In vitro assessment of the role of endoplasmic reticulum stress in sunitinib-induced liver and kidney toxicity

Highlights

• Endoplasmic reticulum stress is crucial for sunitinib-induced hepatotoxicity.
• Mitochondrial damage/oxidative stress contributes to the hepatotoxicity mechanism.
• Nephrotoxicity mechanism seems to be different from hepatotoxicity.

Abstract

Sunitinib, a multi-_targeted tyrosine kinase inhibitor, is prescribed for the treatment of metastatic gastrointestinal stromal tumors, advanced metastatic renal cell carcinoma, and pancreatic neuroendocrine tumors. Hepatotoxicity and nephrotoxicity are significant adverse effects of sunitinib administration; however, there is limited information regarding the molecular mechanisms of these adverse effects. The aim of the present study was to elucidate the role of endoplasmic reticulum stress in hepatotoxicity and nephrotoxicity induced by sunitinib. In addition to endoplasmic reticulum stress, oxidative stress and mitochondrial membrane potential were evaluated to investigate the molecular mechanism more comprehensively. Findings revealed that sunitinib exposure significantly increased the reactive oxygen species levels and decreased the Nrf2 gene expression and GSH/GSSG ratio, suggesting oxidative stress induction in normal hepatocyte (AML12) and normal kidney (HK-2) cell lines. Endoplasmic reticulum stress markers, including ATF4, CHOP, IRE1α, XBP1s and ATF6 mRNA expressions, were upregulated in AML12 cells. Furthermore, enhanced intracellular calcium levels also indicate endoplasmic reticulum stress in hepatocytes. In contrast, sunitinib exposure did not alter endoplasmic reticulum-related gene expression levels and intracellular calcium levels in HK-2 cells. In terms of mitochondrial membrane potential and caspase-3 activity, sunitinib induced mitochondrial membrane damage and increased caspase-3 activation not only in AML12 cells but also in HK-2 cells. The research findings indicate that sunitinib may induce cytotoxic effects in hepatocytes through mechanisms involving oxidative stress, endoplasmic reticulum stress, and mitochondrial damage. However, in the kidney, the toxicity mechanism is different from that of liver, and the endoplasmic reticulum stress does not seem to be involved in this mechanism.

Introduction

Sunitinib, an oral small molecule multi-_targeted tyrosine kinase inhibitor (TKI), has been demonstrated to enhance the survival of patients diagnosed with gastrointestinal stromal tumors, pancreatic neuroendocrine tumors, and metastatic renal cell carcinoma. Treatment with sunitinib is associated with the development of hepatotoxicity, which has the potential to be fatal (Shi et al., 2020), as well as nephrotoxicity, posing a significant threat to the health of patients (Xiao et al., 2019). There have been several clinical case reports on liver and kidney injury caused by sunitinib. One meta-analysis revealed that elevated liver enzymes were present in 40 % of 5.658 sunitinib-treated patients with metastatic renal cell carcinoma (Ibrahim et al., 2013). In clinical trials, it was found that 40–60 % of patients treated with sunitinib experienced elevated serum transaminase levels. Specifically, 2–5 % of these cases reached grade 3 or 4 (Shah et al., 2013) with severe liver damage. Food and Drug Administration (FDA) has issued a black-box warning regarding a rare but life-threatening hepatotoxicity following treatment with sunitinib (Shi et al., 2020). Although sunitinib has been implicated in causing severe or life-threatening liver damage, there is limited information regarding the underlying mechanism of this adverse effect. Mitochondrial dysfunction is the primary molecular mechanism that will be suggested. Indeed, sunitinib has been shown to increase mitochondrial reactive oxygen species (ROS) production, impair mitochondrial membrane potential and oxygen consumption, and induce hepatocyte apoptosis in both in vivo and in vitro studies (Paech et al., 2018, Paech et al., 2017). Furthermore, Tang et al. suggested that sunitinib induces hepatocyte cell death, and the ROS/mitogen-activated protein kinases (MAPKs) signaling pathway might play a role in sunitinib-induced hepatotoxicity (Tang et al., 2022). Guo et al. revealed that the ROS/ nuclear factor erythroid 2-related factor 2 (Nrf2) pathway contributes to mitochondrial apoptosis and sunitinib-induced hepatotoxicity (Guo et al., 2021). In addition to hepatotoxicity, sunitinib's nephrotoxic effects present a notable risk to the health of patients and may constrict the clinical use of the drug. It has been revealed that treatment with sunitinib can result in 15.86 % renal failure, 5.90 % proteinuria, and 2.28 % hematuria in patients (Xiao et al., 2019). The current understanding of the molecular mechanisms underlying sunitinib-induced kidney injury is also limited, similar to hepatotoxicity.

Endoplasmic reticulum (ER) is vital for maintaining cellular homeostasis by regulating the synthesis, folding, and transport of proteins, as well as contributing to lipid metabolism and calcium homeostasis (Schwarz and Blower, 2016). The activation of the unfolded protein response (UPR) in cells is triggered by the accumulation of unfolded or misfolded proteins within ER, leading to ER stress. Three transmembrane receptors, namely inositol-requiring enzyme 1α (IRE1α), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6), orchestrate the management of UPR in ER. They trigger relevant downstream signal transduction pathways in order to enhance the protein-folding capacity of ER. Although UPR is an adaptive mechanism, uncontrolled or excessive ER stress is a significant factor that may lead to cellular damage and, ultimately, cell death. ER stress (Szegezdi et al., 2006). Emerging evidence suggests that ER stress is a contributing factor in the pathogenesis of numerous human diseases, including liver injury, neurodegenerative disorders, metabolic syndromes, and autoimmune conditions and plays a significant role in the development of drug-induced liver injury and kidney toxicity (Foufelle and Fromenty, 2016, Lin et al., 2008). Furthermore, it is essential to emphasize the association between ER stress and mitochondrial damage in drug-induced toxicity (Pu et al., 2023). ER and mitochondria play a collaborative role in apoptosis signaling through close contact sites known as mitochondria-associated ER membranes (MAMs). MAMs act as a central platform for different signaling pathways critical for maintaining cellular homeostasis. In the presence of mitochondrial damage, disruption of the redox balance within ER occurs, leading to impaired ER functions and the initiation of ER stress (Marchi et al., 2018). Studies have also indicated that drug-induced ER stress can lead to mitochondrial dysfunction by impairing mitochondrial membrane potential, respiration, and other mitochondrial structure and functions (Xiao et al., 2020).

Recent in vitro and in vivo studies have revealed that sunitinib can lead to mitochondrial dysfunction and impair mitochondrial structure and liver functions (Guo et al., 2021, Paech et al., 2018). Although the role of mitochondria in sunitinib-induced hepatotoxicity has been recently investigated, a significant knowledge gap exists regarding the involvement of ER stress in these toxic effects. In terms of nephrotoxicity, it is unclear whether mitochondria and/or ER stress contribute to the mechanism of sunitinib-induced injury. Therefore, the main objective of this study is to evaluate the potential contribution of ER stress to the development of hepatotoxicity and nephrotoxicity induced by sunitinib in order to enhance our understanding of these adverse outcome pathways. To test this hypothesis, we used AML12 mouse hepatocytes and HK-2 human proximal tubule cells as in vitro models. Our primary focus was identifying whether sunitinib induces ER stress through alterations in the expression of ER stress-related mRNAs (ATF4, CHOP, IRE1, XBP1s and ATF6) and cytosolic calcium ion (Ca2+) levels. In addition to the assessment of ER stress, the investigation encompassed the evaluation of oxidative stress parameters, including ROS levels, GSH/GSSG ratio and Nrf2 mRNA expression, the examination of mitochondrial dysfunction via mitochondrial membrane potential and the measurement of caspase-3 activity. Furthermore, drug incubations were carried out in the presence of a well-established ER stress inhibitor, 4-phenylbutyrate (4-PBA), a chemical chaperone that facilitates protein folding and trafficking (Zhang et al., 2016) to assess the involvement of ER stress in the sunitinib-induced cellular toxicity. The present study is the first to elucidate the role of ER stress in hepatotoxicity and nephrotoxicity associated with sunitinib. Hence, these novel findings will significantly enhance our understanding, and the investigation of the underlying mechanisms could lead to the development of new strategies for the prevention and treatment of significant adverse effects caused by sunitinib.