependent nephrotic syndrome CD-1 mouse model

Magnesium is a useful marker of CsA toxicity independent of CsA blood levels. Serum indoxyl sulfate was elevated in patients with CsA nephrotoxicity. Fractional excretion Blood Magnesium

Renal transplant recipients

These proteins play important roles in the tubular epithelium and glomerular cells in establishing the pro-fibrotic environment. Biomarkers of acute CsA nephrotoxicity: KIM-1, TNF-α, fibronectin, and elevated microalbuminuria; late increases in urinary osteopontin and TGF-β indicate chronic CsA nephrotoxicity. miR-494 plays a major role in promoting CsA nephrotoxicity.

(Umino et al., 2010)

(Naqvi et al., 2005)

(Camara et al., 2001)

Urinary RBP level is a good parameter to predict progressive CsA nephrotoxicity in heart transplant patients. Magnesium is a promising biomarker of CsA nephrotoxicity. Serum indoxyl sulfate is an early marker for detecting chronic CsA nephrotoxicity. Heart transplant patients with high RBP levels had worse renal prognostic and renal failure. Urine Retinol binding protein (RBP)

Heart transplant patients

Major findings Experimental model Sample Biomarker

Table 2 Summary of the potential biomarkers for the diagnosis of CsA nephrotoxicity.

Significance

References

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accelerate autophagy: this excessive autophagic activity eventually led to the apoptotic death of the kidney cells. Therefore, it seems that CsA activates oxidative stress and autophagy. Autophagy in this context attenuates or promotes the CsA nephrotoxicity.

the brain around the hematoma. Their in vitro study with oxygen hemoglobin-treated primary rat cortical neurons showed that melatonin prevented the opening of the mitochondrial permeability transition pore, which uncouples oxidative phosphorylation and leads to mitochondrial swelling and damage. Gill et al. (2012) also speculated that CsA may exert its neuroprotective effects in asphyxiated piglets by preventing the opening of the mitochondrial permeability transition pore opening, thereby reducing oxidative stress and brain cell apoptosis and preserving energy homeostasis. The possibility that CsA can prevent mitochondrial permeability transition pore opening is supported by the recent study of Yu et al. (2016). First, when HepG2 cells or mice are treated with glucose oxidase (GOX), it not only generates ROS in the hepatic cells, it causes the cytoplasmic p53 to translocate to the mitochondria. This induces the mitochondrial permeability transition pore to open, which in turn causes the hepatic cells to undergo apoptosis. Yu et al. (2016) then showed that CsA co-treatment of the GOX-treated hepatic cells and mice blocks GOX-induced apoptosis. First, they found that it failed to inhibit the ROS generation induced by GOX, which indicates that CsA does not serve as an antioxidant. However, CsA treatment did block the translocation of p53 to the mitochondria, thereby inhibiting the mitochondrial permeability transition and suppressing ROS-mediated cell apoptosis. The possibility that CsA can protect against apoptosis is supported by the recent study of Baky et al. (2016). When they subjected rat pups to a closed head injury and then treated them with CsA, it significantly reduced not only the lipid peroxidation and inflammation in the brain but also the levels of the brain apoptotic biomarkers. In summary, oxidative stress is an important effect of exposure of CsA. Like toxic agents such as mycotoxins (Wu et al., 2014a; Wu et al., 2017a; b), CsA induces ER stress activity and subsequently triggers phenotypic changes and death in tubular cells, thereby inducing nephrotoxicity. Significantly, CsA increases mitochrondrial ROS production, alters the redox balance, and induces lipid peroxidation: these effects promote CsA nephrotoxicity. However, recent studies show that CsA-induced ROS production itself is not the direct cause of this toxicity. CsA also damages the mitochondria, reduces the mitochondrial ΔΨm, and increases mitochondrial permeability transition pore opening, thereby inducing renal cell apoptosis and nephrotoxicity. CsA treatment decreases the antioxidant capacity of the kidney, which sensitizes the kidney to various stresses and thus promotes nephrotoxicity. Oxidative stress is a major factor of CsA nephrotoxicity. However, what causes the oxidative stress initially? What is the initiating event that has CsA at the epicentre in this context? All these questions are quite important for the fully understanding of the mechanism of oxidative stress in the CsA nephrotoxicity. The proposed mechanism by which oxidative stress contributes to CsA nephrotoxicity is shown in Fig. 2.

4.1. CsA-induced autophagy may protect against CsA nephrotoxicity Unlike the studies on oxidative stress, studies on the role of autophagy in CsA nephrotoxicity only started in the last 10 years. They show that autophagy is a very complex process (Wu et al., 2017a; Liu et al., 2017b; c; Wang et al., 2017a; b; Liu et al., 2018) and it actually remains unclear how exactly autophagy contributes to chronic CsA nephropathy. To the best of our knowledge, Pallet et al. (2008c) were the first to report a study on this question: they found that when primary human renal tubular cells were subjected to CsA, they underwent autophagy, as indicated by the production of autophagosomes and the cellular expression of microtubule-associated protein light chain 3II (LC3II), which is a marker of autophagy. Significantly, when autophagy was inhibited, apoptosis increased. Pallet et al. (2008c) also showed that CsA-induced autophagy is dependent on ER stress since salubrinal, which protects against ER stress, suppressed LC3II expression. Similarly, when rat pituitary GH3 cells were treated with CsA, it increased the expression of the autophagy markers LC3I and LC3II in a dose-dependent manner (Yoo and Jeung, 2010). Moreover, this effect associated with ER stress, as indicated by a fall in the expression of the antioxidant catalase and elevation of the expression of ER luminal binding protein and inositolrequiring enzyme 1-α, which are markers of ER stress. Notably, Pallet et al. (2008c) also found that the kidney of CsA-treated rats expressed LC3II. This suggests that autophagy also occurs in vivo. Since it is easily detected, it could serve as a biomarker of CsA-induced nephrotoxicity (Yoo and Jeung, 2010). Several lines of evidence suggest that the autophagic response to ER stress is cytoprotective because it remove superfluous or injured organelles and/or prevents the accumulation of misfolded proteins (HøyerHansen and Jaattela, 2007; Pallet and Anglicheau, 2009). Thus, CsAinduced autophagy may protect kidney cells from nephrotoxicant-induced renal injury by the same mechanisms. The protective role of autophagy in CsA treatment is supported by several other in vitro models. First, Ciechomska et al. (2013) showed that when malignant glioma cells are treated with CsA, they undergo both apoptosis and autophagy. The apoptosis is promoted by ER stress while the autophagy is induced by inhibition of the mTOR/p70S6K1 pathway. Notably, when the autophagy effectors ULK1, Atg5, or Atg7 were silenced, the levels of active caspase-3 and -7 and PARP degradation rose; the cells also exhibited greater CsA-induced death (Ciechomska et al., 2013). Similarly, when rat pituitary GH3 cells were treated with CsA, while the majority underwent apoptosis, some underwent autophagy (Kim et al., 2014). This suggests that autophagy attenuates apoptosis, thereby protecting cells from CsA-induced death (Kim et al., 2014). Notably, CsA-induced autophagy may also prevent other diseases. For example, Chandler et al. (2015) showed that CsA treatment can prevent the opacification of the posterior capsule of the lens by inducing autophagy. Interestingly, the very recent study by Fakharnia et al. (2017) showed that CsA can also decrease autophagy (and apoptosis): when rats receive a global cerebral ischemia-reperfusion injury, their cerebral tissues exhibit opening of the mitochondrial permeability transition pore and undergo both apoptosis and autophagy. The effect of the injury on the mitochondria is mediated by cyclophilin D. CsA is an inhibitor of cyclophilin D: when the rats are treated with CsA before the injury, they exhibit lower levels of the autophagy-associated proteins LC3I, LC3II, and beclin-1 as well as apoptotic markers (Fakharnia et al., 2017). Recently, Yadav et al. (2015) showed that transmembrane BAX inhibitor motif containing 6 (TMBIM6) protects human kidney cells from

4. Role of autophagy in CsA nephrotoxicity Autophagy is a lysosomal degradation pathway that is essential for survival, differentiation, development, and homeostasis (Levine and Klionsky, 2004; Wu et al., 2017a). While it helps to protect organisms against pathologies such as infections, cancer, neurodegeneration, aging, and heart disease (Mizushima et al., 2008), its ability to digest cellular components and even its prosurvival functions can be detrimental to the cell or organism in certain experimental settings (Hua et al., 2017). Several lines of evidence show that autophagy participates in the pathogenesis of nephrotoxicity. For example, when Song et al. (2017) treated rat tubular cells with lead, which is a known nephrotoxicant, it blocked autophagy by inducing lysosomal membrane permeabilization, thereby impairing the function of the lysosomes. Moreover, when kidney cells are treated with two natural compounds, namely, alisol A 24-acetate and alisol B 23-acetate, the PI3K/Akt/ mTOR signaling pathway is inhibited (Wang et al., 2017a). Since this pathway suppresses autophagy, the natural compounds appeared to 896

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Fig. 2. Proposed mechanisms by which oxidative stress leads to cyclosporine A nephrotoxicity.

of CsA on autophagy may depend on the incubation time and/or the cell type. Notably, as discussed in the section on oxidative stress, CsA exhibits a similar paradox in terms of apoptosis: while CsA inhibits mitochondrial-mediated apoptosis in the kidney, it can also induce mitochondrial apoptotic cell death in the same organ. Since ER stress appears to participate in the pathogenesis of kidney diseases in general, autophagy may help to ameliorate kidney injuries far more commonly than was previously thought. Thus, deciphering the biological pathways that induce autophagy in kidney diseases is of great importance because this information may aid the identification of early biomarkers of kidney injury and new therapeutic options (Pallet and Anglicheau, 2009).

CsA-induced nephrotoxicity by enhancing autophagy and activating lysosomes. Specifically, when TMBIM6 was overexpressed in CsAtreated human kidney cells, it increased LC3II expression, lysosomal activity, and cell viability and suppressed the ER stress response. The TMBIM6-induced autophagy associated with PRKAA activation and mTORC1 suppression. TMBIM6 knockout mice were more susceptible to CsA nephrotoxicity than wild-type mice. Thus, TMBIM6 protects kidney cells from CsA-induced nephrotoxity by enhancing autophagy and upregulating lysosomal activity via the PRKAA-mTORC1 pathway. The above studies suggest that CsA activates oxidative stress and autophagy. Autophagy attenuates ER stress-induced toxicity, including nephrotoxicity. At present, the mechanism(s) that underlie the latter mechanism remain poorly understood. In particular, it is not clear which factors drive a CsA-treated cell to either undergo apoptotic death or autophagy and thus cell survival. However, it is possible that the duration of CsA exposure shapes this balance. Han et al. (2008) showed that while short-term CsA treatment activates the ER stress response (as shown by elevated levels of BiP mRNA and protein), long-term CsA treatment reduces ER stress. It is possible that factors that regulate protein expression (possibly also the expression of autophagy-related proteins) determines the balance between the activation and inactivation of the ER stress response, which in turn determines whether the cells do or do not undergo apoptotic cell death. Notably, several studies show that in non-renal cells, CsA largely inhibits autophagy by blocking the opening of the mitochondrial transition pore (Arrington et al., 2006; Elmore et al., 2001). However, it should be noted that in these studies, the cells were exposed to CsA for less than 1 h. By contrast, in the study of de Arriba et al. (2013), which showed that CsA generates mitochondrial transition pores in renal cells, autophagy was only analyzed after 24–48 h of treatment. These discrepancies suggest that the effect

4.2. CsA-induced autophagy may promote CsA nephrotoxicity Conversely, numerous studies show that autophagy also promotes CsA nephrotoxicity (Lim et al., 2012, 2014; Xiang et al., 2013; Lai et al., 2015). For example, when mice are chronically treated with CsA, their kidneys not only exhibited oxidative renal injury, they also displayed increased expression of LC3II and beclin-1 (Lim et al., 2012). The expression of active caspase-3 was also increased and colocalized with LC3II in the injured areas of the CsA-treated kidneys. The kidneys also exhibited p62 accumulation, which indicates poor autophagic clearance. Significantly, co-treatment with antioxidants decreased autophagosome formation and improved the clearance of toxic protein aggregates (Lim et al., 2012). This suggests that chronic CsA nephrotoxicity involves excessive autophagosome production and poor autophagic clearance. Thus, the regulation of autophagy may be an important therapeutic target for preventing the chronic allograft dysfunction that is caused by long-term CsA treatment in renal-transplant 897

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molecule of CsA that contributes to CsA nephrotoxicity (Jin et al., 2017), and the pathway involving the fibrogenic cytokine TGF-β1 promotes CsA-induced renal fibrosis (Islam et al., 2001). There is also evidence that other signaling pathways, including those that involve Ca2+, Akt, Nrf-2, and protein kinase A (PKA), participate in CsA nephrotoxicity. In this section, we will discuss the role that these signaling pathways, and their crosstalks, play in the molecular mechanisms that underlie CsA nephrotoxicity. The specific functions of the major signaling pathways in CsA-induced nephrotoxicity are shown in Fig. 4.

recipients. Since activation of the signaling pathways MAPK, PI3K, and STAT3 contribute to the CsA nephrotoxicity (Yang et al., 2003; Kirk, 2012). Moreover, the pathways p38/MAPK, ERK/MAPK, PI3K/AKT, and STAT3 have close relationship with autophagy and nephrotoxicity (Choi et al., 2018). It is very possible that CsA first activates these pathways and subsequently induces the autophagy and promotes the nephrotoxicity. Nevertheless, the exact mechanisms in this context are not yet clear (Lai et al., 2015). Notably, a subsequent study by Lim et al. (2014) showed that when CsA-treated mice were concurrently treated with Korean red ginseng (KRG), renal function improved and fibrosis was reduced. Moreover, the expression of LC3II, beclin-1, and the number of autophagic vacuoles decreased significantly and autophagic clearance improved, as shown by less accumulation of p62 and ubiquitin. In addition, p62, ubiquitin, and LC3II now colocalized with each other. KRG treatment also reduced the expression of both active caspase-3 and LC3II in the injured area. While the mechanisms underlying the protective effects of KRG are not clear, KRG promoted the autophagy-downregulating AKT/ mTOR pathway. It also reduced the expression of AMP-activated protein kinase, which promotes autophagy. Several other studies also suggest that CsA-induced autophagy may promote CsA nephrotoxicity. Cheng et al. (2008) reported that CsA decreases the survival of renal tubular cells and that this nephrotoxicity associates with inhibition of the phosphorylation of mTOR and AktSer473. Since Akt phosphorylation activates mTOR and inhibits both programmed cell death (including apoptosis) and autophagy (Deng et al., 2017), it is likely that the downregulation of the AKT/ mTOR pathway in the renal cells suppresses autophagy. That this reduces renal cell survival is supported by the fact that Akt is believed to be an important factor for cell survival (Liu et al., 2017c). In addition, several other studies (Xiang et al., 2013; Lai et al., 2015, 2017) showed that CsA treatment induces rat renal dysfunction, tubulointerstitial inflammation, and fibrosis, and that these phenomena were accompanied by increased expression of LC3II, caspase-3, and the DNA damage biomarker 8-hydroxy-2 deoxyguanosine. In summary, increasing lines of evidence suggest that autophagy is involved in CsA nephrotoxixity. A large number of studies show that autophagy protects renal tubular cells from CsA-induced nephrotoxicity. Moreover, the autophagy marker LC3II is easily detected in CsA nephrotoxicity and could serve as a biomarker. TMBIM6 may prevent CsA-induced nephrotoxity by upregulating autophagy through the PRKAA-mTORC1 pathway. Multiple studies show that CsA-induced autophagy can alleviate the deleterious effects of ER stress, which is connected to the oxidative stress mechanisms. Thus, CsA may activate two Janus-faced pathways: one induces ER stress and activates the oxidative stress pathway, thereby inducing CsA nepharotoxicity, and the other induces autophagy, which reduces CsA-induced ER stress and inhibits oxidative stress, thereby attenuating nephrotoxicity. However, since several studies show that autophagy can also enhance CsA nephrotoxicity, it seems that the role that autophagy plays in CsA nephrotoxicity may be much more complicated than was originally thought. Thus, at present, it remains unclear whether autophagy prevents or enhances CsA nephrotoxicity. Further mechanistic studies on autophagy in CsA nephrotoxicity are warranted. The proposed mechanisms by which autophagy participates in CsA nephrotoxicity are shown in Fig. 3.

5.1. MAPK pathway The MAPK family consists of ubiquitous proline-directed proteinserine/threonine kinases that participate in the signal-transduction pathways that control intracellular events and major developmental changes in organisms (Johnson and Lapadat, 2002). Emerging evidence shows that the MAPK signaling pathway is involved in the pathogenesis of CsA nephrotoxicity. The group of Ryan in University College Dublin, Ireland (Martin-Martin et al., 2010) examined the ability of CsA to alter the transepithelial electrical resistance (TER) of kidney cells. TER is a measure of paracellular permeability and renal epithelial barrier function. They first showed that CsA increases the TER in Madin-Darby canine kidney (MDCK) cells, thereby decreasing paracellular permeability, and that ERK1/2 cascade activation plays a pivotal role in this phenomenon (Kiely et al., 2003). Their subsequent study (MartinMartin et al., 2010) showed that subcytotoxic doses of CsA also decreased the paracellular permeability of LLC-PK1 porcine proximal tubular epithelial cells. This change associated with increased membrane localization of the tight junction protein claudin-1 and ERK1/2 phosphorylation. Thus, CsA modulates the renal epithelial barrier function by altering the expression and localization of claudin-1 via the ERK1/2 signaling pathway (Martin-Martin et al., 2010). The same group (O'Connell et al., 2011) later showed that CsA-treated human mesangial cells exhibit ROS and oxidative stress and that this is mediated by the ERK1/2 pathway. Thus, CsA activates this MAPK pathway, thereby promoting CsA-induced glomerular dysfunction and causing nephrotoxicity. MAPK family members also play important roles in ischemia-reperfusion injury in the kidneys and the remarkable ability of CsA pretreatment to protect kidneys from this injury. Thus, Yang et al. (2003) showed that when rats undergo ischemia-reperfusion injury, their kidneys exhibit increased expression of the MAPKs ERK, JNK, and p38, especially in the renal tubules of the outer medulla. However, CsA preconditioning alters this expression pattern: ERK expression is further upregulated while JNK and p38 expression are decreased (Yang et al., 2003). Smad proteins are pivotal intracellular effectors of TGF-1: they act as transcription factors and play important roles in apoptosis (Derynck and Zhang, 2003; Xu et al., 2016). There seems to be a crosstalk between the MAPK and Smad pathways that regulates CsA-induced apoptosis in renal proximal tubular cells: Iwayama et al. (2011) showed that CsA induces the nuclear translocation of p-Smad2/3 and apoptosis, and that this required the upregulation of ERK and p38 (but not JNK). The presence of crosstalk between the MAPK and Smad pathways was shown by co-treating the cells with epidermal growth factor (EGF). EGF treatment alone activates ERK and p38 (but not JNK). When CsAtreated cells are co-treated with EGF, it inhibits the CsA-induced nuclear translocation of p-Smad2/3 and apoptosis. Since inhibiting p38 eliminated this protective effect of EGF (ERK inhibition had no effect), it appears that crosstalk between Smad and p38/ERK differentially regulates apoptosis in CsA-induced renal proximal tubular cell injury. Notably, Wang et al. (2013) showed that CsA also promotes in vitro trophoblast migration via the MAPK/ERK/NF-κB and Ca2+/calcineurin/NFAT pathways. In addition, the review of Matsuda and Koyasu (2000) reported that CsA is a highly specific inhibitor of T-cell activation because it blocks the activation of the JNK and p38 signaling

5. Signaling pathways that participate in CsA nephrotoxicity While the molecular mechanisms that underlie CsA nephrotoxicity are still not fully understood, increasing lines of evidence suggest that various signaling pathways participate in this toxic process. For example, the p38, ERK, and JNK MAPK subfamilies have been shown to participate in CsA nephrotoxicity (Martin-Martin et al., 2010; Iwayama et al., 2011), CsA decreases NO expression and thereby reduces renal vascular tone (Shihab et al., 2003), NF-κB is an important target 898

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Fig. 3. Mechanisms by which cyclosporine A induces autophagy and the relationships between autophagy, endoplasmic reticulum (ER) stress, and cyclosporine A nephrotoxicity.

extracellular matrix protein synthesis, and interstitial inflammatory cell infiltration (Yoon and Yang, 2009). CsA has a proinflammatory effect on the microvascular endothelium and iNOS may participate in this effect: CsA treatment of human intestinal microvascular endothelial cells increased their binding to leukocytes, and this is associated with the inhibition of iNOS (Rafiee et al., 2002). The fact that CsA induces dysfunction in intestinal endothelial cells may help explain why it is not effective as a long-term treatment for chronically active inflammatory bowel disease. Thus, CsA may induce endothelial cell inflammation by blocking iNOS. In addition, Shihab et al. (2003) showed that the kidneys of rats with chronic CsA nephrotoxicity exhibit upregulation of VEGF and its KDR/Flk-1 receptor, and that this is elevated by a co-treatment that blocks NOS activity. Conversely, the CsA-induced increase in VEGF expression is suppressed by co-treatment with L-arginine, which increases NOS activity. Thus, CsA increases renal production of VEGF by blocking NOS. These studies show that the NO pathway plays an important role in ameliorating CsA-associated vasoconstriction, thereby attenuating CsA nephrotoxicity. However, there is controversy in the field. Some studies show that by reducing NO levels, CsA blocks the ability of NO to maintain vascular tone. As a result, nephrotoxicity occurs readily. By contrast, other studies show that NO synthesis is enhanced during CsA nephrotoxicity, and this counterbalances the predominantly preglomerular vasoconstriction. This controversy has not yet been discussed in the literature but it certainly suggests that NO participates in CsAinduced renal injury in complex ways. We suspect, however, that in the kidney, CsA predominantly inhibits NO synthesis and induces renal endothelium-dependent apoptosis and nephrotoxicity. Studies on the

pathways in T cells that are triggered by antigen recognition. Thus, the p38, ERK, and JNK MAPK subfamilies are all involved in CsA nephrotoxicity. Specifically, the ERK1/2 cascade plays an important role in the CsA-induced mechanisms that generate nephrotoxicity, namely, the reduction of kidney cell paracellular permeability, ROS production, and oxidative stress. In ischemia-reperfusion injury in the kidneys, CsA protects by upregulating ERK and downregulating JNK and p38 expression. Since the MAPK signaling pathways are known to participate in many cell functions and diseases (Johnson and Lapadat, 2002), it is likely that the MAPK pathways play other, as yet undetermined, roles in the regulation of the CsA nephrotoxicity. Since few studies have examined these pathways in CsA nephrotoxicity, deeper and more systematic analyses are warranted. 5.2. NO pathway CsA nephrotoxicity is characterized by renal vasoconstriction that often progresses to chronic injury with irreversible structural renal damage (El-Bassossy and Eid, 2018). Renal vasoconstriction is attributed to an imbalance in the release of vasoactive substances, including decreased production of NO (Mukai et al., 2017). NO is generated from L-arginine by NOS isoforms. There are at least three NOS isoforms, namely, neuronal NOS, inducible NOS (iNOS), and endothelial NOS (eNOS) (Aliancy et al., 2017). In the kidney, NO is a vasoactive factor that plays a key role in maintaining vascular tone (Liu et al., 2017b), in part by inhibiting the formation of endothelin, which is a peptide that constricts blood vessels (Yoon and Yang, 2009). NO also decreases glomerular thrombosis and ischemia, mesangial cell proliferation, 899

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Fig. 4. Functions of the major signaling pathways in cyclosporine A-induced nephrotoxicity. On CsA treatment, the ERK1/2 pathway in kidney cells becomes highly activated; this increases transepithelial electrical resistance (TER) due to the induction of claudin-1 expression. These pathways decrease the paracellular permeability of kidney cells. ERK1/2 also activates ROS and oxidative stress and promotes CsA-induced glomerular dysfunction and the subsequent nephrotoxicity. EGFinduced activation of p38 ameliorates CsA-induced activation of Smad and apoptosis. The vasoactivity of NO plays a key role in maintaining vascular tone. CsA inhibits NO synthesis and induces vascular endothelial growth factor (VEGF), thereby increasing renal arterial pressure. Conversely, CsA also enhances NO levels by increasing eNOS mRNA expression and inhibits the expression of endothelin-1, thereby counterbalancing preglomerular vasoconstriction. While NF-κB contributes to CsA nephrotoxicity, the NF-κB pathway mediates the ability of Klotho expression to ameliorate the nephrotoxicity of CsA. TGF-β1 is a fibrogenic cytokine; its expression is induced by ERK1/2 and this contributes to CsA-induced renal fibrosis. p38 increases TGF-β1-induced TER. The TGF-β1/IL-2/COX-2 pathway is involved in CsA-induced renal tubular atrophy and interstitial fibrosis. CsA amplifies the Ca2+ pathway by inhibiting the plasma membrane Ca2+ pump, thereby causing proximal tubule injury. CsA causes nephrotoxicity via the PTEN/Akt signaling pathway. CsA activates Nrf-2 pathway and prevents the oxidative stress. The roles of the JAK/STAT pathways, including the STAT3 pathway, in CsA nephrotoxicity remain poorly understood (Martin-Martin et al., 2010; Shihab et al., 2003; Jin et al., 2017; El-Gowelli et al., 2014; Calderaro et al., 2003).

This effect of CsA may explain why CsA effectively treats rheumatoid arthritis. In addition, CsA significantly suppresses NF-κB p65 and IκBα expression and NF-κB DNA-binding activity in cardiac xenografts. This associates with prolongation of the survival of the xenografts (Yang et al., 2009). Xu et al. (2011) showed that CsA augments the growth of A431 epidermoid carcinoma xenograft tumors by activating TGF-β-activated kinase 1 and inducing the transcriptional activation of NF-κB (as shown by IKKβ-mediated phosphorylation-dependent degradation of IκB and the resulting nuclear translocation of p65). Thus, NF-κB is an important target molecule of CsA. NF-κB p65 can be modulated by Klotho and contributes to CsA nephrotoxicity. NF-κB is also involved in the ability of CsA to inhibit the expression of key costimulatory cell-surface molecules on DCs. CsA prolongs the survival of cardiac xenografts by suppressing NF-κB activity. Further studies on the roles of NK-κB in CsA nephrotoxicity are warranted.

exact functions of NO in CsA nephrotoxicity are needed. 5.3. NF-κB pathway Compared with other pathways, there is relatively little information on the role of NK-κB pathway in CsA nephrotoxicity. Very recently, Jin et al. (2017) reported that Klotho, an anti-aging protein, ameliorates CsA-induced nephropathy by downregulating kidney inflammation via the PDLIM2/NF-κB p65 signaling pathway. Specifically, when mice transfected with a Klotho-expressing adenovirus were treated with CsA, they exhibited less CsA nephrotoxicity than the wild-type mice; this associated with decreased NF-κB p65 expression, increased expression of PDLIM2, and reduced production of inflammatory cytokines and iNOS. Moreover, treatment of primary human renal proximal tubule epithelial cells with soluble Klotho protein and LPS reduced their NF-kB p65 expression and increased their PDLIM2 expression. Co-treatment with PDLIM2 siRNA blocked the ability of Klotho to inhibit the expression of NF-kB p65 (Jin et al., 2017). Several studies suggest that NF-κB is a target molecule of CsA and that CsA-induced inhibition of NF-κB in non-T cells (Nakahara et al., 2003). CsA inhibits the IL-17 production of CD4+ T cells and this effect is mediated by the IL-15-activated NF-κB pathway (Cho et al., 2007).

5.4. TGF-β1 pathway TGF-β1 is a fibrogenic cytokine that contributes to CsA-induced renal fibrosis. In rats with CsA nephrotoxicity, TGF-β1 mRNA expression in the kidney was markedly increased (Islam et al., 2001). Upregulated renal TGF-β1 expression was also found in patients with CsA 900

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hyperexpression could be an effective way to inhibit CsA-associated renal fibrosis. Moreover, TGF-β1 usually exerts its CsA-induced pathological effects on the kidney via the MAPK pathway, particularly ERK and p38. p38/TGF-β1/ERK1/2 plays an important role in the CsA-induced impairment of renal paracellular permeability. The TGF-β1/IL-2/ COX-2 crosstalking pathway also participates in CsA-induced renal tubular atrophy and interstitial fibrosis. Moreover, the oxidative stress induced by CsA involves endothelin-1/TGF-β1 signaling. Thus, TGF-β1 crosstalks closely with other signaling pathways, thereby contributing to CsA nephrotoxicity.

nephrotoxicity (Khanna et al., 2002). These data show that while CsA is well known for its ability to inhibit the expression of proinflammatory cytokines, it also stimulates TGF-β1 expression. Moreover, this high expression of TGF-β1 may contribute to CsA-induced renal disease (Islam et al., 2001). Since TGF-β1 is a fibrogenic cytokine, this suggests that blocking TGF-β1 hyperexpression could prevent CsA-associated renal fibrosis, (Leonarduzzi et al., 2001). Slattery et al. (2005) also hypothesized that TGF-β1 promotes EMT in renal cells, and that this may play a major role in the development of CsA-induced renal fibrosis. Indeed, they showed that when human tubular cells are treated with CsA and develop EMT, they express TGF-β1. The signaling pathway that leads to TGF-β1 expression involves the activation of protein kinase Cβ, which leads to the activation of ERK. Feldman et al. (2007) also showed that when MDCK cells are treated with CsA, they develop renal epithelial barrier dysfunction that involves crosstalk between TGF-β1 and ERK. They also found that treating the cells with TGF-β1 in the absence of CsA also elevated TER, and that inhibiting the p38 pathway attenuates this effect. This suggests that p38 regulates CsA-induced TGF-β1 expression. Interestingly, this research group showed that inhibiting ERK also blocked the ability of TGF-β1 to induce TER (MartinMartin et al., 2011). Thus, the crosstalking p38/TGF-β1/ERK1/2 pathway seems to play an important role in CsA-induced modulation of renal paracellular permeability. The group of Prof. El-Mas (El-Gowelli et al., 2014) found that the TGF-β1/IL-2/COX-2 signaling pathway also participates in CsA-induced renal tubular atrophy and interstitial fibrosis. Papachristou et al. (2009) also showed that endothelin-1/TGF-β1 signaling participates in CsAinduced renal dysfunction: when human tubular cells were treated with CsA, they produced endothelin-1, which is known to contribute in general to renal vascular derangement and to promote the synthesis and activation of TGF-β1 and adhesion molecules (Lüscher and Barton, 2000; Leask, 2009). Furthermore, the El-Mas group found that CsAinduced renal oxidative damage in rats is due to excessive production of lipid peroxides that seems to be driven by upregulated endothelin-1/ TGF-β1 signaling and downregulated COX-2 expression (Helmy et al., 2015). CsA-treated mesangial cells exhibit upregulated extracellular matrix protein synthesis in vitro, and CsA treatment associates with glomerulosclerosis in vivo: when mesangial cells were treated with pharmacologically relevant concentrations of CsA, it upregulated the expression of TGF-β1 in a time- and dose-dependent manner (Waiser et al., 2002). CsA also upregulated the type I and II TGF-β1 receptors by autocrine mechanisms. In addition, CsA induced plasminogen activator inhibitor type-1 and fibronectin expression, and antibodies to TGF-β1 blocked this expression (Waiser et al., 2002). Organ-transplant recipients are prone to the development of multiple aggressive and metastatic non-melanoma skin cancers (Mittal and Colegio, 2017). When nude mice bearing human squamous cell carcinoma (SCC) xenograft tumors are treated with CsA, the skin cancers become invasive and aggressive tumors (Walsh et al., 2011). This change associated with increased tumor TGF-β1 expression and elevated EMT. Thus, CsA activated the TGF-β1 signaling pathway, which enhanced the expression of proteins that promote the EMT. In addition, Chen et al. (2015a) showed that by activating the TGF-β1/Smad3 signaling pathway, CsA promotes the proliferation and migration of rat gingival fibroblasts and their production of collagen. This helps explain why CsA treatment induces gingival overgrowth. Interestingly, CsA can also inhibit TGF-β1 expression (Eickelberg et al., 2001). Lung fibrosis is a fatal condition that is characterized by excessive extracellular matrix deposition that associates with increased TGF-β1 activity (Mackinnon et al., 2012). Eickelberg et al. (2001) showed that CsA inhibits TGF-β1-induced signaling in lung fibroblasts, and that this effect associated with CsA-induced activation of JunD. In summary, the TGF-β1 signaling pathway plays an important role in CsA nephrotoxicity. Since TGF-β1 is a fibrogenic cytokine that is responsible for CsA-induced renal fibrosis, preventing TGF-β1

5.5. Ca2+ pathway The role of Ca2+ in CsA nephrotoxicity is relatively less well understood than the role of other signaling molecules. However, several in vitro and in vivo studies show that it participates in CsA renal injury (Calderaro et al., 2003; da Costa et al., 2003). Thus, Calderaro et al. (2003) showed that when the LLC-PK1 porcine proximal tubule-like cell line is treated with CsA, it amplifies Ca2+ signaling after exposure to agents that trigger intracellular Ca2+ release. This effect is achieved by CsA-mediated inhibition of the plasma membrane Ca2+ pump, which prevents Ca2+ export. Thus, CsA increases the sensitivity of the renal cells to events that induce Ca2+ signaling. This augmentation of Ca2+ signaling, which plays an essential role in many cellular functions, is likely to contribute significantly to the side effects induced by CsA. da Costa et al. (2003) also showed that when rat proximal tubules were exposed to CsA, their Ca2+ levels rose and tubule injury was detected. In addition, increasing lines of evidence suggest that the immunosuppressive effects as well as the main side effects of CsA may reflect extensive changes in the cellular Ca2+ pathway (Wei et al., 2011; Wang et al., 2013). It is noteworthy that CsA is a typical calcineurin (Ca2+-calmodulin-activated phosphatase) inhibitor (Gooch et al., 2017; Jia et al., 2016). Whether the CsA-induced Ca2+ signaling is involved in the inhibition of calcineurin is not fully understood.

5.6. Akt pathway Kuwana et al. (2008) showed that when mice are treated with the nephrotoxin cisplatin, Akt connects with PI3K to mediate the renal tubular injury. Chen et al. (2015b) also found evidence that Akt participates in CsA nephrotoxicity. Thus, when human proximal tubular epithelial cells were treated with CsA, they exhibited EMT along with elevated microRNA-21 expression. Transfection with the microRNA-21 precursor rapidly increased phosphorylated Akt expression and downregulated PTEN expression. While CsA also downregulated Smad7 downregulation and upregulated TGF-β1 in the renal cells, such changes were independent of microRNA-21. These changes were also observed in a mouse model of CsA-induced allograft fibrosis. Therefore, microRNA-21 may mediate CsA nephrotoxicity via the PTEN/Akt signaling pathway. The Akt pathway may also contribute to the increased propensity of organ-transplant recipients to develop skin cancers such as basal cell carcinomas and SCCs (Santana et al., 2017; Hashemi-Sadraei and Hanna, 2017). When nude mice bearing a human SCC (epidermoid carcinoma A431) were treated with CsA, the tumors became aggressive (Arumugam et al., 2012). This effect was abrogated by blocking both the Akt and p38 signaling pathways: the growth of the tumors dropped by > 90% and the residual tumors exhibited lower expression of phosphorylated Akt and p38. They were also less invasive and more highly differentiated. The drop in phosphorylated Akt expression was also accompanied by a significant reduction in phosphorylated mTOR expression. Notably, PI3K and Akt often crosstalk (Ersahin et al., 2015). Thus, studies on the PI3K/Akt pathway in CsA nephrotoxicity are needed. 901

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Similarly, erdosteine shows treatment potential on CsA nephrotoxicity by reducing its effect on lipid peroxidation and glomerular ROS production (Uz et al., 2012). Mariee and Abd-Ellah (2011) further showed that docosahexaenoic acid (DHA) supplementation can attenuate the oxidative damage in rat kidney tissues (Mariee and Abd-Ellah, 2011). Moreover, DHA treatment significantly restored NO level. Khan et al. (2011) showed that pituitary adenylate cyclase-activating polypeptide (PACAP) ameliorated CsA-induced renal tubular injury, inhibited TGFβ1 expression, and prevented tubulointerstitial fibrosis in CsA-exposed murine kidneys. Ouyang et al. (2014) reported that 2-deoxy-D-glucose has protective effect on CsA nephrotoxicity. This effect is due to the reduction of CsA-induced ROS. Recently, nebivolol, was reported to attenuate CsA-induced impairment of renal functions, oxidative stress and restored the balance in renal NO system (Hewedy and Mostafa, 2016). Very recently, valsartan was shown to ameliorate CsA nephrotoxicity by diminishing CsA-induced renal Klotho downregulation and oxidative stress (Raeisi et al., 2017). A long term oral treatment with vardenafil prevented Cs-Amediated rat kidney damage (Essiz et al., 2015). Other antioxidants including hydrocortisone (Ciarcia et al., 2012), hydroxytyrosol (Capasso et al., 2008), vitamin E (de Arriba et al., 2013; Damiano et al., 2015), and vitamin D analogs (Tomasini-Johansson et al., 2017) also show very promising anti-oxidative potential caused by CsA. Some plant extracts also show good capacity to reduce the CsA nephrotoxicity in animals. For example, treatment of rats with epicatechin ameliorated the CsA nephrotoxicity by decreasing lipid peroxidation and enhancing the antioxidants enzyme activities (Al-Malki and Moselhy, 2011). However, dietary epicatechin does not get absorbed into plasma in significant amounts due to extensive first pass metabolism, which is why this study used i.p. injection (Mizuma and Awazu, 2004). This would require a human to inject the compound each day, which would raise compliance issues. Olive leaf extract also has some ameliorating potential on CsA nephrotoxicity in rats (Mostafa-Hedeab et al., 2015). Apocynin protects the kidney function from toxic effects induced by CsA via the inhibition of NADPH oxidase activity (Ciarcia et al., 2015). Very recently, Lai et al. (2017) showed that schisandrin B alleviated of CsA nephrotoxicity by reducing ROS levels through suppressing activities of oxidative stress, apoptosis, and autophagy. Spirulina is a promising renoprotective natural agent against renal injury induced by CsA (Aziz et al., 2018). Spirulina ameliorates CsA-mediated kidney oxidative stress through decreasing glutathione, lipid peroxidation, and nitrite kidney content while increasing SOD activity (Aziz et al., 2018). Curcumin exhibits protective effect against CsA-induced rat nephrotoxicity. Huang et al. (2018) showed that curcumin increases renal antioxidant capacity and reduces the Bax/Bcl-2 ratio, subsequently improves CsA-induced renal failure and renal tubular deformation and cell vacuolization. Together, many natural compounds and drugs have the protective effect against CsA nephrotoxicity. Normally, most of these compound show antioxidative effects and reduce the levels of ROS and lipid peroxidation, some can also enhance the antioxidants enzyme activities. However, it is noteworthy that most of these positive conclusions are raised from the animal (rats and mice) studies and the additional studies are needed to clarify whether these drugs and natural compounds are included in clinical practice.

5.7. Nrf-2 pathway Relatively little is known about the role of the Nrf-2 pathway in CsA nephrotoxicity. However, CsA strongly activates the Nrf-2 pathway in renal proximal tubular epithelial cells in vitro (Hamon et al., 2014). This study is also of interest because the authors generated a differential equation model of the Nrf-2 pathway that they calibrated quantitatively with an omics data subset. In addition, when human tubular cells are co-treated with CsA and Schisandrin B (a natural compound) (Lai et al., 2017), the CsA-induced cellular oxidative stress is reduced; this effect was achieved by Schisandrin B-mediated translocation of Nrf2 to the nucleus and the downstream expression of HO-1, which is a Nrf2 target gene (it also a target gene of NF-κB) (Cao et al., 2017). Notably, CsA also stimulates HO-1 expression in human gingival fibroblasts (Chin et al., 2011). Moreover, siNrf-2 treatment of the cells reduced the CsAstimulated HO-1 mRNA expression. The inhibition of ERK also significantly decreased CsA-stimulated Nrf-2 nuclear translocation and HO-1 mRNA expression. Thus, CsA-stimulated HO-1 expression is mediated by the activation of ERK and Nrf-2 plays a protective role against CsA-induced gingival fibrosis by modulating collagen turnoverrelated genes. 5.8. STAT3 pathway EGF receptor-tyrosine kinase inhibitors (EGFR-TKIs) have dramatically prolonged the overall survival of non-small cell lung cancer (NSCLC) patients who have a tumor mutation that activates EGFR. However, primary or acquired resistance eventually leads to therapeutic failure (Chen et al., 2017). Recently, Shou et al. (2016) showed that CsA significantly increased the anti-cancer effect of the EGFR-TKI gefitinib in EGFR-TKI-sensitive and -resistant NSCLC cells. CsA promoted gefitinib-induced apoptosis by inhibiting the STAT3 pathway. This was supported by the fact that siRNAs against STAT3 had the same effect as CsA: they enhanced gefitinib-induced apoptosis in lung cancer cells. Moreover, the NSCLC patients who had high levels of p-STAT3 responded much less well to EGFR-TKIs. Notably, STAT3 activation is known to be one of the pivotal mechanisms of EGFR-TKI resistance and several studies show that cyclophilins, which are inhibited by CsA, contribute to STAT3 activation (Bauer et al., 2009; Kirk, 2012). Thus, combining EGFR-TKIs with CsA or another STAT3 inhibitor is a promising approach to improving the efficacy of EGFR-TKIs in advanced NSCLC. While the role of the STAT pathway in CsA nephrotoxicity has not yet been reported, several studies show that the JAK/STAT pathway crosstalks closely with MAPK (Wu et al., 2014b, 2017a; b). Since MAPK plays a well documented role in CsA nephrotoxicity (see MAPK pathway section), the potential role of the JAK/STAT pathway in CsA nephrotoxicity should be an interesting topic for further study. 5.9. PKA pathway When the LLC-PK1 and MDCK cell lines were treated with CsA, the PKA pathway was activated (França et al., 2014). This caused DNA fragmentation. Inhibiting the PKA signaling pathway in MDCK cells significantly reduced CsA-induced DNA fragmentation. Both CsAtreated lines also produced IL-6 in a PKA-dependent manner. When PKA was inhibited in the CsA-treated cells, their NO production rose (França et al., 2014). Thus, inhibiting PKA may help to increase the vasodilator activity of NO and therefore prevent CsA nephrotoxicity.

7. Conclusions Nephrotoxicity is the most frequent and clinically important complication of CsA use. It is particularly common in renal-transplant patients. CsA-induced apoptosis correlates with the oxidative stress, ER stress, and autophagy that CsA causes. The identification of potential clinical biomarkers of CsA nephrotoxicity is currently a subject of considerable interest because these markers will facilitate the rapid identification of this disease, thereby preventing the development of irreversible damage. Accurate biomarkers will also improve the rational

6. Therapeutical strategies for CsA nephrotoxicity It is well known from the above studies that oxidative stress is a key effect of exposure of CsA. Thus, some antioxidant compounds seem to have the therapeutical potential for this toxicity. Ceftriaxone was reported to be suppress the CsA-induced oxidative stress in rat kidney by modulation of oxidative and antioxidant system (Yilmaz et al., 2011). 902

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antioxidant supplementation with CsA treatment may help to prevent its severe renal, hepatic, or neurological and cardiovascular side effects in transplant or non-transplant patients (Lee, 2010; Lai et al., 2017). Fourth, an analysis of structure-activity relationships may promote the development of non-nephrotoxic analogues of CsA. The excellent clinical results that have been obtained during the prolonged learning phase with CsA are encouraging. A more judicious use of CsA in the future should give rise to improved outcomes. Finally, from the above studies that it seems that CsA hits 100% of the biological targets directly, is that really plasuible, and what are the missing links or the molecular initiating events to say? Are these all off target or are they related to primary pharmacology? All these questions should be carefully considered for the future studies.

use of CsA. The biomarkers should be involed in the mechanisms of CsA nephrotoxicy. However, the underlying function of the biomarkers in the CsA nephrotoxicity is still poorly understood. Oxidative stress is an important toxic effect of CsA. CsA induces ER stress and subsequently triggers human tubular phenotypic changes and death. Importantly, CsA increases mitochondrial ROS production, which modifies the redox balance and induces lipid peroxidation: this leads to CsA nephrotoxicity. CsA treatment also decreases the antioxidant capacity of the kidney: this may sensitize the kidneys to various stresses, thereby promoting the development of nephrotoxicity. However, the upstream event that reguate the CsA-induced oxidative stress is not fully understood. At present, the evidence suggests that while CsA activates autophagy by inducing ER stress and the oxidative stress pathway, this autophagic response has complicated Janus-faced functions in CsA nephrotoxicity. Specifically, some studies show that CsA-induced autophagy appears to attenuate CsA nephrotoxicity, while others suggest that it promotes CsA nephrotoxicity. The factors that drive one or the other function of CsA-induced autophagy and thus cause cell death or survival remain unclear. It is possible that the duration of CsA treatment plays an important role in this active balance. Additional mechanistic studies of the role of autophagy in CsA nephrotoxicity are needed. Many signaling pathways are involved in the molecular mechanisms that underlie CsA nephrotoxicity. Identifying the signaling pathways that contribute to CsA nephrotoxicity is of great importance because this information could lead to the development of early biomarkers of kidney injury. The ERK1/2 pathway plays an important role in the activation of ROS and oxidative stress, and it promotes CsA-induced glomerular dysfunction and the subsequent nephrotoxicity. Crosstalk between p38/ERK and Smad differentially regulates apoptosis in CsAinduced renal proximal tubular cell injury. NF-κB is an important target of CsA and the ability of the anti-aging protein Klotho to ameliorate CsA nephrotoxicity is mediated by its downregulation of this transcription factor. TGF-β1/IL-2/COX-2 is involved in CsA-induced renal tubular atrophy and interstitial fibrosis while endothelin-1/TGF-β1 signaling enhances CsA-induced oxidative stress. CsA amplifies the Ca2+ signaling pathway in a tubule-like cell line by inhibiting the plasma membrane Ca2+ pump; this causes Ca2+-dependent rat proximal tubule injury. Currently, it is unclear that whether the CsA-induced Ca2+ signaling is involved in the mechanism of inhibition of calcineurin. The NO pathway prevents CsA-associated vasoconstriction and thereby helps attenuate CsA-induced nephrotoxicity. However, whether CsA increases or decreases NO expression is not yet clear. Some studies show that CsA significantly decreases NO levels and that this loss of NO protection reduces the vascular tone and makes the kidney more susceptible to CsA nephrotoxicity. However, other studies show that NO synthesis is enhanced during CsA nephrotoxicity and that this counterbalances the predominantly preglomerular vasoconstriction. We suspect that overall, CsA in the kidney inhibits NO synthesis and induces renal endothelium-dependent apoptosis and nephrotoxicity. However, studies on the NO levels in CsA nephrotoxicity and its exact functions in this context are needed. Given that chronic CsA nephropathy increases the rate of late allograft loss and chronic renal failure, a comprehensive understanding of the molecular mechanisms that underlie chronic CsA-induced renal injury is essential to improve the long-term outcomes of CsA-treated patients. Future questions that should be addressed include the ideal dosing schedule of additional immunosuppressive agents, the distinction between nephrotoxicity and rejection, and the precise mode of ADME studies. Second, CsA can be viewed as the prototype of a new generation of immunosuppressive agents that open new perspectives in the field of immunoregulation. The ability to synthesis the compound may permit future biochemical manipulations that increase its immunobiological specificity and decrease its toxicity. Third, exogenous antioxidants, including natural antioxidants, have been shown to inhibit the adverse actions of CsA (Fletcher et al., 2005). Thus, the

Conflicts of interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 31602114; 31601679; 31572575), the Yangtze Fund for Youth Teams of Science and Technology Innovation (2016cqt02), and the project of Czech Science Foundation No. GA1719968S. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.fct.2018.06.054. References Aliancy, J., Stamer, W.D., Wirostko, B., 2017. A review of nitric oxide for the treatment of glaucomatous disease. Ophthalmol. Ther 6 (2), 221–232. Al-Malki, A.L., Moselhy, S.S., 2011. The protective effect of epicatchin against oxidative stress and nephrotoxicity in rats induced by cyclosporine. Hum. Exp. Toxicol. 30 (2), 145–151. Amore, A., Emancipator, S.N., Cirina, P., Conti, G., Ricotti, E., Bagheri, N., Coppo, R., 2000. Nitric oxide mediates cyclosporine-induced apoptosis in cultured renal cells. Kidney Int. 57 (4), 1549–1559. Arrington, D.D., Van Vleet, T.R., Schnellmann, R.G., 2006. Calpain 10: a mitochondrial calpain and its role in calcium-induced mitochondrial dysfunction. Am. J. Physiol. Cell Physiol. 291, 1159–1171. Arumugam, A., Walsh, S.B., Xu, J., Afaq, F., Elmets, C.A., Athar, M., 2012. Combined inhibition of p38 and Akt signaling pathways abrogates cyclosporine A-mediated pathogenesis of aggressive skin SCCs. Biochem. Biophys. Res. Commun. 425 (2), 177–181. Ay, H., Uzun, G., Onem, Y., Aydinoz, S., Yildiz, S., Bilgi, O., Topal, T., Atasoyu, E.M., 2007. Effect of hyperbaric oxygen on cyclosporine-induced nephrotoxicity and oxidative stress in rats. Ren. Fail. 29 (4), 495–501. Aziz, M.M., Eid, N.I., Nada, A.S., Amin, N.E., Ain-Shoka, A.A., 2018. Possible protective effect of the algae spirulina against nephrotoxicity induced by cyclosporine A and/or gamma radiation in rats. Environ. Sci. Pollut. Res. Int. 25 (9), 9060–9070. Baky, N.A., Fadda, L., Al-Rasheed, N.M., Al-Rasheed, N.M., Mohamed, A., Yacoub, H., 2016. Neuroprotective effect of carnosine and cyclosporine-A against inflammation, apoptosis, and oxidative brain damage after closed head injury in immature rats. Toxicol. Mech. Meth. 26 (1), 1–10. Bauer, K., Kretzschmar, A.K., Cvijic, H., Blumert, C., Löffler, D., Brocke-Heidrich, K., Schiene-Fischer, C., Fischer, G., Sinz, A., Clevenger, C.V., Horn, F., 2009. Cyclophilins contributes to Stat3 signaling and survival of multiple myeloma cells. Oncogene 28 (31), 2784–2795. Benway, C.J., Iacomini, J., 2018. Defining a microRNA-mRNA interaction map for calcineurin inhibitor induced nephrotoxicity. Am. J. Transplant. 18 (4), 796–809. Burdmann, E.A., Andoh, T.F., Yu, L., Bennett, W.M., 2003. Cyclosporine nephrotoxicity. Semin. Nephrol. 23 (5), 465–476. Busauschina, A., Schnuelle, P., van der Woude, F.J., 2004. Cyclosporine nephrotoxicity. Transplant. Proc. 36 (2 Suppl), 229S–233S. Caires, A., Fernandes, G.S., Leme, A.M., Castino, B., Pessoa, E.A., Fernandes, S.M., Fonseca, C.D., Vattimo, M.F., Schor, N., Borges, F.T., 2018. Endothelin-1 receptor antagonists protect the kidney against the nephrotoxicity induced by cyclosporine-A in normotensive and hypertensive rats. Braz. J. Med. Biol. Res. 51 (2), e6373. Calderaro, V., Boccellino, M., Cirillo, G., Quagliuolo, L., Cirillo, D., Giovane, A., 2003. Cyclosporine A amplifies Ca2+ signaling pathway in LLC-PK1 cells through the inhibition of plasma membrane Ca2+ pump. J. Am. Soc. Nephrol. 14 (6), 1435–1442.

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