Hydrogen sulde protects from cisplatin-induced acute kidney injury via attenuating inammation activated by necroptosis in canine

Background Cisplatin, as an effective anti-cancer drug, has signicant effects on a variety of solid tumors such as ovarian cancer and malignant lymphoma. However, cisplatin has strong nephrotoxicity, which greatly limits its clinical application. Unfortunately, the way to validly inhibit cisplatin-induced kidney damage remain poor understood currently. Hydrogen sulde (H 2 S) was considered to be the third new gas signal molecules, involved in various physiological functions of the body. This present study was designed to investigate the effect of H 2 S on cisplatin-induced acute kidney injury (AKI) and the involved mechanisms in canine. Results Cisplatin-inject canine developed severe AKI symptom as indicated by renal dysfunction and pathological changes. Whereas H 2 S attenuated the serum creatinine (Scr), blood urea nitrogen (Bun) level and renal tubular damage. Cisplatin induced necroptosis and regulated the corresponding protein expression of RIPK1, RIPK3, PARP1, and then activated inammatory factors expression such as TNF-α, NF-κB, IL-1β in canine kidney tissues. Cisplatin also triggered oxidative stress and affected energy metabolism. However, H 2 S signicantly improved necroptosis and inammation, manifested by increasing Cas8 activity and the expression of anti-inammatory factors such as IL-4, IL-10 and IFN-γ. At the same time, the antioxidant capacity and energy metabolism levels of canine kidney were notably improved. Conclusion Collectively, our results suggest that H 2 S protect kidney from cisplatin-induced AKI via mitigating necroptosis and inammation. Besides, H 2 S improved the level of energy metabolism and displayed a potency in cisplatin-induced oxidative stress. These provided a new idea for reducing cisplatin nephrotoxicity.


Introduction
Acute kidney injury (AKI), as an independent risk factor for mortality, is a clinical syndrome characterized by rapid decline of renal function. As one of the most common complications in hospitalized patients, AKI increases the risk of death by 10 to 15 times and result in a mortality rate of 50%, which it accounts for approximately 2 million deaths per year worldwide [1,2]. There have a large-scale multicenter epidemiological survey of critically ill patients showed that AKI induced by drug nephrotoxicity accounted for 19% [3]. Cisplatin (cis-diamminedichloroplatinum II, CDDP), as a well-known chemotherapy drug, has a signi cant therapeutic effect on various solid tumors. However, the use of cisplatin is frequently limited by various signi cant side effects especially nephrotoxicity. As well as tubular cell injury and death, cisplatin can cause in ammation of kidney tissue [4,5]. It has been reported that 30%-40% of cisplatintreated patients developed AKI [6]. Unfortunately, in the prevention of cisplatin nephrotoxicity, some people have tried to supplement magnesium, and some have tested antioxidants and cisplatin transport blockers in animal experiments, but the results are not satisfactory [7,8].
Necroptosis is one of the cell death modes of AKI, which is a type of programmed cell death mediated by receptor interacting protein kinase (RIPK) signaling [9]. RIPK1 is a key factor in the initiation of necroptosis, it combines with tumor necrosis factor receptor 1 (TNFR1), TNFR1-associated death domain protein (TRADD) and TNFR-associated factor 2 (TRAF-2) via death domain to form complex I that can induce necroptosis via forming RIPK1/RIPK3/MLKL necrosome in the absence of caspase-8 (Cas8) [10,11]. In recent years, more and more studies have provided that necroptosis is associated with in ammation. For instance, Welz et al. have found that RIPK3 gene de ciency prevented the development of in ammation and cell death in both the small intestine and colon of mice [12]. Murakami et al. have identi ed that programmed necrosis promoted in ammation by regulating the release of intracellular damage-associated molecular pattern in mice with retinal degeneration [13]. Additionally, RIPK3 can activate glutamate-ammonia ligase, thereby increasing the decomposition of glutamate, and further mitochondrial glutamate catabolism leads to local free ammonia accumulation and increases ROS expression [13,14]. This suggest that necroptosis may be associated with oxidative stress.
Hydrogen sul de (H 2 S) was considered to be the third endogenous signaling gaseous transmitter along with nitric oxide (NO) and carbon monoxide (CO), which plays an important role in various tissues in both health and disease [15]. In fact, H 2 S was initially identi ed as a harmful exogenous gas with pungent smell, which can cause damage to kinds of tissues and organs of the body [16]. Until 1996, Abe and Kimura discovered that H 2 S can be produced by a series of enzymatic reactions in mammals [17], the physiological function of H 2 S has been gradually recognized. In general, H 2 S is synthesized from Lcysteine via three enzymes: cystathionine-β-synthase (CBS), cystathionine-β-lyase (CSE) and 3mercaptopyruvate sulfurtransferase (3-MST) [18]. 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 H 2 S played an important role in in ammation. For example, H 2 S can induce apoptosis of neutrophils to reduce in ammation [19]. Administration of H 2 S to rats with colitis can down-regulate the expression of pro-in ammation cytokine tumor necrosis factor α (TNF-α), whereas inhibiting the synthesis of H 2 S in healthy rats induced in ammation in the small intestine and colon [20]. Furthermore, Chen et al. have showed that exogenous administration of NaHS could alleviate airway in ammation [21]. In addition, some recent reports have highlighted that H 2 S displayed signi cant antioxidant properties, which could up-regulate the expression of key antioxidant enzymes and remove ROS [22,23]. King et al. have reported that the level of oxidative stress in mice with myocardial infarction was increased after CSE knockout, and the myocardial injury was aggravated, both of which were alleviated via exogenous H 2 S [24].
Improving the pathogenesis of cisplatin-induced AKI is of great signi cance to the clinical application of cisplatin drugs. In our present study, we built a canine AKI model with cisplatin, and examined whether H 2 S attenuates cisplatin nephrotoxicity, and explored how H 2 S protects kidney from cisplatin nephrotoxicity. To our knowledge, previous studies of cisplatin-induced AKI have focused on mice, there are fewer reports about AKI in dogs and the effect of H 2 S on AKI. This study revealed a possible mechanism of H 2 S alleviating cisplatin-induced AKI in dogs, which will provide more possibilities for clinically reducing the side effects of cisplatin.

Preparation of animals
All procedures used in this experiment were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural. Twenty-four adult male beagles (8-12 kg) were divided randomly into three groups (n = 6 per group, the remaining two dogs in each group were standby for any unexpected condition). The laboratory staff cleaned the kennel regularly to ensure the environment is good; the kennel temperature is controlled at 18-26℃. All dogs were given free access to standard food and water during the study. Dogs in the cis group were injected with 5 mg /kg body weight cisplatin, and control dogs received an injection of equal volume of saline. As for H + cis group, dogs were injected with 50 mM NaHS solution (1 mg / kg / h) 30 min before cisplatin injection (5 mg / kg) and then inject once daily. Dogs were anesthetized 72 h after the cisplatin injection. The left kidney tissues and blood were quickly collected, blood sample was collected for blood urea nitrogen (Bun) and serum creatinine (Scr) measurement, the upper half of left kidney was quickly removed and xed in 10% phosphate-buffered formalin for hematoxylin-eosin staining, and the lower half of left kidney was quickly removed and frozen in liquid nitrogen and then stored at − 80 °C.

Serum analysis
The Bun and Scr levels were evaluated using a UniCel DxC800 Synchron chemistry system (Bekman, USA). The renal injury model was considered to be established when Bun and Scr levels in the cisplatintreated group increased by twice as much as those in the C group.

Histopathological examination
The canine left kidney tissues were rapidly xed in 10% formaldehyde for at least 24 h and were embedded in para n for microscopic examination. From the prepared para n blocks, sections (5-µm thick) were cut, obtained and stained with hematoxylin and eosin (H&E) for light microscopic observation.

Detection of antioxidant levels
The kidney tissues were homogenized (1:10 w/v) with a glass Te on homogenizer (Heidolph SO1 10R2RO) in physiological saline. The homogenate was centrifuged at 700 × g for 30 min at 4 °C to obtain the supernatant to measure the activities of SOD, GSH, CAT as well as MDA, H 2 O 2 and NO content levels by the detection kits (Nanjing Jiancheng Bioengineering Institute, China), according to the manufacturer protocols.

Detection of ATPase
The activities of Na + -K + -ATPase, Ca 2+ -Mg 2+ -ATPase, and Ca 2+ -ATPase were determined using the appropriate assay kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's protocol using 10% tissue homogenates. The activities of Na + -K + -ATPase, Ca 2+ -Mg 2+ -ATPase and Ca 2+ -ATPase were measured by quantifying the inorganic phosphorus (Pi) production from the conversion of ATP to ADP at 660 nm using the molybdenum blue spectrophotometric method and were expressed as U/mg.prot. When one type of ATPase was tested, the inhibitors of other types of ATPase were added to depress the hydrolysis of phosphate radicals.

Quantitative real-time PCR analysis
Total RNA from canine kidney tissues was extracted using using Trizol reagent according to the manufacturer's protocol. The concentration and purity of the total RNA were determined spectrophotometrically at 260 / 280 nm (Gene Quant 1300/100, General Electric Company, USA).
Quantitative real-time PCR was performed on a Light Cycler® 480 System (Roche, Basel, Switzerland) after reverse transcription by using the fast qPCR kit (RR047A, Takara). All of the primers (Table. 1) were designed by Premier Software (PREMIER Biosoft International, USA) for q-PCR. The relative mRNA level was calculated according to the method of 2 −ΔΔCt , accounting for gene-speci c e ciencies was normalized to the mean mRNA expressions of GAPDH.

Immuno uorescence staining
The kidney sections was treated with 0.01 M sodium citrate buffer (PH 6.0) by a microwave-based antigen retrieval technique for 20 min at 95℃ was used followed by 10 min 3% H 2 O 2 to block endogenous peroxidase activity, incubated with RIPK1 (1:500; Bioss, China), RIPK3 (1:500; Bioss, China) antibodies for 24 h at 4℃ and secondary antibodies for 30 min at 37℃. After staining with DAB the slides were visualized with microscope.

Western Blot analysis
The protein samples were separated by 8%, 10% and 12% SDS-PAGE and were transferred to PVDF membranes (Merck Millipore, USA, Cat# ISEQ. 00010, LOT# R6PA4145H). The membranes were blocked with 5% skim milk for 3 h at 37 °C and were incubated for 14 h at 4 °C, with the following diluted . After washing three times for 15 min each with PBST, the membranes were incubated for 2 h at 37 °C with peroxidase-conjugated secondary antibodies against rabbit IgG (Santa Cruz Biotechnology, Argentina, Cat# sc-2357, RRID: AB_628497). After washing three times for 15 min each, the bound antibodies were visualized by chemiluminescence using the ECL-plus reagent (GE Healthcare, Buckinghamshire, UK). The GAPDH content was analyzed as the loading control using a rabbit polyclonal antibody.

Statistical analysis
Statistical analyses of all data were performed using GraphPad Prism (version 8.0, GraphPad Software Inc., San Diego, CA, USA). The signi cant values (P < 0.05) were obtained by One-way ANOVA. All data displayed normal distribution and passed the test for equal variance. The data are expressed as the mean ± SD, and the differences were considered to be signi cant if P < 0.05.

H 2 S attenuated cisplatin-induced renal injury in canine
We tested six samples in each group and found that the content of Bun and Scr increased signi cantly after cisplatin treatment (P < 0.01), while H 2 S could improve the change (Fig. 1A). In addition, we observed canine kidney tissues stained by hematoxylin and eosin (H&E) in the C group, H + cis group and cis group. The histopathological changes in renal tissues are shown in Fig. 1B. The kidney tissues in the corresponding C groups displayed normal morphologies, including the glomeruli and tubulesballoon are normal, the saccule cavity is obvious, and the vessel wall is clear. However, some features of renal pathological damage appeared in the cis group, after cisplatin administration, the canine kidney tissue showed glomerular brosis (black arrow), tubular atrophy (red arrow), as well as renal interstitial hyperplasia (green arrow). Besides, we also found the in ltration of in ammatory cells in renal tissue (yellow arrow). Obviously, in the hydrogen sul de group, renal pathological damage was relieved, but there were still some changes compared with the normal group, including renal balloon occlusion (white arrow) and brinoid necrosis of vascular wall (blue arrow).

The antioxidant capacity in canine kidney tissues
The results of antioxidant activity of canine kidney tissues were as follows. Compared with the corresponding C groups, the activitis of SOD, GSH and CAT in cis groups were signi cant decreased (P < 0.05 or P < 0.01), after the addition of H 2 S, the above antioxidant enzyme activities were restored ( Fig. 2A,   B and C). In addition, there were no signi cant difference between the C group and H + cis group about the contents of MDA, H 2 O 2 and NO (P > 0.05), whereas, the above substances in cis group enhanced compared to the corresponding C group and H + cis group (P < 0.01) (Fig. 2D, E and F).

ATPase activities and energy metabolism-related expressions in canine kidney tissues
ATPase activity results appear in Fig. 3A-C. The activity of all ATPase were weakened after cisplatin treated. Notably, the most decreased was Ca 2+ -ATPase activity in cis group, which decreased by about 28% (Fig. 3C). Besides, there was also signi cant difference in Ca 2+ -Mg 2+ -ATPase activity under cisplatin (P < 0.01) (Fig. 3B). Whereas in H + cis group, all ATPase activity recovered. At the same time, the expression of some energy metabolism genes was also detected. As shown in Fig. 3D-I, the expression levels of pyruvate kinase (PK), uncoupling protein 1 (UCP1), succinate dehydrogenase (SDH), pyruvate dehydrogenase complex (PDHX) and lactate dehydrogenase (LDH) were markedly decreased (P < 0.01) after cisplatin treatment. Moreover, the expression of all energy metabolism related genes in H + cis group were elevated signi cantly compared to that cis group (P < 0.01). Among them, the relative expression of PK and the protein expression of LDH in the H + cis group was slightly higher than that C group.

The relative expressions of necroptosis-related genes in canine kidney tissues
The effect of cisplatin on the relative expression of necroptosis related genes and the role of H 2 S in canine kidney tissues are shown in Fig. 4. Cisplatin treatment signi cantly increased the mRNA and protein levels of necrosis genes including RIPK1, RIPK3 and PARP1 (P < 0.01) (Fig. 4A-C), while Cas8, as genes that induce apoptosis and inhibit necrosis, were decreased signi cantly (P < 0.01) (Fig. 4D). Among them, the most increased was PARP1 mRNA expression in cis group, which about doubled compared to the corresponding C group (Fig. 4C). However, after H 2 S treatment, the levels of all necrosis genes decreased, at the same time, the levels of Cas8 enhanced. Obviously, the protein expression levels of RIPK1 and PARP1 in H + cis group decreased slightly compared to that cis group (P > 0.05) (Fig. 4A, C), only decreased by about 5% and 3% respectively. Furthermore, the mRNA expression of TAK1, TAB2 and TAB3 in cis group were enhanced signi cantly compared to that C group (P < 0.01), whereas H 2 S treatment, their mRNA expression levels decreased signi cantly (P < 0.01) (Fig. 4E-G).

The level of in ammatory response in canine kidney tissues
As shown in Fig. 5, there were signi cant difference in the mRNA expression level of pro-in ammatory genes (including IL-1β, IL-6, TNF-α, NF-κB, COX2 and iNOS) after cisplatin treatment (P < 0.01). Among them, the most increased was the COX2 mRNA expression level in cis group, which was about 5.5 times of the corresponding C group (Fig. 5E). Besides, the pro-in ammation genes mRNA expression levels in the H + cis group were down-regulated obvious compared to that cis group (P < 0.01). Furthermore, the mRNA expression levels of anti-in ammatory genes (including Hsp60, Hsp70, IL-10, IL-4 and IFN-γ) in cis group were decreased signi cantly compared to that corresponding C group (P < 0.01), while H 2 S could improve this change (Fig. 5G-K). Moreover, the protein expression level trend of in ammation related genes were consistent with that of mRNA expression level.

Discussion
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 H 2 S protected against cisplatin-induced canine kidney injury by limiting necroptosis, in ammation and oxidative stress. Moreover, our study con rmed that cisplatin reduces the energy metabolism of the kidney tissues, whereas H 2 S 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 de ciency 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 H 2 S and found it can weaken the expression of RIPK1, RIPK3 and PARP1, at the same time enhanced Cas8 activity. These suggest that H 2 S relieve cisplatin-induce necroptosis of canine kidney.
Furthermore, there are reports have demonstrated that necroptosis plays an important role in in ammation and is involved in multiple in ammatory diseases. Vince et al. have illustrated that activation of RIPK3 can generate bioactive interleukin-1β (IL-1β) that is a potent in ammatory cytokine [32]. The research of Welz et al. suggested that inhibiting RIPK3-induced necrosis can prevent the in ammation 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 in ammatory factors, thereby inducing in ammation [33,34]. There are other reports have shown that RIPK1 triggered a second wave of cell death in AKI, while RIPK1 may regulate in ammation in a way unrelated to cell death [35,36]. In the present study, we observed through optical microscopy that H 2 S alleviate the pathological damage of canine kidney caused by cisplatin, such as glomerular brosis, interstitial hyperplasia and renal tubular atrophy, etc. Further detection at the molecular level shown that H 2 S reduced the expression of pro-in ammatory factors (including IL-1β, IL-6, TNF-α, NF-κB, COX2, iNOS) and increased several antiin ammatory 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 in ammation. There are studies have revealed that Hsp70 could suppress RIPK1dependent necroptosis [37]. Zonneveld et al. have reported that a recently epitopes derived from Hsp60 can ease in ammation 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 ( . In this setting, extenuating oxidative stress-induced necroptosis via H 2 S seem to be an effective way to against renal in ammation. In the current study, we found that H 2 S restored the activity of antioxidant enzymes (including SOD, GSH and CAT) and decreased the total content of MDA, H 2 O 2 and NO, which suggested that H 2 S 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, Ca 2+ -Mg 2+ -ATPase and Ca 2+ -ATPase. Our results shown that cisplatin reduced the level of canine kidney energy metabolism, and H 2 S could improve this situation.

Conclusion
In summary, here we have demonstrated that H 2 S had a powerful protective effect on cisplatin-induced AKI via enhancing the antioxidant capacity and the level of energy metabolism, as well as improving cell necroptosis and in ammation (Fig. 6). These nding provide new and valuable clues for the treatment of cisplatin nephrotoxicity and AKI. Simultaneously, our study enrich the understanding of the H 2 S effect on necroptosis and in ammation, which may provide new insight into the physiological role of H 2 S. Declarations SW and EX designed the experiments, SW, RG, HJ, YF, MH, SJ and TG performed the experiments. SW and XR analyzed the experimental data. SW and YL wrote this paper. All authors read and approved the nal manuscript. Availability of data and materials All data generated or analyzed during this study are available from the corresponding author by request.
Ethics approval and consent to participate Not applicable.

Con ict of interest
The authors declare that there are no con icts of interest.  Table   Table. 1 mRNAs Primer Sequences    represented as the mean ± SD (n = 6), # P < 0.05, # # P < 0.01 presented a signi cant difference; * P < 0.05, ** P < 0.01 presented a signi cant difference compared to the C group.