Hydrogen sulfide protects against cisplatin-induced experimental nephrotoxicity in animal models: a systematic review and meta-analysis

Introduction

Cisplatin, as an efficient chemotherapy drug, induces DNA crosslinks, causes DNA damage, and ultimately exerts its anti-cancer effect (Ghosh, 2019). Since its discovery in the late 1960s, cisplatin and its derivatives have been widely used in the treatment of various solid tumors, including lung cancer (Mascaux et al., 2000), mammary cancer (Rodler et al., 2023), bladder cancer (Kamat et al., 2016a), and ovarian cancer (Markman, 1994). However, cisplatin can cause multiple organ toxicity in the process of tumor treatment, among which kidney toxicity is the most common (Ries & Klastersky, 1986). Despite the clinical use of pre-treatment hydration therapy to reduce multi-organ toxicity caused by cisplatin, acute kidney injury (AKI) still occurs in approximately 30% of patients (Latcha et al., 2016). The nephrotoxic effects of cisplatin are cumulative and dose-dependent, mainly through the transport of organic cationic transporter-2 and copper transporter-1 in the basolateral membrane of the proximal tubule to intracellular accumulation (Guo et al., 2018; Sharaf, El Morsy & El-Sayed, 2023), resulting in proximal tubular injury, oxidative stress, inflammation, DNA damage, mitochondrial dysfunction, and vascular injury (Pabla et al., 2009; Yan et al., 2016), and ultimately AKI. Once AKI occurs and is not under control, the disease will progress into chronic kidney disease (CKD) eventually (Jiang et al., 2020; Xiao et al., 2016; Zhang et al., 2023a). At present, there are no effective drugs or methods to treat kidney damage caused by cisplatin. Therefore, seeking a novel strategy has become a hot topic in the prevention and treatment of cisplatin-induced AKI (cis-AKI).

In the last twenty years, extensive research has been conducted on hydrogen sulfide (H₂S) due to its significant functions in preserving cellular membrane stability, controlling cellular differentiation and growth, safeguarding the integrity of mitochondrial DNA, engaging in signal transmission processes, and governing programmed cell death (Cuevasanta et al., 2012; Kamat et al., 2016b; Zhang et al., 2019; Zhong et al., 2010). This makes it the third naturally occurring gas molecule, following carbon monoxide and nitric oxide. Evidence has shown that endogenous H₂S is closely involved in the physiological functions and pathological changes of the kidney (Ahmad et al., 2012; Yuan et al., 2019). According to animal experimental research reports, increasing the amount of H₂S with exogenous H₂S donors can alleviate the renal toxicity caused by cisplatin (Ahangarpour et al., 2014; Cao et al., 2018a), mainly through anti-inflammatory, antioxidant, autophagy, and other mechanisms (Ahangarpour et al., 2014; Sun et al., 2020; Yuan et al., 2019).

Although H₂S has been proven to be protective against cisplatin-induced nephrotoxicity in animal studies, there is no approved H₂S medication available for patients yet. Thus, conducting meta-analyses on published animal studies to validate H₂S efficacy in cis-AKI is of significance for future clinical investigations. To shed light on the future applications of H₂S in oncology patients with cis-AKI, we reviewed and summarized current publications and performed a meta-analysis on the current findings about the protective effect of H₂S against nephrotoxicity caused by cisplatin in animal models through in vivo studies.

Materials and Methods

Results

Study selection process

The process of retrieving literature was illustrated in Fig. 1. A total of 282 articles were initially gathered from PubMed/MEDLINE, Web of Science, Cochrane Library, and Scopus databases. Following the removal of duplicates and exclusion criteria, a meta-analysis was conducted on 13 literature (Abdel-Daim et al., 2019; Ahangarpour et al., 2014; Ahmad, 2022; Cai et al., 2024; Cao et al., 2018a, 2018b; Chiarandini Fiore et al., 2008; Elkhoely & Kamel, 2018; Jiang et al., 2024; Karimi et al., 2017; Sun et al., 2020; Wang, Liu & Liu, 2022; Yuan et al., 2019). The characteristics and experimental protocols of the included studies were listed in Table 1. Eight exogenous H₂S donors were administered to elevate systemic H₂S levels in animal models. These included synthetic compounds (ADT-OH, NaHS, Na₂S₄, GYY4137) and natural plant extracts (Allicin, diallyl disulfide (DADS), diallyl sulfide (DAS), diallyl trisulfide (DATS)), which either directly release H₂S or were metabolized into H₂S in vivo. Four administration routes were used: intra-peritoneal injection, intravenous injection, oral administration, and gavage. Certainly, different donor drugs and animal species require adjustment and optimization of the corresponding drug dose.

Table 1:

Main characteristics of included studies.

Reference	Species	Dosage (cisplatin, i.p)	Treatment duration (day) (cispaltin)	Administration (H2S)	Donor	Dosage (H2S)	Treatment duration (day) (H2S)	Sample (cis vs cis+H2S)
Cai et al. (2024)	C57BL/6 mice	25 mg/kg	4	po	ADT-OH	37 mg/kg	4	6 vs 6
Cai et al. (2024)	C57BL/6 mice	25 mg/kg	4	po	GYY4137	100 mg/kg	4	6 vs 6
Cai et al. (2024)	C57BL/6 mice	25 mg/kg	4	po	NaHS	5.6 mg/kg	4	6 vs 6
Jiang et al. (2024)	BALb/c mice	20 mg/kg	5	i.p	DATS	20 mg/kg	20	8 vs 8
Wang, Liu & Liu (2022)	beagle	5 mg/kg	3	i.v	NaHS	1 mg/kg/h	3	6 vs 6
Ahmad (2022)	Wistar rat	5 mg/kg	7	i.p	NaHS	56 µmol/kg	35	6 vs 6
Sun et al. (2020)	C57BL/6 mice	25 mg/kg	3	i.p	NaHS	5.6 mg/kg	4	5 vs 5
Sun et al. (2020)	C57BL/6 mice	25 mg/kg	3	i.p	GYY4137	100 mg/kg	4	5 vs 5
Sun et al. (2020)	C57BL/6 mice	25 mg/kg	3	i.p	Na2S4	500 µg/kg	4	5 vs 5
Yuan et al. (2019)	C57BL/6 mice	16 mg/kg	4	i.p	NaHS	5.6 mg/kg	4	8 vs 8
Abdel-Daim et al. (2019)	Wistar rat	7 mg/kg	7	po	Allicin	10 mg/kg	14	8 vs 8
Elkhoely & Kamel (2018)	SD rat	3.5 mg/kg	4	i.g	DAS	50 mg/kg	4	6 vs 6
Elkhoely & Kamel (2018)	SD rat	3.5 mg/kg	4	i.g	DAS	100 mg/kg	4	6 vs 6
Cao et al. (2018a, 2018b)	SD rat	7 mg/kg	6	i.p	Na2S4	5.6 mg/kg	7	8 vs 8
Cao et al. (2018a)	C57BL/6 mice	25 mg.kg	3	i.p	NaHS	5.6 mg/kg	4	9 vs 9
Cao et al. (2018b)	C57BL/6 mice	25 mg.kg	3	i.p	GYY4137	100 mg/kg	4	9 vs 9
Karimi et al. (2017)	SD rat	5 mg/kg		i.p	NaHS	200 µg/kg	15	8 vs 8
Ahangarpour et al. (2014)	SD rat	6 mg/kg	6	i.p	NaHS	100 µmol/kg	6	10 vs 10
Chiarandini Fiore et al. (2008)	SD rat	10.5 mg/kg	4	i.g	DADS	292.5 mg/kg (1 day) 146.25 mg/kg (3 days)	4	8 vs 8

DOI: 10.7717/peerj.19481/table-1

Note:

(DADS: diallyl disulfide, DAS: diallyl sulfide, DATS: diallyl trisulfide, ip: intraperitoneal injection, ig: intragastric, iv: intravenous injection, po: peros(Latin)- oral administration).

Risk of bias assessment

Using the SYRCLE bias risk tool to evaluate the quality of animal studies revealed that all studies had unknown bias in the blind design of grouping, intervention, outcome assessment, inclusion, and exclusion of reports. Among the 13 included pieces of literature, nine had low bias, four had unknown bias, and no literature had high bias (Fig. 2 and Fig. S1).

Renal function recovery after H₂S treatment on cis-AKI

Serum creatinine and urea nitrogen were measured in 19 studies. Renal function was significantly improved after H₂S donors postprocessing, with creatinine (SMD = −2.96, 95% CI [−3.72, −2.19], Z = 7.54, p < 0.00001; Fig. 3) and urea nitrogen (SMD = −2.73, 95% CI [−3.68, −1.78], Z = 5.62, p < 0.00001; Fig. 4). The overall effect size was accompanied by a high degree of heterogeneity (I² = 73%, p < 0.00001, and I² = 75%, p < 0.00001, respectively).

Effects of H₂S on oxidative stress, inflammation and apoptosis

Superoxide dismutase (SOD) was measured in six studies. A meta-analysis of these studies showed that H₂S treatment was associated with significantly increased SOD levels (SMD = 2.90, 95% CI [1.36–4.43], Z = 3.70, p = 0.0002; Fig. 5), the heterogeneity was statistically significant (I² = 79%, p = 0.0001). Additionally, nine separate research inquiries examining malondialdehyde (MDA) concentrations demonstrated that subjects who underwent H₂S adaptation exhibited considerably reduced MDA levels when compared to those who received no treatment (SMD = −2.85, 95% CI [−3.98 to −1.71], Z = 4.91, p < 0.00001; Fig. 5), with considerable variation observed across all included studies (I² = 74%, p = 0.0003). As for glutathione (GSH), statistically reported by four studies, as well as SOD activity, treatment of H₂S induced a significant elevation (SMD = 4.82, 95% CI [2.58–7.06], Z = 4.21, p < 0.0001; I² = 66%, p = 0.03, Fig. 5).

In contrast, cotreatment of cisplatin with H₂S exhibited a significant decrease of these pro-inflammatory markers compared to the cis group as follows: IL-1β (SMD = −4.41, 95% CI [−5.84 to −2.97], Z = 6.03, p < 0.00001), NF-κB (SMD = −4.26, 95% CI [−5.46 to −3.07], Z = 6.99, p < 0.0001), TNF-α (SMD = −4.43, 95% CI [−7.72 to −1.14], Z = 2.64, p = 0.008, Fig. 5; SMD = −4.84, 95% CI [−6.22 to −3.45], Z = 6.85, p < 0.00001, Fig. 5).

H₂S treatment also reduced apoptosis and tissue damage, such as a notable decrease in cleaved-caspase-3 level (SMD = −1.48, 95% CI [−2.06 to −0.89], Z = 4.94, p < 0.00001, Fig. 5) and renal tubule injury (SMD = −7.29, 95% CI [−9.14 to −5.44], Z = 7.72, p < 0.00001, Fig. 6). Unfortunately, a suitable meta-analysis could not be conducted due to the lack of quantitative data available for the TUNEL-positive cells.

Discussion

Currently, this is the first attempt to conduct a meta-analysis on H₂S efficacy for cisplatin-induced experimental nephrotoxicity. This meta-analysis assessed the consistency of results in animal studies on using H₂S for treating cis-AKI. According to all 13 articles reviewed and analyzed, H₂S donors effectively mitigated renal toxicity induced by cisplatin and significantly re-stored renal function after cisplatin treatment. Therefore, H₂S could be a new approach for treating cis-AKI, theoretically. This meta-analysis may shed light on establishing preclinical and clinical investigation guidelines for treating human cis-AKI with H₂S donors.

Although cis-AKI is not rare in oncological patients clinically, there are limited prevention and treatment methods available, except for adequate hydration (Launay-Vacher et al., 2008). According to a meta-analysis on clinical prevention of cisplatin nephrotoxicity, magnesium supplementation could be a forward-thinking and cost-effective preventive strategy (Hamroun et al., 2019), which was supported by several animal studies (Solanki et al., 2014; Yokoo et al., 2009). On the other hand, a meta-analysis conducted by Emre Aydıngöz et al. (2023) also shows a protective effect of H₂S on ischemia-reperfusion injury (IRI), also a common AKI model, demonstrating that H₂S effectively prevents or improves renal IRI before, during, or after ischemia and during the reperfusion phases (Emre Aydıngöz et al., 2023).

In the renal system, H₂S biosynthesis is primarily regulated by three enzymes: cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST) (Cirino, Szabo & Papapetropoulos, 2023). These enzymes are abundantly localized in the brush border and cytoplasm of glomerular endothelial cells, proximal tubules, distal tubules, and peritubular capillary epithelial cells, establishing the kidneys as a critical site for endogenous H₂S production involved in physiological regulation (Lu et al., 2022). Subsequent studies identified CBS and CSE as the predominant H₂S-synthesizing enzymes in renal tissues, with CBS predominantly expressed in proximal tubular cells of the outer cortex, while CSE exhibited predominant localization in the inner cortex and outer medulla (Cirino, Szabo & Papapetropoulos, 2023). In cis-AKI, impairment of endogenous H₂S-synthesizing enzymes played a pivotal role in renal pathophysiology. Experimental evidence demonstrated that cisplatin treatment significantly downregulated CSE expression in proximal tubular cells, a phenomenon mechanistically linked to excessive ROS accumulation (Cao et al., 2018b; Jiang et al., 2024). ROS may directly damage enzyme structures or indirectly suppress transcriptional activity through pro-inflammatory pathways such as NF-κB activation (Elkhoely & Kamel, 2018). Animal studies further corroborated that cisplatin administration markedly reduced renal H₂S levels, while pharmacological inhibition of CSE using DL-propargylglycine (PAG) exacerbated tubular necrosis, inflammatory infiltration, and oxidative damage (Ahmad, 2022; Cao et al., 2018b). These findings underscored the critical disruption of endogenous H₂S biosynthesis in driving cis-AKI progression. Notably, plasma H₂S concentrations also declined during cis-AKI, reflecting systemic depletion of this cytoprotective molecule. The reduction in both renal and circulating H₂S levels correlated with impaired antioxidant defenses, amplified mitochondrial dysfunction, and enhanced susceptibility to cisplatin-induced apoptotic signaling cascades (Ahmad, 2022; Cai et al., 2024; Cao et al., 2018b). The suppression of H₂S-synthesizing enzymes served as a central mechanism linking cisplatin toxicity to renal injury.

Our analysis has explored the therapeutic potential and underlying mechanisms of H₂S donors in cisplatin-associated nephrotoxicity, including their ability to inhibit the upregulation of inflammatory factors, reduce ROS production, and alleviate apoptosis and death of tubule cells. NaHS improved mitochondrial energy metabolism by sulfhydrating sirtuin 3 (SIRT3) on both CXXC zinc finger motifs, increasing SIRT3 expression and enhancing its deacetylase activity (Yuan et al., 2019). There is also a direct interaction between H₂S and p47phox, which inhibits NADPH oxidase activity and reduces ROS accumulation by persulfide p47phox (Cao et al., 2018b). Moreover, by inhibiting the ROS/mitogen-activated protein kinase signaling pathway, the polysulfide donor Na₂S₄ promoted nuclear factor erythroid two-related factor 2 translocation into the nucleus, reduced NOX activation, and alleviated renal toxicity (Cao et al., 2018a). Drawing on our previous research, we also found that GYY4137, a long-acting H₂S donor, provides a stable concentration of H₂S in solution for more than 24 h, exerting a strong protective effect on diabetic renal damage via affecting multiple ROS-associated enzymes (Chen et al., 2023; Yu et al., 2021). Na₂S_4, NaHS, and GYY4137 similarly reduced the production of inflammatory cytokines and kidney inflammation caused by cisplatin. This mechanism of action is associated with S-sulfhydrylation of signal transduction and transcriptional activator3 (STAT3) and inhibitor kappaB kinaseβ (IKK-β), leading to phosphorylation and decreased expression of pro-inflammatory genes (Sun et al., 2020). ADT-OH, a new sustained-release donor of H₂S, is found to reduce cisplatin-induced RTEC cell death and mitochondrial dysfunction in HK-2 cells compared to control cells, indicating its potential for treating cisplatin-induced acute kidney injury (Cai et al., 2024). Aside from these potential mechanisms, H₂S also reduces lipid deposition, inhibits ferroptosis, and protects renal tubular cells (Cai et al., 2024; da Costa Marques et al., 2022; Zhang et al., 2023b). These studies also showcased that H₂S donor molecules curtailed inflammation and enhanced mitochondrial function in animal models of cis-AKI.

The high heterogeneity, with I² = 73% on Scr and I² = 75% on BUN, respectively, may be due to the differences in animal species, sample size, and methodology among the studies. In terms of experimental animals, only one study used dogs as experimental subjects (Wang, Liu & Liu, 2022), and the rest used rats (eight studies) or mice (10 studies). The lack of significance in animal species subgroup analysis may be attributed to this factor (SMD = −2.96, 95% CI [−3.72 to −2.19], p = 0.38, I² = 0%, Fig. S4). Due to the lack of data from large mammals, there is still a long way to go for subsequent clinical translation. There were different effect ranges for different H₂S donors seen in our study. This suggested that different donors may have varying degrees of renal protection, and further research is needed to identify the most suitable donor for specific applications. We also observed significant differences in the release curve (Jiang et al., 2024), physicochemical and biological characteristics, time of action, and potential therapeutic applications of H₂S donors in animals (Cai et al., 2024). These differences highlighted the complexity of H₂S donor research and the need for a deeper understanding of their mechanisms of action. NaHS was the most commonly used donor in eight studies, accounting for 51% of the total. The NaHS group demonstrated the narrowest 95% CI and a low level of heterogeneity in subgroup analyses of donors (95% CI [−2.65 to −1.53], I² = 20%, Fig. S3). Moreover, based on the above analysis, GYY4137, used in three studies, stands out among other H2S donors due to its sustained release characteristics. As mentioned above, ADT-OH is more effective than other H₂S donors and offers protection against cisplatin-induced renal tubular cell apoptosis, oxidative stress, and mitochondrial dysfunction in vitro (Cai et al., 2024). Allicin is the main bioactive compound in garlic, which can be decomposed into DADS, DAS, and DATS (Bautista et al., 2005; Borlinghaus et al., 2014). I. vivo, red blood cells converted these compounds into H₂S, producing experimental results similar to those of other artificial H₂S donors (Benavides et al., 2007). With the deepening understanding of various garlic extracts, researchers gradually integrate their pharmacological properties, such as anti-inflammatory (Zhu et al., 2022), immune regulation (Arreola et al., 2015), pathogen infection resistance (Hall et al., 2017), antioxidant stress (Vazquez-Prieto et al., 2011), and organ protection (Abdel-Daim et al., 2020). There were five studies using garlic extracts in this meta-analysis (Abdel-Daim et al., 2019; Chiarandini Fiore et al., 2008; Elkhoely & Kamel, 2018; Jiang et al., 2024), and the final results confirmed their inhibitory effect on cisplatin-induced nephrotoxicity (SMD = −7.27, 95% CI [−10.37 to −4.16], p = 0.0009, I² = 79%, Fig. S3). As the four garlic extracts were only utilized in 1–2 studies, we amalgamated them into a single major category that was applied for initial subgroup analysis, showing that natural H₂S donors possess equivalent therapeutic potential to synthetic donors. To ensure precision, separate subgroup analyses of these four extracts were also conducted, and their renal protective effects remained unchanged despite high heterogeneity (I² = 85.6%, p < 0.00001, Fig. S9). However, it is worth noting that there may be some structural limitations on those natural donors in clinical translation, and by-products during the release of H₂S (El-Saber Batiha et al., 2020). More accurate extraction and modifications of those compounds remain to be established.

There are common issues in meta-analyses of animal studies, such as high heterogeneity and funnel plot asymmetry. Despite subgroup analysis on H₂S donor type, species, and administration methods, the heterogeneity is not significantly reduced in the current study, probably due to potential intra-study bias, inadequate experimental design, sampling error, and other factors. These limitations may have weakened the generalizability of the study findings and increased the risk of translating the preclinical experiments into clinical studies.

Conclusions

H₂S exhibits significant nephroprotective effects on animal experimental AKI models, making it a promising candidate for the treatment of AKI induced by various causes. The beneficial effects are likely attributed to their ability to reduce oxidative stress, inflammation, and possible cell deaths. The broad applications of H₂S on cis-AKI animal models seen in the current analysis, signify its potential as a game-changer in renal medicine. The consistent and positive results encourage further exploration into the mechanisms of H₂S-mediated renal protection. Well-designed preclinical investigations for H₂S as a drug to prevent cisplatin-induced nephrotoxicity are necessary. More attention should be paid to the beneficial effects of sustained-released H₂S donors and extracts from natural sources in the prevention and treatment of cis-AKI in the future.

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