The Role Of Anaphylatoxin C5a In The Pathogenesis Of Cisplatin-induced Nephrotoxicity In Mice

Cisplatin is one of the most potent chemotherapeutic agents available that is widely used to treat a variety of malignancies, including ovarian, lung, head, and neck cancers, as well as testicular and bladder tumors. Unfortunately, at high doses, cisplatin induces cumulative and dose-dependent nephrotoxicity, a major side effect that restricts maximization of therapeutic effects. In clinical practice, approximately one third of patients experience renal dysfunction after treatment with cisplatin. Cisplatin nephrotoxicity is likely caused by a combination of multiple mechanisms, involving DNA damage, caspase activation, mitochondrial dysfunction and formation of reactive oxygen species. However, the exact molecular and cellular mechanisms by which cisplatin induces nephrotoxicity remain unclear. Recently, results from multiple studies strongly implicate the importance of inflammatory mechanisms in the pathogenesis of cisplatin-induced nephrotoxicity. Importantly, urinary levels of complement terminal complexes (C5b-9), the final end-product of complement activation, is increased in cisplatin-treated patients and is associated with nephrotoxicity. These findings, coupled with the important role of complements in immune and inflammatory responses, have led us to hypothesize that activation of the complement system contribute to cisplatin-induced nephrotoxicity.Materials and Methods Pathogen-free, 6-to 8-week-old male C5-deficient mice (C5KO), C5aR knockout mice (C5aRKO), CD59ab double knockout mice (CD59KO) and corresponding control mice were treated with cisplatin (dissolved in saline, 20μg/g body wt ip) or saline and sacrificed 72 hours post injection. In some groups, C5KO mice were injected with C5 or C5a proteins 10 min before cisplatin administration. Blood was collected immediately on the day of sacrifice for creatinine and blood urea nitrogen assay. The kidneys were either immediately fixed with 4% formaldehyde and 2% glutaraldehyde for histology measurement or snap-frozen and kept at -80℃until molecular biology examination.ResultsOur data showed that cisplatin treatment significantly elevated serum BUN and creatinine levels, increased the percentage of apoptotic tubular cells and renal neutrophil infiltration and oxidative/nitrosative stress in wild-type mice, which were significantly reduced in C5KO mice. Pretreatment with purified C5 restored sensitivity to cisplatin-induced nephrotoxicity in C5KO mice. These results suggested that C5 was an important mediator of increased apoptosis, inflammatory cell infiltration, and oxidative/nitrosative stress observed with cisplatin-induced nephrotoxicity. During complement activation, C5 is cleaved into two smaller fragments, C5a and C5b. The C5b fragment participates in the formation of membrane attack complexes (MACs), while C5a functions as an anaphylatoxin. In order to determine whether C5a was indeed the protective factor in cisplatin-induced renal injury in C5KO mice, C5a was injected to C5KO mice before cisplatin administration. We found injection of C5a also restored cisplatin-induced nephrotoxicity in C5KO mice as well as administration of C5. Furthermore, similar to C5KO mice, C5aRKO mice were also resistant to cisplatin-induced nephrotoxicity. In wild-type mice, administration of cisplatin triggered the increased renal expression of multiple cytokines and caspases. This induction was diminished in C5KO mice, which was restored by pretreatment with C5 or C5a proteins. Moreover, we also demonstrated that C5a or C5aR deficiency diminished cisplatin-induced cytokine and caspase gene expression, as well as p-STAT3 and NF-kB activation in kidneys from mice. Interestingly, renal injury induced by cisplatin was similar between wild-type and CD59KO mice, and the formation of MACs by cisplatin in the kidney was diminished in C5KO mice, but not in C5aRKO mice. Taken together, these findings indicated that C5a contributed to cisplatin-induced nephrotoxicity through a signaling pathway downstream from C5a receptor binding.ConclusionIn conclusion, our findings suggest that C5a plays an important role in the pathogenesis of cisplatin nephrotoxicity. Likely, C5a binds to C5aR, leading to induction of proinflammatory cytokines and inflammation. The formation of MACs does not appear to contribute to the nephrotoxicity of cisplatin based on our study results. C5aR blockade could be a novel strategy in attenuating cisplatin-induced kidney injury.