Cisplatin is a powerful anticancer drug, its hydrated or hydroxylated metabolite are mainly excreted through the kidney. Due to nephrotoxicity, the clinical application of cisplatin has been limited. Unfortunately, there is currently no effective way to prevent the kidney damage caused by cisplatin. In the present study, we demonstrated that H2S protected against cisplatin-induced canine kidney injury by limiting necroptosis, inflammation and oxidative stress. Moreover, our study confirmed that cisplatin reduces the energy metabolism of the kidney tissues, whereas H2S can improve this situation.
Necroptosis is a type of programmed necrosis, which is of central pathophysiological relevance in a variety of disease states such as myocardial infarction [25], atherosclerosis [26] and ischemia-reperfusion injury [27]. TNFR regulation is the classic pathway of necroptosis, during this process two complexes are formed. Complex I is mainly composed of TRADD, RIPK1, TRAF2 and TRAF5, if RIPK1 is ubiquitinated, it will bind to transforming growth factor-β activated kinase 1 (TAK1), TAK1-binding protein 2 (TAB2), TAB3 and further activate nuclear factor kappa-B (NF-κB) to inhibit cell death [28]. On the other hand, if RIPK1 is deubiquitinated, it will form complex II with RIPK3, TRADD, Cas8 that can initiate necroptosis under conditions of inactivation of Cas8 [29]. Previous studies have shown that necroptosis is involved in various pathological conditions of the kidney. Newton et al. have shown that RIPK3 deficiency can improve kidney ischemia-reperfusion injury in mice [30]. Xu et al. have found that knocking out mice necroptosis key genes RIPK1 and RIPK3 can attenuate the damage caused by cisplatin to the kidney [31], which indicates that necroptosis is one of the main mechanism of cisplatin-induced AKI. Therefore, inhibiting the expression of key necrosis factors (such as RIPK1 and RIPK3) may be a way to alleviate cisplatin nephrotoxicity. In this regard, we evaluated the renoprotective effect of H2S and found it can weaken the expression of RIPK1, RIPK3 and PARP1, at the same time enhanced Cas8 activity. These suggest that H2S relieve cisplatin-induce necroptosis of canine kidney.
Furthermore, there are reports have demonstrated that necroptosis plays an important role in inflammation and is involved in multiple inflammatory diseases. Vince et al. have illustrated that activation of RIPK3 can generate bioactive interleukin-1β (IL-1β) that is a potent inflammatory cytokine [32]. The research of Welz et al. suggested that inhibiting RIPK3-induced necrosis can prevent the inflammation of intestinal epithelial cells in mice [12]. Moreover, there are several studies have indicated that the necroptosis induced by RIPK3 promotes the production of some cytokines and inflammatory factors, thereby inducing inflammation [33, 34]. There are other reports have shown that RIPK1 triggered a second wave of cell death in AKI, while RIPK1 may regulate inflammation in a way unrelated to cell death [35, 36]. In the present study, we observed through optical microscopy that H2S alleviate the pathological damage of canine kidney caused by cisplatin, such as glomerular fibrosis, interstitial hyperplasia and renal tubular atrophy, etc. Further detection at the molecular level shown that H2S reduced the expression of pro-inflammatory factors (including IL-1β, IL-6, TNF-α, NF-κB, COX2, iNOS) and increased several anti-inflammatory factors (including IL-4, IL-10, IFN-γ) activities, as well as heat shock protein 60 (Hsp60) and Hsp70. Hsp as a highly conserved cellular stress proteins synthesized in organism, are related to necroptosis and inflammation. There are studies have revealed that Hsp70 could suppress RIPK1-dependent necroptosis [37]. Zonneveld et al. have reported that a recently epitopes derived from Hsp60 can ease inflammation via reducing TNF-α and increasing IFN-γ [38], which are consistent with our results.
Previous studies have indicated that cisplatin could cause renal oxidative stress and induce damage kidney, in more details, the change was that the content of malondialdehyde (MDA) increased and the activity of glutathione (GSH) decreased [39]. Besides, Waly et al. have shown that cisplatin induced oxidative stress in human kidney (HEK 293) cells via reducing the activities of superoxide (SOD), GSH and catalase (CAT) [40]. Additionally, Zhang et al. have demonstrated that RIPK3 mediates oxidative stress could induce necroptotic cell death and inflammation [41]. In this setting, extenuating oxidative stress-induced necroptosis via H2S seem to be an effective way to against renal inflammation. In the current study, we found that H2S restored the activity of antioxidant enzymes (including SOD, GSH and CAT) and decreased the total content of MDA, H2O2 and NO, which suggested that H2S could raise antioxidant capacity in cisplatin-induced canine AKI.
In addition to the above, there are many other important factors that transmit and execute necrotic signals. A recent study have indicated that glycolytic pyruvate played a novel anti-necroptotic role in ischaemic stress of mice gut [42]. Another report have shown that the activities of ATPase were inhibited and several energy metabolism related genes expressions decreased during necroptosis [43], which suggested energy metabolism seems to be related to necroptosis. Here we detected the expression of energy metabolism related genes (including PK, SDH, UCP1, PDHX and LDH) and the activities of Na+-K+-ATPase, Ca2+-Mg2+-ATPase and Ca2+-ATPase. Our results shown that cisplatin reduced the level of canine kidney energy metabolism, and H2S could improve this situation.