Calcineurin inhibitor induced nephrotoxicity leading to kidney failure

K.H. Liang, Department of Nephrology, University Medical Center Utrecht

E. Sendino Garví, Department of Pharmaceutical Sciences, Utrecht University

A.M. van Genderen, Department of Pharmaceutical Sciences, Utrecht University

R. Masereeuw, Department of Pharmaceutical Sciences, Utrecht University

Tacrolimus (TAC) and cyclosporin A (CsA) are widely used calcineurin inhibitors (CNI) whose therapeutic efficacy (i.e. immunosuppression) is limited by nephrotoxicity, particularly in kidney transplant recipients. This adverse outcome pathway (AOP) outlines the mechanistic sequence of events leading from CsA or TAC exposure to chronic kidney injury. Despite the molecular initiating event remains unknown, the first recorded key event is the activation of nuclear factor kappa B (NF-κB) and induction of NADPH oxidase 2 (NOX2). These upstream events promote increased production of reactive oxygen species (ROS), a well-established response observed in vitro and in vivo. Elevated ROS levels drive oxidative stress, characterized by depletion of antioxidant capacity and increased oxidative damage markers. Oxidative stress leads to mitochondrial dysfunction, including loss of membrane potential, impaired respiration, and reduced ATP production. These mitochondrial changes are accompanied by metabolic disturbances, such as alterations in TCA cycle intermediates, amino acid pathways, and fatty acid metabolism, described across cell-based systems and in vivo studies. Metabolic impairment and cellular stress are associated with increased TGF-β signalling, a central event consistently linked to CNI-induced tubular injury and fibrosis. Persistent activation of TGF-β pathways contributes to extracellular matrix deposition, tubular atrophy, and interstitial fibrosis. These tissue-level alterations ultimately culminate in decreased kidney function, reflected clinically by reduced GFR and progression toward chronic kidney disease. This AOP provides a structured framework to integrate mechanistic evidence, identify key data gaps, and support the development of  new approach methodology-based approaches for assessing CNI nephrotoxicity.

Calcineurin inhibitor induced nephrotoxicity leading to kidney failure

This AOP describes the sequence of key events (KE’s) that link the usage of CNIs to nephrotoxicity and the adverse outcome kidney failure. CNIs are immunosuppressive drugs that inhibit calcineurin by binding to immunophilins, such as cyclophilin and FK-binding proteins (1). Calcinuerin is a crucial enzyme for activating T-cells. Blockage of the enzyme leads to reduction of interleukin-2 and T-cell proliferation. Currently, the two most commonly used systemic CNIs are TAC and CsA, typically administered  orally. Indication for systemic CNI treatment is solid organ transplantation and are used to effectively manage several autoimmune disorders, including lupus nephritis, idiopathic inflammatory myositis and interstitial lung disease. CNIs are associated with both acute and chronic kidney damage due to its narrow therapeutic window (2, 3). Nephrotoxicity secondary to CNIs occurs in liver (52%), heart (20–75%) and kidney (76–94%) transplant recipients (4). Risk factors for CNI toxicity include volume depletion, diuretic use, older donor age, exposure to high CNI doses, concomitant use of nephrotoxic drugs (particularly NSAIDs), concomitant use of CYP3A4/5 or P-glycoprotein (ABCB1) inhibitors, and genetic polymorphisms affecting CYP3A4/5 and P-glycoprotein function. CNI are also available in a topical form, which is used for mild-to-moderate atopic dermatitis. However, because of the route of exposure, nephrotoxicity is not an issue for topical CNIs.  

​​​​​​​Molecular Initiating Event: Unkown

As CNI-induced nephrotoxicity involves several mechanisms, a well-defined MIE cannot be found. CNI-induced nephrotoxicity can be a result of vasoconstriction of the afferent arteriole, leading to ischemia in the kidney (2). However, there is also a direct toxic effect, such as increased oxidative stress, of CNI’s on tubular epithelial cells, as has been shown in multiple studies (5-7). In tubular epithelial cells, the influx transporter, if existing, of CNI’s is unknown, and the only efflux transporter that has been identified to transport CNI’s is P-glycoprotein (ABCB1). Identifying the initial interaction of CNI with kidney cells, and consequently the MIE, remains challenging.

​​​​​​​​​​​​​​Key Event 1: Activation of NF-kB and NOX2

NF-κB, a downstream target of CNI, is activated in tubular cells after treatment with TAC and CsA (8). The pathway of NF-κB activation is not exactly known, but it could be activated by angiotensin II (9), degradation of I-κBα (10) or upstream kinases: c-Jun N-terminal kinases (JNK) and Janus Kinase (JAK)(11). A transcriptomics analysis of mouse cortical proximal tubular cells treated with TAC and CsA showed significant activation of NF-κB and pro-inflammatory cytokines which are strongly associated with kidney disease, such as monocyte chemoattractant protein -1 (MCP-1) and Rantes (11). Inhibition of Nf-κB after CNI exposure in several in vivo studies have shown attenuation of nephropathy, indicating an important role of the factor in the development of CNI nephrotoxicity (12, 13). Additionaly, activation of NF-κB leads to upregulation of NADPH oxidases (NOX) in the tubule (14).

The primary function of NOX are generating reactive oxidative species (ROS) by transferring one electron to dioxygen leading to the product superoxide. Isoforms NOX1, NOX2 and NOX4 are expressed in the kidney and known sources of ROS production (15).  NOX2 in particular is associated with CsA-induced kidney fibrosis and chronic nephrotoxicity (16, 17). Furthermore, NOX2 was significantly increased in the tubule and interstitial cells after immunohistochemical analyses of kidney biopsies from liver transplant recipients with CNI nephrotoxicity after exposure to TAC or CsA (16). Moreover, TAC increases ROS via NOX in the glomerulus, especially in endothelial glomerular cells (18).

​​​​​​​Key Event 2: ROS production

Mitochondria generate about 90% of cellular ROS, making them the primary source of ROS in renal proximal tubules (19). The kidney is highly metabolically active, abundant in mitochondira, and, therefore, particularly susceptible to ROS. When ROS production exceeds the cell’s antioxidant capacity, oxidative stress occurs, damaging cellular components and contributing to chronic kidney disease (CKD) progression (20).

In a proximal tubular cell line derived from a healthy human adult male kidney (human kidney-2 (HK-2) cells), TAC increased the intracellular ROS levels by 1.67 fold compared to the control group (21). Exposure to TAC (30 µM and 60 µM, for 24 h) in induced pluripotent stem cell (iPSC) derived  kidney organoids significantly increased ROS levels detected by immunofluorescence staining with MitoSOX Red (22). Moreover, damaged mitochondria with spherical shapes and cristolysis were observed in the kidney organoids after TAC exposure, and quantification with Mitrotracker showed increased mitochondrial stress (22).  

In addition to increasing ROS, CNI also disrupt antioxidant defense systems leading to imbalances in the redox environment, amongst which decreasing glutathione (23). Moreover, CsA exposure damages the inner and outer mitochondrial membrane, resulting in mitochondrial permeability transition pores. The exact mechanism has yet to be elucidated, but it has been suggested that oxidative stress may alter thiol groups of membrane proteins, leading to misfolding and clustering of these proteins and resulting in opening of membrane pores (23).  Subsequently, apoptotic mediators, such as cytochrome c, are released into the cytosol. Moreover, increased ROS might lead to increased expression of dynamin related protein 1, a GTPase that regulates mitochondrial fission and decreased expression of mitofusin 2  and optic atrophy protein 1 (Opa1), both important proteins in the mitochondrial fusion process (24, 25). Consequently, increased fission and decreased fusion takes place in the mitochondria, dysregulating the apoptotic pathways (26, 27).

​​​​​​​​​​​​​​Key Event 3: Oxidative stress

Oxidative stress, defined by an imbalance between pro-oxidant and antioxidant systems, is harmful to cellular health due to the excessive production of ROS and reactive nitrogen species  (28). Evidence strongly indicates this to be a KE in TAC-induced nephrotoxicity (7, 29-31). A study in (porcine-derived) LLC-PK1 cells demonstrated an increase in hydrogen peroxide, a direct indicator of oxidative stress upon TAC treatment with increased expression of pro-oxidant enzymes and reduced activity of antioxidant pathways, resulting in cellular damage and apoptosis (7).

In vitro studies using kidney cell lines and kidney organoids offer deeper insights into the cellular and molecular mechanisms of TAC-induced oxidative stress. Kidney proximal tubular epithelial cells exposed to TAC exhibit upregulation of oxidative stress markers and mitochondrial dysfunction (32). These findings are supported by several other studies, which reported downregulation of antioxidants and upregulation of NOX2 after TAC in vitro and in vivo. Furthermore, treatment with coenzyme Q10 has been shown to alleviate TAC-induced kidney dysfunction by preventing ROS production and improving mitochondrial respiration in HK-2 cells (33).

In vivo studies in rodents further clarify the mechanisms by which TAC induces oxidative stress (34). Further, in an intracellular metabolomics analyses, mice treated with CNIs showed increased oxidative stress by either decreased glutathione or decreased glutathione peroxidase activity in the kidney cells (7). Rabbits exposed to TAC showed increased oxidative stress caused by impairments in antioxidant response (35). Additionally, the anti-oxidant cilastatin counteracted TAC-induced nephrotoxicity in rat proximal tubular cells, resulting in reduced oxidative DNA damage (33). Overall, the use of antioxidants and oxidative stress inhibitors has shown potential in mitigating the oxidative damage caused by TAC in vitro and in vivo, suggesting possible therapeutic strategies to counteract its nephrotoxic  effects (36).

Human studies have also indicated that patients undergoing TAC therapy exhibit decreased antioxidant defenses (37, 38) including reduced glutathione levels. Additionally, increased levels of malondialdehyde, indicating a process called lipid peroxidation, as a result of oxidative stress have been found in patients. Collectively, these findings underscore oxidative stress as an early key event in TAC exposure, potentially initiating the cascade of events that eventually leads to kidney failure.

​​​​​​​Key Event 4: Mitochondrial dysfunction

The kidney, with its high energy demands for reabsorption processes across different nephron segments, relies heavily on mitochondria. Mitochondrial dysfunction is a significant factor, leading to impaired ATP production, increased ROS levels, metabolic deficiencies and damage that directly impact kidney function (39).

In vitro studies using HK-2 cells have shown that TAC exposure causes a loss of mitochondrial membrane potential. These effects were accompanied by decreased oxygen consumption rates and impaired ATP synthesis, further confirming mitochondrial dysfunction (33). Also kidney organoids treated with TAC exhibit significant mitochondrial damage, including loss of mitochondrial membrane potential, increased ROS production, and activation of autophagy as a cellular response to mitochondrial stress (40). Additionally, coenzyme Q10, a mitochondrial-targeted antioxidant, could partly mitigate the mitochondrial dysfunction and oxidative stress induced by TAC (41).

In vivo studies have shown that TAC administration in mice results in increased mitochondrial ROS production and decreased antioxidant defenses, exacerbating mitochondrial dysfunction and kidney injury (42). Electron microscopy of kidney tissues from TAC-treated mice revealed a reduction in both the number and volume of mitochondria, along with structural abnormalities (33), which was also demonstrated in rats, with the remaining mitochondria appearing fragmented (fission and fusion) (33). These findings suggest that TAC not only impairs mitochondrial function but also affects mitochondrial biogenesis and morphology.

Long-term exposure of human patients to TAC leads to progressive kidney failure, characterized by interstitial fibrosis, tubular atrophy, and inflammation (43), all of this potentially linked to mitochondrial damage. In agreement, ultrastructural analysis of kidney biopsies from patients treated with TAC revealed mitochondrial swelling, cristae disruption, and increased ROS production (44).

​​​​​​​​​​​​​​Key Event 5: Metabolic impairment

Cell metabolism impairment is a critical event in the pathway of TAC-induced nephrotoxicity, encompassing various disruptions that contribute to kidney damage and dysfunction (42, 45). Within mitochondria, the TCA cycle, fueled by glycolysis-derived pyruvate, produces energy-rich nucleotides and accumulates coenzyme NADH+ in the presence of oxygen (46, 47). NADH is processed by the mitochondrial respiration chain complex (subunits I-IV), along with cytochrome C and coenzyme Q10, creating a gradient that converts ADP into ATP, which is then transported into the cytosol (48).

In vivo studies have provided additional insights into the mechanisms of TAC-induced nephrotoxicity. For instance, in a mouse model, TAC administration resulted in significant alterations in kidney metabolism, including changes in metabolites such as L-valine and D-glucose, indicative of disrupted mitochondrial function (42, 45). Treatment with non-toxic doses of TAC showed impairments in the TCA cycle, including increased folate metabolism, citric acid metabolism, and glutathione synthesis, indicating a metabolic switch as a reaction to oxidative stress (G) (42, 45). In proximal tubule cells, L-carnitines were the most differentially accumulated metabolites upon TAC exposure in. Other in vitro studies using kidney cell lines have also demonstrated the impact of TAC on cellular metabolism including mitochondrial function disruption leading to decreased ATP production and increased ROS production (49, 50). These metabolic changes induce cellular stress and apoptosis, contributing to tubular dysfunction and fibrosis. Additionally, they found that TAC inhibits key metabolic enzymes, such as pyruvate dehydrogenase and citrate synthase, further impairing cellular energy metabolism. These findings highlight the direct effects of TAC on kidney cell metabolism and its role in nephrotoxicity development.

Furthermore, another study found TAC-induced metabolic impairment involved alterations in amino acid metabolism, with decreased levels of essential amino acids such as valine and leucine (7). These changes impair protein synthesis and cellular repair mechanisms, further exacerbating kidney damage.

In clinical settings, TAC-induced nephrotoxicity is often observed in kidney transplant recipients (50). Studies have shown that TAC can cause metabolic disturbances, including hyperglycemia and dyslipidemia, which are risk factors for CKD (51). TAC impairs glucose metabolism by reducing insulin secretion and increasing insulin resistance, leading to hyperglycemia (52). Additionally, the same study found TAC linked to alterations in lipid metabolism, resulting in elevated levels of cholesterol and triglycerides. These metabolic changes exacerbate kidney injury by promoting oxidative stress and inflammation within the kidney.

​​​​​​​Key Event 6: Aberrant TGF-βeta

Exposure to TAC or CsA has been associated with significant alterations in TGF-β expression in the kidney, contributing to cytoxicity and fibrosis (53). An increased TGF-β expression has been observed with an aberrant activation of the TGF-β receptor stimulating the smad-mediated production of extracellular matrix (ECM) components, including collagen and fibronectin, features of fibrosis (49, 54). In transplant recipients, long-term treatment with TAC or CsA resulted in elevated intrarenal expression of TGF-β, collagen, fibronectin, matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinases-2 (TIMP-2), and osteopontin, with TAC showing more pronounced effects than CsA (53). The TGF-β-inducible gene-H3 is a product induced by TGF-β1 and plays a role in the fibrotic response. In vitro studies have demonstrated that TAC induces fibroblast-to-myofibroblast transition via a TGF-β-dependent mechanism, which is an indication of fibrosis (55). This process involves the inhibition of the calcineurin (Cn)/nuclear factor of activated T cells (NFAT) axis, induction of TGF-β1 ligand secretion, and receptor activation in kidney fibroblasts.

Furthermore, anti-TGF-β treatment in animal models has been shown to prevent nephrotoxicity induced by CNIs, highlighting the therapeutic potential of targeting the TGF-β pathway to mitigate kidney fibrosis (56).

​​​​​​​Key Event 7: Increased kidney fibrosis

TAC-treated kidneys exhibit significant evidence of kidney fibrosis, including increased expression of alpha-smooth muscle actin (α-SMA), indicating fibroblast activation (55, 57). Nitric oxide modulation has been proposed to affect fibrosis by altering TGF-β1 expression, leading to increased matrix deposition and decreased matrix degradation through increased plasminogen activator inhibitor-1 (58). In addition to increased TGF-β expression, macrophage infiltration and cellular proliferation are critical cellular players contributing to tubulointerstitial fibrosis (59). Cellular proliferation occurs both in the tubular and interstitial regions, starting in the medulla and progressing to fibrotic areas. Macrophages, which produce pro-fibrotic cytokines such as TGF-β and platelet-derived growth factor, may infiltrate during the early phases of fibrosis (60). This infiltration is potentially related to increased expression of osteopontin (OPN), a chemoattractant for macrophages, following CsA exposure (61).

In vitro studies on PTECs have shown that both TAC and CsA induce significant OPN mRNA expression (62). Treatment of mice with TGF-β resulted in increased intra-renal OPN mRNA and protein expression. In TAC-treated animals, a parallel increase in OPN and TGF-β mRNA was observed. Anti-TGF-β antibody treatment in vitro inhibited both TGF-β and OPN mRNA expression in PTECs, and similar results were obtained in vivo with CsA-treated mice (63). In support, OPN-deficient mice exhibit less severe CsA nephrotoxicity, characterized by reduced interstitial collagen deposition and macrophage infiltration, compared to control mice (61). Macrophage influx has been correlated with apoptosis in kidney tissue of CsA-treated rats (64). Similarly, OPN expression has been associated with increased microvascular injury in CsA nephrotoxicity, suggesting it could be an early marker of CNI toxicity (65).

​​​​​​​​​​​​​​Key Event 8: Apoptosis

TAC and CsA have been directly related to cellular damage that activate apoptotic pathways (5, 6), which is primarily driven by oxidative stress and direct toxic effect on tubular cells (7). Additionally, TAC can activate the Fas system, a critical pathway in apoptosis, leading to increased cell death in kidney tissues (66). Treatment with TAC and CsA activates the endoplasmic reticulum (ER) stress pathway, leading to the upregulation of CHOP (C/EBP homologous protein), a key mediator of ER stress-induced apoptosis (67). These cellular events culminate in the activation of caspase-12 and caspase-3 (68), executing the apoptotic process. Furthermore, in a study involving mice, TAC administration led to tubular atrophy and interstitial fibrosis, with a marked increase in apoptotic cells in the kidney cortex (66). In the clinic, biopsies from kidney transplant recipients on TAC therapy revealed increased apoptotic markers, such as caspase-3 activation and DNA fragmentation (64). These findings suggest that TAC-induced apoptosis contributes to the deterioration of kidney function in humans.

​​​​​​​Adverse Outcome: Kidney failure

Kidney biopsies taken from patients exposed to TAC and CsA revealed several clinical manifestations of nephrotoxicity. These include mild arteriolopathy, striped interstitial fibrosis, glomerular congestion, tubular microcalcification and arterial hyalinosis (69). Other than histological abnormalities, decline in kidney function is often observed in patients (70). Kidney function can be estimated by measuring serum creatinine levels. Creatinine is a waste product in the body that is freely filtered at a constant rate and minimally reabsorbed by the kidney. Using serum creatinine, the estimated glomerular filtration rate (eGFR) can be calculated with a formula that adjusts for age and sex. Kidney failure, e.g. end-stage kidney disease, is defined as an eGFR below 15 mL/min/1.73 m². Next to declined eGFR, other indicators for declined kidney function are decreases in urea clearance, leading to increased blood urea nitrogen (70). Urea is the end product of protein metabolism formed in the liver and is excreted, reabsorbed and transported by the kidneys. Studies have reported that 9.5% to 16.5% of patients develop kidney failure as a result of continuous CNI usage (71, 72).

This adverse outcome pathway (AOP) is developed as part of the ‘Virtual Human for Safety Assessment (VHP4Safety)’ consortium. The mission of the consortium is to improve the prediction of the potential harmful effects of chemicals and pharmaceuticals based on a holistic, interdisciplinary definition of human health by developing the Virtual Human Platform and accelerating the transition from animal-based testing to innovative safety assessment. The Virtual Human Platform integrates data on human physiology, chemical characteristics and perturbations of biological pathways, for the first time in an inclusive and integrated manner. This project is funded by the Dutch Research Council (NWO) programme entitled the ’Dutch Research Agenda: Research on Routes by Consortia (NWA-ORC).

Within the VHP consortium, in vitro case studies are used to feed the Virtual Human Platform with newly generated data. This AOP focuses on nephrotoxicity caused by tacrolimus (TAC), cyclosporin A (CsA) and other calcineurin inhibitors leading to kidney failure.

In this section, we assessed the AOP using the Brasdford-Hill criteria (73), including the AOP’s biological plausibility and assessment of the empirical evidence: dose-response and temporal concordance. We also assessed the overall KEs within the AOP, including their domain of applicability and essenciality.

Biological Plausibility

Currently, there is no evidence supporting the intracellular transport of calcineurin inhibitors, specifically TAC and CsA. TAC has been documented to be secreted from proximal tubule cells in the kidney via the P-glycoprotein efflux pump, encoded by the ABCB1/MDR1 gene (74). However, it remains unclear whether this transport is exclusively mediated by P-glycoprotein or involves other transporters. This gap in knowledge regarding the cellular transport mechanisms of these agents presents a significant challenge in elucidating the initial triggering event and subsequent KEs and relationships (KERs). Although the MIE could not be identified, it has been suggested that NF-κB and NOX2 activation are among the earliest key events in nephrotoxicity induced by CNI (14, 16, 17). Exposure to these nephrotoxic agents results in increased expression of both NF-κB and NOX2. The activation of NF-κB and NOX2 promotes the generation of ROS by transferring an electron from NADPH to molecular oxygen within the mitochondrial respiratory chain.

In this AOP, although the sequence of KEs is depicted linearly, we propose that the KEs of ROS production, oxidative stress, and mitochondrial dysfunction are fundamentally interconnected and dynamic processes that occur simultaneously. This simultaneity complicates the assessment of whether one event precedes another or if they occur concurrently. Given that critical metabolic processes occur in the mitochondria, mitochondrial dysfunction results in metabolic impairment (7). Studies have identified the metabolic pathways of arginine, amino acids, and pyrimidine as the most affected by TAC exposure in both in vitro and in vivo models (7). Metabolic impairment due to CNI exposure has been associated with aberrant TGF-β signaling and subsequent fibrosis in kidney cells and animal models (49). Evidence suggests that fibrosis may arise from either increased TGF-β levels or malfunctions in the TGF-β receptor signaling pathway, or a combination of both (49). Increased fibrosis has been observed alongside the presence of pro-apoptotic and apoptotic cells in in vitro, animal, and human studies (64, 75). However, apoptosis can be triggered by various stressors, including oxidative stress, mitochondrial dysfunction, and increased fibrosis, complicating its placement within the AOP framework. Ultimately, increased apoptosis leads to the loss of functional kidney tissue and eventual kidney failure.

​​​​​​​Empirical evidence: Dose-response and temporal concordance

CNI induced kidney damage in patients is associated with the dosage of CNI. A mean reduction in TAC dosage of 41% (range 11–89) led to a 86% (range 45–100) reduction in serum creatinine within 1–14 days (76). To decrease CNI usage, combining CNI with or switching to other immunosuppressive drugs seems to be able to partly restore kidney function. Switching from CNI to sirolimus for example led to an increase of 27% of eGFR (from 34 to 42 ml/min/1.73m²) in adult liver transplant recipients (77). Adding immunosuppressant mycophenolate mofetil to reduce CNI doses led to decreased serum creatinine levels from 2.63+/-0.39 to 1.74+/-0.34 mg/dl after one month and was maintained within a follow-up period of 4.8 years in adult liver transplant recipients (78). In the pediatric heart transplant population, improvement of kidney function after decreasing CNI by 50% and adding mycophenolate was also observed. At 1 year post-intervention, GFR was increased by 67% from 46.5 to 77.6mL/min/1.73 m²a nd remained stable during the mean follow-up of 26.3 months (79).

In animal models, male Sprague-Dawley rats dosed daily with CsA (2.5 or 25 mg/kg/day), TAC (0.6 or 6 mg/kg/day) for 1-28 days, a significant increase in blood urea nitrogen was observed in rats treated with CsA (high dose) or TAC (high dose) for 14 and 28 days (80). In another study, significant impairment of eGFR was seen in Sprague-Dawley rats treated with doses of CsA as low as 5 mg/kg/day (81). CsA 7.5 mg/kg/day caused a significant reduction in effective kidney plasma flow, and at 10 mg/kg/day filtration fraction declined significantly, again, implying that CNI-induced kidney failure is dose-dependent (81). In vitro models also showed that several KE’s in this AOP are dose-dependent. First of all, Jin et al. showed a dose-response in cell viability when HK-2 cells were exposed to TAC (82). Additionally, increased ROS production and acecelerated apoptosis were found in those cells in a dose-dependent manner. In human mesangial cells, CsA exposure led to a dose-dependent loss of viability, and only after 48 hours a decrease in cell proliferation was observed, suggesting that CsA nephrotoxicity is also time-dependent (83). In PTEC, the oxygen consumption rate and activity of the electron transport chain complexes I, II, IV were dose-dependently decreased after 48 hour exposure to CsA whereas glycolysis, measured by the extracellular acidification rate is dose-dependently increased, which both could be an indicator of mitochondrial dysfunction (84). In conclusion, aforementioned in vitro, in vivo and in clinical results imply that CNI treatment is associated with several KE’s and kidney failure in a dose- and time-dependent manner.

The AOP described here aims to provide a basic mechanistic framework for developing in vitro assays that could accurately predict quantitatively nephrotoxicity safety assessments. These assays could be part of an integrated testing strategy to minimize the need for repeated dose toxicity studies. Generating quantitative data by measuring all KEs in a single model after exposure to various concentrations of CNI could also accelerate the development of computational predictive methods. With potential overlap between KEs in this AOP with other nephrotoxic agents, the current AOP could offer additional connections for extensive networks to model nephrotoxicity and kidney failure.

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