Hydrogen sulfide protects from acute kidney injury via attenuating inflammation activated by necroptosis in dogs

Acute kidney injury (AKI), an independent risk factor for mortality, is a clinical syndrome characterized by a rapid decline of renal function in humans. As one of the most common complications in hospitalized patients, AKI increases the risk of death by 10–15 times and has a mortality rate of 50%, which accounts for approximately 2 million deaths per year worldwide [1, 2]. In dogs, acute kidney injury is a common disease reported in veterinary clinics, and effective prevention and timely treatment of this disease can prevent substantial kidney damage. A survey revealed that 19% of the AKI reported are induced by drug nephrotoxicity in humans [3]. Dialysis is crucial for the removal of toxic byproducts of drug metabolism. In addition, a report stated that the mortality rate of AKI is 47%–61% in dogs [4]. Therefore, the development of an effective method to prevent canine AKI is crucial.

Necroptosis is one of the cell death modes in AKI; it is a type of programmed cell death mediated by receptor interacting protein kinase (RIPK) signaling [5]. RIPK1 is a key factor in necroptosis initiation; it combines with tumor necrosis factor receptor (TNFR) 1, TNFR1-associated death domain (TRADD) protein, and TNFR-associated factor (TRAF)-2 through the death domain to form complex I, which can induce necroptosis through the formation of the RIPK1/RIPK3/mixed lineage kinase domain-like necrosome in the absence of caspase-8 (Cas8) [6, 7]. In recent years, many studies have revealed that necroptosis is associated with inflammation. For instance, Welz et al. [8] demonstrated that RIPK3 gene deficiency prevented inflammation and cell death in both the small intestine and colon of mice. Murakami et al. [9] revealed that programmed necrosis promoted inflammation through the regulation of the release of intracellular damage-associated molecular patterns in mice with retinal degeneration. Additionally, RIPK3 can activate glutamate-ammonia ligase, thereby increasing glutamate decomposition, and mitochondrial glutamate catabolism leads to local free ammonia accumulation and increases reactive oxygen species (ROS) expression [9, 10]. Thus, necroptosis may be associated with oxidative stress.

Hydrogen sulfide (H₂S) is the third endogenous gaseous signal molecule after nitric oxide and carbon monoxide, which plays a crucial role in various tissues in both health and disease [11]. H₂S was initially identified as a harmful exogenous gas with a pungent smell that can damage various tissues and organs of the body [12]. In 1996, Abe and Kimura [13] discovered that H₂S can be produced by a series of enzymatic reactions in mammals; since then, the physiological function of H₂S has been gradually identified. In general, H₂S is synthesized from L-cysteine by three enzymes: cystathionine-β-synthase, cystathionine-β-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase [14]. These three enzymes are widely distributed in the cardio-cerebrovascular system, liver, and kidney, as well as in the cells of other tissues. Several studies have reported that H₂S plays a crucial role in inflammation. For example, H₂S can induce neutrophil apoptosis to reduce inflammation [15]. H₂S administration to rats with colitis downregulates the expression of proinflammation cytokine tumor necrosis factor-alpha (TNF-α), whereas the inhibition of H₂S synthesis in healthy rats induces inflammation in the small intestine and colon [16]. Furthermore, Chen et al. [17] showed that exogenous administration of sodium hydrosulfide (NaHS) can alleviate airway inflammation. In addition, some recent reports have highlighted that H₂S has significant antioxidant properties, which can upregulate the expression of key antioxidant enzymes and remove ROS [18, 19]. King et al. [20] reported that after CSE knockout, the oxidative stress level in mice with myocardial infarction increased and myocardial injury aggravated, both of which were alleviated by exogenous H₂S.

Alleviation and prevention of AKI pathogenesis are of great significance in the veterinary field. In this study, we developed a canine AKI model with cisplatin, examined whether H₂S attenuates cisplatin nephrotoxicity, and explored the mechanism by which H₂S protects the kidney from cisplatin nephrotoxicity. To our knowledge, studies on cisplatin-induced AKI have focused on mice, and few reports are available regarding AKI in dogs and the effect of H₂S on AKI. This study revealed a possibility of H₂S alleviating cisplatin-induced AKI in dogs, which will provide more possibilities for clinically reducing the side effects of drugs.

AKI is the acute decline of renal function in a short time due to various reasons. Dialysis and kidney transplantation are effective AKI treatments, but they are difficult to apply in veterinary clinics because of their high cost. Therefore, effective methods for AKI prevention are of great significance in the field of veterinary medicine. In the present study, we demonstrated that H₂S protected against cisplatin-induced canine kidney injury through the inhibition of necroptosis, inflammation, and oxidative stress. Moreover, our study confirmed that cisplatin reduces energy metabolism in canine kidney tissues, whereas H₂S improves this situation (Fig. 7).

Fig. 7
The schematic diagram of protective effect of H₂S on canine AKI induced by cisplatin. Cisplatin induces necroptosis of canine renal tissues and triggers inflammation, as well as reduces renal antioxidant capacity and energy metabolism. These are notably improved by H₂S.
H₂S, hydrogen sulfide; AKI, acute kidney injury; NF-κB, nuclear factor kappa B; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; SOD, superoxide; CAT, catalase; RIPK, receptor interacting protein kinase; PK, pyruvate kinase; LDH, lactate dehydrogenase; UCP, uncoupling protein; SDH, succinate dehydrogenase; MDA, malondialdehyde.

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Necroptosis is a type of programmed necrosis which is of central pathophysiological relevance in various diseases such as myocardial infarction [22], atherosclerosis [23], and ischemia-reperfusion injury [24]. TNFR regulation is the classic pathway of necroptosis, during which two complexes are formed. Complex I is mainly composed of TRADD, RIPK1, TRAF2, and TRAF5. If RIPK1 is ubiquitinated, it binds to TAK1, TAB2, and TAB3 and further activates NF-κB to inhibit cell death [25]. Conversely, if RIPK1 is deubiquitinated, it forms complex II with RIPK3, TRADD, and Cas8, which can initiate necroptosis under conditions of Cas8 inactivation [26]. Studies have shown that necroptosis is involved in various pathological conditions of the kidney. Newton et al. [27] demonstrated that RIPK3 deficiency can improve kidney ischemia-reperfusion injury in mice. Xu et al. [28] revealed that knocking out mice necroptosis key genes RIPK1 and RIPK3 can attenuate the damage caused by cisplatin to the kidney, which indicates that necroptosis is one of the main mechanisms of cisplatin-induced AKI. Therefore, inhibiting the expression of key necrosis factors (such as RIPK1 and RIPK3) may alleviate cisplatin nephrotoxicity. In this regard, we evaluated the renoprotective effect of H₂S and found that it can weaken the expression of RIPK1, RIPK3, and PARP1, and simultaneously, it can enhance Cas8 activity. Thus, H₂S can relieve cisplatin-induced necroptosis of the canine kidney.

AKI often manifests as an inflammation of the kidney tissue [29]. TNF-α is a pro-inflammatory factor mainly produced by macrophages, which can induce substances such as IL and interferon to cause inflammation [30]. Moreover, TNF-α can activate the NF-κB pathway, thereby inducing the production and release of pro-inflammatory factors IL-1 and IL-6. Gong et al. [31] demonstrated that the protein expressions of TNF-α and IL-6 in rat kidneys were significantly upregulated on the third day after cisplatin injection into rats. Furthermore, studies have demonstrated that necroptosis plays a crucial role in inflammation and is involved in multiple inflammatory diseases. Vince et al. [32] illustrated that the activation of RIPK3 can generate bioactive IL-1β, which is a potent inflammatory cytokine. Welz et al. [8] suggested that the inhibition of RIPK3-induced necrosis can prevent the inflammation of intestinal epithelial cells in mice. Moreover, several studies have indicated that necroptosis induced by RIPK3 promotes the production of some cytokines and inflammatory factors, thereby inducing inflammation [33, 34]. Studies have demonstrated that RIPK1 triggered a second wave of cell death in AKI, whereas RIPK1 potentially regulated inflammation in a way unrelated to cell death [35, 36]. In the present study, optical microscopy observation revealed that H₂S alleviates the pathological damage to the canine kidney caused by cisplatin. Further detection at the molecular level showed that H₂S reduced the expression of pro-inflammatory factors (including IL-1β, NF-κB, and TNF-α).

Studies have indicated that cisplatin can cause renal oxidative stress and induce damage kidney; in detail, the content of MDA increased, and the activity of glutathione (GSH) decreased [37]. Furthermore, Waly et al. [38] showed that cisplatin induced oxidative stress in human kidney (HEK 293) cells through the reduction of the activities of SOD, GSH, and CAT. Additionally, Zhang et al. [39] demonstrated that RIPK3 mediates oxidative stress, which can induce necroptotic cell death and inflammation. In this setting, extenuating oxidative stress-induced necroptosis through H₂S seems to be effective against renal inflammation. In this study, we found that H₂S restored the activity of antioxidant enzymes (including SOD and CAT) and decreased the total content of MDA, which suggested that H₂S could increase antioxidant capacity in cisplatin-induced canine AKI.

Additionally, many other crucial factors transmit and execute necrotic signals. A recent study indicated that glycolytic pyruvate played a novel anti-necroptotic role in ischemic stress of mice gut [40]. Another report revealed that ATPase activities were inhibited, and several energy metabolism–related gene expressions decreased during necroptosis [41], which suggested that energy metabolism is related to necroptosis. Furthermore, Yang et al. [42] observed an imbalance of energy metabolism in canine kidney tissues in a lipopolysaccharide-induced canine septic AKI. 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 showed that cisplatin reduced the level of canine kidney energy metabolism, and H₂S can mitigate this condition.

In summary, we successfully established a cisplatin-induced kidney injury model in dogs and demonstrated that H₂S has a powerful protective effect on cisplatin-induced AKI through the enhancement of the antioxidant capacity and energy metabolism level, as well as the reduction of cell necroptosis and inflammation. These findings provide new and valuable clues for the treatment of canine AKI and are of great significance for AKI prevention in veterinary clinics. Simultaneously, our study enriched the understanding of the H₂S effect on necroptosis and inflammation, which may provide new insights into the physiological role of H₂S.

Supplementary Fig. 1

Scr and Bun levels in dogs. In the pre-test, 12 adult healthy beagles were randomly divided into four groups: C group, H group, H+cis group and cis group. Blood samples were taken 72 h after cisplatin injection to test the levels of Scr and Bun. Results showed that there was no significant difference in Scr and Bun between H group and corresponding C group, indicating that the injection of NaHS solution had no effect on canine kidney. Therefore, in order to respect and protect the experimental animals, the hydrogen sulfide group was not set in our formal experiment.