{'fraction_non_alphanumeric': 0.03996647635011262, 'fraction_numerical': 0.020742758367817297, 'mean_word_length': 4.918164910105394, 'pattern_counts': {'":': 0, '<': 13, '<?xml version=': 0, '>': 2, 'https://': 8, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Hydrogen sulfide protects from acute kidney injury via attenuating inflammation activated by necroptosis in dogs

Background The treatment of acute kidney injury (AKI), a common disease in dogs, is limited. Therefore, an effective method to prevent AKI in veterinary clinics is particularly crucial. Objectives Hydrogen sulfide (H2S) is the third gaseous signal molecule involved in various physiological functions of the body. The present study investigated the effect of H2S on cisplatin-induced AKI and the involved mechanisms in dogs. Methods Cisplatin-injected dogs developed AKI symptoms as indicated by renal dysfunction and pathological changes. In the H2S-treated group, 50 mM sodium hydrosulfide (NaHS) solution was injected at 1 mg/kg/h for 30 min before cisplatin injection. After 72 h, tissue and blood samples were collected immediately. We performed biochemical tests, optical microscopy studies, analysis with test kits, quantitative reverse-transcription polymerase chain reaction, and western blot analysis. Results The study results demonstrated that cisplatin injection increased necroptosis and regulated the corresponding protein expression of receptor interacting protein kinase (RIPK) 1, RIPK3, and poly ADP-ribose polymerase 1; furthermore, it activated the expressions of inflammatory factors, including tumor necrosis factor-alpha, nuclear factor kappa B, and interleukin-1β, in canine kidney tissues. Moreover, cisplatin triggered oxidative stress and affected energy metabolism. Conversely, an injection of NaHS solution considerably reduced the aforementioned changes. Conclusions In conclusion, H2S protects the kidney from cisplatin-induced AKI through the mitigation of 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.


INTRODUCTION
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 2/14 https://vetsci.org 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, TNFR1associated 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 (H 2 S) 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]. H 2 S 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 H 2 S can be produced by a series of enzymatic reactions in mammals; since then, the physiological function of H 2 S has been gradually identified. In general, H 2 S 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 H 2 S plays a crucial role in inflammation. For example, H 2 S can induce neutrophil apoptosis to reduce inflammation [15]. H 2 S administration to rats with colitis downregulates the expression of proinflammation cytokine tumor necrosis factor-alpha (TNF-α), whereas the inhibition of H 2 S 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 H 2 S 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 H 2 S.
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 H 2 S attenuates cisplatin nephrotoxicity, and explored the mechanism by which H 2 S 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 https://doi.org/10.4142/jvs.22064

H2S improves AKI in dogs
3/14 https://vetsci.org H 2 S on AKI. This study revealed a possibility of H 2 S alleviating cisplatin-induced AKI in dogs, which will provide more possibilities for clinically reducing the side effects of drugs.

Preparation of animals
All procedures used in this experiment were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University (SRM-11). In total, 24 adult male beagles (8-12 kg) were divided randomly into three groups: control group (C), hydrogen sulfide + cisplatin group (H+cis), and cisplatin group (cis) (n = 8 per group, six were used for the experiments, and the remaining two were on standby for any unexpected condition). The laboratory staff cleaned the kennel regularly to ensure a healthy environment; the kennel temperature was controlled at 18°C-26°C. All dogs had access to standard food and water ad libitum during the study. Dogs in the cis group were injected with 5 mg/kg body weight cisplatin [21], and dogs in the C group were injected with an equal volume of saline. In the H+cis group, dogs were injected with 50 mM NaHS solution (1 mg/kg/h) 30 min before cisplatin injection (5 mg/kg). Dogs were anesthetized 72 h after the cisplatin injection. Blood and a small part of the left kidney tissues were quickly collected. Blood samples were collected for blood urea nitrogen (Bun) and serum creatinine (Scr) measurement. A portion of the collected kidney tissue was quickly removed and fixed in 10% phosphate-buffered formalin for hematoxylin and eosin (H&E) staining; the remaining tissue was quickly removed and frozen in liquid nitrogen and then stored at −80°C.

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

Histopathological examination
The canine left kidney tissues were rapidly fixed in 10% formaldehyde for at least 24 h and then embedded in paraffin for microscopic examination. From the prepared paraffin blocks, 5-µm-thick sections were obtained and stained with H&E for light microscopic observation.

Detection of antioxidant levels
The kidney tissues were homogenized in physiological saline (1:10 w/v) with a glass Teflon homogenizer (Heidolph SO1 10R2RO). The homogenate was centrifuged at 700 × g for 30 min at 4°C to obtain the supernatant to measure the activities of superoxide (SOD) and catalase (CAT) as well as malondialdehyde (MDA) levels by using detection kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's protocols.

Detection of ATPase
The activities of Na + -K + -ATPase, Ca 2+ -Mg 2+ -ATPase, and Ca 2+ -ATPase were determined by using 10% tissue homogenates with appropriate assay kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol. Inorganic phosphorus produced during the conversion of adenosine triphosphate to adenosine diphosphate was quantified using the molybdenum blue spectrophotometric method at 660 nm and expressed as U/mg.prot. When one type of ATPase was tested, the inhibitors of the other types of ATPase were added to depress the hydrolysis of phosphate radicals. https://doi.org/10.4142/jvs.22064

Quantitative reverse-transcription polymerase chain reaction (qPCR) analysis
Total RNA from canine kidney tissues was extracted 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). qPCR was performed on a Light Cycler 480 System (Roche, Switzerland) after reverse transcription by using the fast qPCR kit (RR047A; Takara, Japan). All of the primers ( Table 1) were designed using Premier Software (PREMIER Biosoft International, USA) for qPCR. The relative messenger RNA (mRNA) level was calculated according to the method of 2 -ΔΔCt , and gene-specific efficiencies were normalized to the mean mRNA expressions of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Immunohistochemistry staining
The kidney sections were treated with 0.01 M sodium citrate buffer (pH 6.0) by using a microwave-based antigen retrieval technique for 20 min at 95°C, followed by 3% H 2 O 2 for 10 min to block endogenous peroxidase activity. Subsequently, they were incubated with RIPK1 (1:500; Bioss, China) and RIPK3 (1:500; Bioss) antibodies for 24 h at 4°C and secondary antibodies for 30 min at 37°C. After staining the slides with 3,3'-diaminobenzidine, they were observed under a microscope.

H2S improves AKI in dogs
phosphate-buffered saline with Tween 20, the membranes were incubated for 2 h at 37°C with peroxidase-conjugated secondary antibodies against rabbit IgG (Cat# sc-2357, RRID: AB_628497; Santa Cruz Biotechnology, Argentina). After washing three times by PBST for 15 min each again, the bound antibodies were visualized through chemiluminescence by using the ECL-plus reagent (GE Healthcare, UK). The GAPDH content was analyzed as the loading control by using a rabbit polyclonal antibody.

Statistical analysis
Statistical analyses of all data were performed using GraphPad Prism (version 8.0; GraphPad Software Inc., USA). Significant values (p < 0.05) were obtained through a one-way analysis of variance. 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 significant if p < 0.05.

H 2 S attenuates cisplatin-induced renal injury in dogs
As shown in Supplementary Fig. 1, after injection of NaHS solution, the levels of Bun and Scr in dogs were not significantly different from those in the control group, indicating that injection of NaHS solution had no effect on the kidneys of dogs. Besides, the content of Bun and Scr increased significantly after cisplatin treatment (p < 0.01), whereas H 2 S had the opposite effect ( Fig. 1A and B) In addition, we observed canine kidney tissues stained with H&E in the C, H+cis, and cis groups. Histopathological changes in renal tissues are presented in Fig. 1C. The kidney tissues in the C group displayed normal morphologies. However, some features indicating renal pathological damage were observed in the cis group; after cisplatin administration, the canine kidney tissue showed degeneration of renal tubular epithelial cells (blue arrow) and amyloidosis (yellow arrow). In addition, numerous inflammatory cells infiltrated the kidney tissue (red arrow). As expected, in the H+cis group, renal pathological damage was relieved, but some changes remained compared with the C group, including a small amount of inflammatory cell infiltration (red arrow).

Antioxidant capacity in canine kidney tissues
The results of antioxidant activity of canine kidney tissues were as follows. Compared with the C group, the activities of SOD and CAT in the cis group significantly decreased (p < 0.01); however, after the addition of H 2 S, the aforementioned antioxidant enzyme activities were restored ( Fig. 2A and B). In addition, no significant difference was observed between the C and H+cis groups in terms of MDA levels (p > 0.05), whereas MDA levels were upregulated in the cis group compared with the C and H+cis groups (p < 0.01; Fig. 2C).

Expression levels of energy metabolism-related genes in canine kidney
The expression levels of PK, UCP1, SDH, PDHX, and LDH markedly decreased (p < 0.01) after cisplatin treatment (Fig. 4). Moreover, the expression of all energy metabolism-related genes in https://doi.org/10.4142/jvs.22064 H2S improves AKI in dogs 6/14 https://vetsci.org the H+cis group increased significantly compared with that in the cis group (p < 0.01; Fig. 4A-F). Among them, the relative expression of PK and the protein expression of LDH in the H+cis group was slightly higher than that in the C group ( Fig. 4A and C).

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. 5. Cisplatin treatment significantly increased the mRNA and protein levels of necrosis genes, including RIPK1 and RIPK3, whereas H 2 S pretreatment markedly reduced the levels of RIPK1 and RIPK3 (p < 0.01) (Fig. 5A-D).
https://doi.org/10.4142/jvs.22064     Moreover, the mRNA and protein expression of PARP1 in the cis group was significantly higher than that in the C and H+cis groups (p < 0.01; Fig. 5E and G). Compared with the C and H+cis groups, the mRNA and protein expressions of Cas8 in the cis group were significantly decreased (p < 0.01; Fig. 5F and G). Furthermore, the mRNA expressions of transforming growth factor-β activated kinase (TAK) 1, TAK1-binding protein (TAB) 2, and TAB3 in the cis group were enhanced significantly compared with those in the C group (p < 0.01), whereas after H 2 S treatment, their mRNA expression levels decreased significantly (p < 0.01; Fig. 5H-J).

Level of inflammatory response in canine kidney tissues
Compared with the C group, the mRNA and protein expressions of NF-κB, IL-1β, and TNF-α in the cis group increased significantly (p < 0.01; Fig. 6). Among them, NF-κB mRNA expression increased the most in the cis group, which was approximately 2.5 times higher than that in the C group (Fig. 6A). After H 2 S pretreatment, the mRNA and protein expressions of NF-κB, IL-1β, and TNF-α decreased. As expected, compared with the cis group, the protein expression of TNF-α in the H+cis group decreased the least, and no significant difference was observed between the two groups (p > 0.05; Fig. 6C).

DISCUSSION
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 https://doi.org/10.4142/jvs.22064 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 H 2 S 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 H 2 S improves this situation (Fig. 7).

H2S improves AKI in dogs
Necroptosis is a type of programmed necrosis which is of central pathophysiological relevance in various diseases such as myocardial infarction [22] [39] demonstrated that RIPK3 mediates oxidative stress, which can induce necroptotic cell death and inflammation. In this setting, extenuating oxidative stress-induced necroptosis through H 2 S seems to be effective against renal inflammation. In this study, we found that H 2 S restored the activity of antioxidant enzymes (including SOD and CAT) and decreased the total content of MDA, which suggested that H 2 S 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, Ca 2+ -Mg 2+ -ATPase, and Ca 2+ -ATPase. Our results showed that cisplatin reduced the level of canine kidney energy metabolism, and H 2 S can mitigate this condition.
In summary, we successfully established a cisplatin-induced kidney injury model in dogs and demonstrated that H 2 S 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 H 2 S effect on necroptosis and inflammation, which may provide new insights into the physiological role of H 2 S.