Abstract
Background and purpose: Increasing evidence suggests that ferroptosis plays a key role in the pathophysiology of acute kidney injury (AKI) induced by cisplatin. The Nrf2 signaling pathway regulates oxidative stress and lipid peroxidation and positively regulates cisplatin-induced AKI (CI-AKI). However, its effect as well as that of its activator leonurine on ferroptosis after CI-AKI remain unclear.
Experimental Approach: The anti-ferroptotic effects of Nrf2 and its activator leonurine were assessed using a mouse model of cisplatin-induced AKI. In vitro, the potential effects of leonurine on erastin- and RSL3-induced HK-2 human PTEC ferroptosis were examined.
Key Results: As expected, Nrf2 deletion induced ferroptosis-related protein expression and iron accumulation in vivo, further aggravating CI-AKI. The Nrf2 activator leonurine prevented iron accumulation and lipid peroxidation and inhibited ferroptosis in vitro, while these effects were abolished in siNrf2-treated cells. Moreover, leonurine potently ameliorated cisplatin-induced renal damage, as indicated by the assessment of SCr, BUN, KIM-1, and NGAL. Importantly, leonurine activated the Nrf2 antioxidative signaling pathway and prohibited changes in ferroptosis-related morphological and biochemical indicators, such as the MDA level, SOD and GSH depletion and GPX4 and xCT downregulation, in CI-AKI. Moreover, Nrf2 KO mice were more susceptible to ferroptosis after CI-AKI than control mice, and the protective effects of leonurine on AKI and ferroptosis were largely abolished in Nrf2 KO mice.
Conclusion and Implications: These data suggest that the renal protective effects of Nrf2 and its activator leonurine on CI-AKI are achieved at least partially by inhibiting lipid peroxide-mediated ferroptosis and highlight the potential of leonurine as a CI-AKI treatment.
Key words : Cisplatin-induced acute kidney injury; Ferroptosis; Nrf2; Leonurine
Abbreviations: Leo, leonurine; CDDP, cisplatin; AKI, acute kidney injury; CI-AKI, cisplatin-induced acute kidney injury; HK-2, Human Kidney-2; SCr, serum creatinine; BUN, Blood Urea Nitrogen; SOD, Superoxide Dismutase; MDA, malondialdehyde; GSH, glutathione; Nrf2, nuclear factor erythroid-2; HO-1, Heme Oxygenase-1; NQO1, NAD(P)H quinone dehydrogenase 1; KIM1, kidney injury molecule-1; NGAL, Neutrophil gelatinase-associated lipocalin; FTH-1, ferritin heavy chain; FTL, ferritin light chain; TFR, transferrin receptor 1; GPX4, glutathione peroxidase 4; XCT, cystine/glutamateantiporter system; SLC7A11, Solute Carrier Family 7 Member 11; ROS, reactive oxygen species; LPO, lipid peroxidation; RSL3:(1S,3R)-RSL3; CCK-8, Cell Counting Kit-8; FBS, fetal bovine serum; DCFH-DA, 2,7-Dichlorodihydrofluorescein diacetate; PBS, phosphate buffered saline; DMSO, Dimethyl sulfoxide; HE, Hematoxylin and eosin staining; PCR, Polymerase Chain Reaction; UUO, unilateral ureteral obstruction.
Introduction
Acute kidney injury (AKI), a common and severe syndrome characterized by tubular damage and sudden loss of renal function within hours, is a global health problem with high morbidity and mortality(Hoste et al., 2018). As an antitumor drug, cisplatin is widely used in the treatment of solid tumors and is one of the main causes of AKI in patients with cancer(Yang et al., 2018). Because the exact molecular mechanisms of CI-AKI are not fully understood, strategies for the prevention and treatment of AKI are limited. Tubular cell injury and death play vital roles in the initiation and progression of AKI(Deng et al., 2021; Linkermann et al., 2014). Previous studies focused on the induction of renal tubular cell apoptosis and necrosis by cisplatin, but specific inhibitors of both apoptosis and necrosis failed to completely prevent or stop CI-AKI(Herzog et al., 2012; Tristao et al., 2012). Ferroptosis, a new type of “regulated cell death”, was recently reported to be involved in the pathological processes of cisplatin-induced AKI (Hu et al., 2020). Thus, elucidating the mechanisms by which cisplatin induces ferroptosis will have important clinical value for alleviating AKI.
In contrast to other regulatory cell death mechanisms, such as apoptosis, necrosis and pyrogen death, ferroptosis is a newly discovered regulatory type of cell death caused by iron-dependent lipid peroxidation (Stockwell et al., 2017) (Deng et al., 2021). In the case of ferroptosis, low FTH-1 expression and TFR overexpression lead to excessive iron accumulation, which in turn promotes ROS production via Fenton reactions (Dixon and Stockwell, 2014). At the same time, the function of system XC is disrupted during ferroptosis, thereby decreasing the production of glutathione (GSH) and inhibiting the antioxidant capacity of GPX4 (Wang et al., 2020) (Maiorino et al., 2018). Thus, ROS have been demonstrated to a central role in the entire ferroptosis process (Park and Chung, 2019). In cisplatin-induced AKI, the generation of excess ROS can cause oxidative stress and lead to tubular cell damage. Nrf2, the most important nuclear transcription factor for antioxidative stress, regulates not only the expression of a series of signaling proteins and enzymes to maintain cellular redox homeostasis but also many important genes related to iron storage and transport (Dodson et al., 2019). Previous studies have highlighted the role of Nrf2 in alleviating lipid peroxidation and ferroptosis and further elucidated the role of the Nrf2-lipid peroxidation-ferroptosis axis in neurodegenerative and cardiovascular diseases(Dodson et al., 2019). Many Nrf2 activators have been reported to alleviate cisplatin-induced nephrotoxicity by inhibiting ROS generation (Fan et al., 2020). In addition, ferrostatin-1, the most potent inhibitor of ferroptosis, was shown to significantly reduce cisplatin-induced HK-2 cell death and attenuate CI-AKI in mice(Deng et al., 2019). Considering the close relationship among ROS, Nrf2 and ferroptosis, we proposed that controlling ferroptosis via Nrf2 activation might improve CI-AKI.
Leonurine, a major active alkaloid compound found only in motherwort, has been successfully extracted and depurated(Li et al., 2020). Leonurine was recently shown to regulate various pathological processes, including oxidative stress, inflammation, fibrosis, and apoptosis, as well as a variety of metabolic disorders. Leonurine protects against LPS-induced acute renal injury and fibrosis induced by unilateral ureteral obstruction (UUO) in mice(Cheng et al., 2015). However, an alleviating effect of leonurine on CI-AKI has not been reported. At the same time, some studies have shown that leonurine can alleviate aging and ischemic stroke in mice by activating the Nrf2 pathway(Chen et al., 2019; Xie et al., 2019), but its effect on ferroptosis has not been investigated. Herein, we first investigated whether the levels of Nrf2 are directly related to ferroptosis sensitivity in mice with CI-AKI. We then investigated the potential of leonurine to protect against ferroptosis in vitro and in vivo. Finally, using Nrf2 siRNA and Nrf2 knockout (KO) mice, we further assessed the underlying mechanisms by which leonurine protects against ferroptosis and AKI in vitro and in vivo.