Neutral ceramidase deficiency protects against cisplatin-induced acute kidney injury

Cisplatin is a commonly used chemotherapeutic for the treatment of many solid organ cancers; however, its effectiveness is limited by the development of acute kidney injury (AKI) in 30% of patients. AKI is driven by proximal tubule cell death, leading to rapid decline in renal function. It has previously been shown that sphingolipid metabolism plays a role in regulating many of the biological processes involved in cisplatin-induced AKI. For example, neutral ceramidase (nCDase) is an enzyme responsible for converting ceramide into sphingosine, which is then phosphorylated to become sphingosine-1-phosphate, and our lab previously demonstrated that nCDase knockout (nCDase−/−) in mouse embryonic fibroblasts led to resistance to nutrient and energy deprivation–induced cell death via upregulation of autophagic flux. In this study, we further characterized the role of nCDase in AKI by demonstrating that nCDase−/− mice are resistant to cisplatin-induced AKI. nCDase−/− mice display improved kidney function, reduced injury and structural damage, lower rates of apoptosis, and less ER stress compared to wild-type mice following cisplatin treatment. Although the mechanism of protection is still unknown, we propose that it could be mediated by increased autophagy, as chloroquine treatment resensitized nCDase−/− mice to AKI development. Taken together, we conclude that nCDase may represent a novel target to prevent cisplatin-induced nephrotoxicity.Supplementary key words: autophagy, ceramide, sphingosine-1-phosphate, renal disease, animal models, cisplatin, sphingosine metabolism, chemotherapy, ER stress, chloroquine

Cisplatin (cis-diamminedichloridoplatinum(II)) is an effective chemotherapeutic for the treatment of many solid organ cancers (1, 2, 3). Unfortunately, the success of cisplatin in treating these cancers is limited by its nephrotoxicity. Thirty percent of patients treated with cisplatin develop cisplatin-induced acute kidney injury (AKI) (4, 5, 6, 7). AKI is defined as a rapid decline in glomerular filtration rate, clinically measured by increases in blood urea nitrogen (BUN) or serum creatinine (SCr) (8). AKI is primarily mediated by renal proximal tubule cell death, inflammation, and impaired microvasculature (9). Development of AKI not only limits the ability to treat cancer patients but also puts patients at risk for long-term renal effects (8, 10, 11). As development of cisplatin-induced AKI is thought to be largely driven by cell death (4, 6), protecting proximal tubule cells from death is an attractive strategy to prevent kidney structural damage and maintain function. Sphingolipid metabolism in the kidney is recognized as a regulator of several cellular processes, including cell death, that play a role in the development of AKI and other renal diseases (12, 13, 14); thus, this connection has been explored by our lab.Sphingolipids are a class of bioactive lipids with a common sphingoid base backbone. Sphingolipid metabolism centers around the formation and breakdown of ceramides (15, 16). Neutral ceramidase (nCDase) is an enzyme involved in sphingolipid metabolism that cleaves ceramide into sphingosine, which is then recycled back to ceramide or phosphorylated to become sphingosine-1-phosphate (S1P) (17, 18, 19, 20). The balance of these three bioactive lipids is regulated by dynamic processes and is thought to play an important role in regulating cellular stress responses (15, 16). In particular, ceramide, sphingosine, and S1P have been implicated in regulation of cell death and autophagy (16). Manipulating the balance of ceramide has also been shown to play a role in cisplatin-induced AKI. Inhibition of ceramide generation protected mice from cisplatin-induced AKI, while inhibition of glucosylceramide synthase, an enzyme that glycosylates ceramide species to generate glycosphingolipids, exacerbated cisplatin-induced AKI (21).Our lab demonstrated that nCDase knockout (nCDase^−/−) in mouse embryonic fibroblasts protected cells from nutrient and energy deprivation–induced cell death (22). This protection was mediated via upregulation of autophagy and mitophagy (22). Autophagy is known to play a protective role in cisplatin-induced AKI by decreasing levels of apoptotic cell death (23, 24). Additionally, nCDase^−/− mice were found to be protected in a model of traumatic brain injury due to preservation of mitochondrial function (25). Mitochondrial dysfunction is also known to be a major mediator of cisplatin-induced AKI (4). Therefore, we hypothesized that nCDase deficiency would prevent development of cisplatin-induced AKI via upregulation of autophagy and inhibition of cell death.In this study, we utilized wild-type and nCDase^−/− C57BL/6 mice in the cisplatin-induced AKI model. Our data demonstrate that loss of nCDase attenuates AKI development following 20 mg/kg cisplatin treatment as evidenced by markers of kidney function, kidney injury, cell death, and kidney pathology. Furthermore, we demonstrate that chloroquine (CQ) treatment exacerbates development of AKI in nCDase^−/− mice. This suggests that nCDase deficiency could be mediating protection from cisplatin-induced AKI by increasing basal autophagy and allowing cells to survive cisplatin-induced injury. These data indicate that nCDase may be a feasible target for prevention of cisplatin nephrotoxicity.