The effects of CORM3 or NaHS on the oxidative stress caused by chronic kidney disease in rats: potential interaction between CO and H2S signaling pathway

Neurotoxicity is implicated as a severe complication of chronic kidney disease (CKD). Accumulation of urea and other toxic compounds leads to oxidative stress, inflammation and destruction of the blood-brain barrier. Carbon monoxide (CO) and hydrogen sulfide (H2S) have been shown to have anti-inflammatory, anti-apoptotic, and anti-proliferative properties. The aims of the present study were evaluated the protective effects of CO-releasing molecule (CORM3) and H2S donor (NaHS) on oxidative stress and neuronal death induced by CKD in the hippocampus and prefrontal cortex by considering interaction between CO and H2S on CBS expression. CORM3 or NaHS significantly compensated deficits in the antioxidant defense mechanisms, suppressed lipid peroxidation and reduced neuronal death in hippocampus and prefrontal cortex and improvement the markers of renal injury that induced by CKD. In addition, CORM3 or NaHS significantly improved CBS expression which were reduced by CKD. However, improving effects of CORM3 on antioxidant defense mechanisms, lipid peroxidation, neuronal death, renal injury and CBS expression were prevented by amino-oxy acetic acid (AOAA) (CBS inhibitor) and reciprocally improving effects of NaHS on all above indices were prevented by zinc protoporphyrin IX (Znpp) (HO-1 inhibitor). In conclusion, this study demonstrated that formation of CO and H2S were interdependently improved CKD-induced oxidative stress and neuronal death, which is may be through increased expression of CBS.


Introduction
Chronic kidney disease (CKD) is considered to be one of the most important public health problems worldwide (Bronas et al. 2017).CKD results in several severe neurological complications such as cognitive dysfunction, stroke, seizures and encephalopathy (Matta et al. 2014).Renal inflammation and oxidative stress play a significant role in the pathogenesis of CKD (Zalba et al. 2006).Elevated levels of oxidative stress markers, deficiency of antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH) and lipid peroxidation have been found in patients with CKD (Kitada et al. 2020).Accumulation of uremic toxins (especially neurotoxins), proinflammatory cytokines, and reactive oxygen species following CKD leads to neurotoxicity and neuronal death in specific areas of the brain, such as the frontal cortex and hippocampus (Jing et al. 2018;Kovalčíková et al. 2018).ROS and pro-inflammatory cytokines impair hippocampal functions such as stabilization of working memory, synaptic plasticity and long-term potentiation, and affect memory and cognition (Wu et al. 2018).Compounds that may activate anti-inflammatory pathways and as well as reducing oxidative stress have important clinical applications in the treatment of neurological complications induced by CKD.
Gasotransmitters have multiple physiological and pathophysiological functions in different organ systems of the body (Olson and Donald 2009).Hydrogen sulfide (H 2 S) is one of the most important neurotransmitters and neuroprotectors playing an essential role in brain function (Paul and Snyder 2015).It is predominantly synthesized in the brain by cystathionine beta-synthase (CBS) (Robert et al. 2003).There is evidence that the administration of H 2 S can effectually protect organs against oxidative stress.H 2 S has 2 distinct and important mechanisms that protect cells against oxidative stress: either it acts as a direct scavenger of ROS or it regulates the antioxidant defense system (Greabu et al. 2016).Our previous study has shown that NaHS (H 2 S donor) increased the expression of Nuclear factor-erythroid 2-related factor 2 (Nrf2), a transcriptional factor that binds to response elements on the promoter region of the HO-1 gene and led to the augmentation of antioxidant defense system (Hamidizad et al. 2022).The expression of CBS can stimulate the expression of several antioxidant enzymes, including SOD and catalases (Hadadha et al. 2015).CBS deficiency leads to a variety of pathological conditions including thrombosis, osteoporosis and fatty liver (Yaghmai et al. 2002;Voskoboeva et al. 2018).According to our previous study, the inhibition of CBS reduced the therapeutic effect of CORM3 on the cognitive impairment induced by CKD and aggravated neuronal injury in the hippocampus and prefrontal cortex (Hamidizad et al. 2022).Carbon monoxide (CO) has appeared to have many of the functions of H 2 S including neurotransmission, anti-proliferative effects and cytoprotective effects moreover, antioxidant effects of CO and CO-releasing molecule (CORM) are published in certain conditions (Queiroga et al. 2015;Ismailova et al. 2018).CO is essentially produced by heme oxygenase (HO) in the brain (Queiroga et al. 2015;Tian et al. 2015).HO-1 is activated in response to various cellular stress conditions, such as oxidative stress and inflammation (Campbell et al. 2021).Exogenous CO (CORM) increases the activation of Nrf2/HO pathways in pathological conditions (Wilson et al. 2014).
There are many reports on the interaction of CO and CBS/ H 2 S (Bannenberg and Vieira 2009).It has been suggested that CBS is a specific CO sensor (Farrugia and Szurszewski 2014).CO can bind to CBS and lead to its inactivation (Kajimura et al. 2010;Farrugia and Szurszewski 2014).Moreover, under physiological conditions, H 2 S/CBS and CO/HO pathways reciprocally inhibit each other (Jin et al. 2006).However, endogenous H 2 S and CO are in synergy with each other in the recurrent febrile seizure model (Han et al. 2006).In the acetic acid-induced gastric ulcer healing model, CO-releasing molecules 2 (CORM2) and H 2 S donor (NaHS) increased the mRNA expression of CBS at the ulcer margin (Magierowski et al. 2018).In cognitive impairment induced by CKD, the improving effects of CORM3 and NaHS on the Nrf2/HO-1 signaling pathways are reciprocally interdependent (Hamidizad et al. 2022).
Considering the antioxidant properties of H 2 S and CO and their interaction, which are reported to be inconsistent, in the present study, we aimed to clarify the interaction between H 2 S and CO on CBS enzyme expression, SOD levels and lipid peroxidation in CKD rats.In this regard, we investigated the therapeutic effects of intracerebroventricular (i.c.v) injection of CORM3 and NaHS on the neurotoxicity of hippocampal and prefrontal cells induced by CKD in rats.

Experimental design
The experiments were conducted on 36 male Wistar rats, weighing between 200 and 250 g.All the ethical codes established by the Committee of Monitoring Laboratory Animals of Tehran University of Medical Sciences, are considered for all experiments on animals.Rats were housed under standard conditions (12 h light-dark cycle; 20-22 °C) with free access to water and food.Rats underwent stereotaxic surgery in the ninth week after CKD induction as previously described (Gava et al. 2012).Stainless-steel guide cannulas (a 23-gauge needle) were placed directed toward the right lateral ventricles.The stereotaxic coordinates (0.9 mm posterior to bregma; 1.5 mm lateral to sagittal suture; 3 mm beneath the brain surface) were selected for intra-cerebro-ventricular (i.c.v) injection (Schwarz et al. 2006).In the tenth week, the rats were randomly divided into six groups: sham (n = 10), five-sixth (5/6) nephrectomy (CKD) (n = 6), CKD + NaHS, (n = 6), CKD + CORM3 (n = 6), CKD + NaHS and Znpp (n = 6), CKD + CORM3 and AOAA (n = 6).Rats in sham and CKD groups were given normal saline (4 µl) by i.c.v injection for one week.Animals in CKD + NaHS groups were treated with NaHS (17 µg in 4 µl) (Liu et al. 2011) and CKD + CORM3 groups were treated with CORM3 (50µmol in 4 µl) (Ling et al. 2017) by i.c.v injection for one week.Moreover, to study the interaction between H 2 S and CO, rats received i.c.v injection of zinc protoporphyrin IX (Znpp), HO-1 inhibitor (75nmol in 4 µl) (Soriano et al. 2012) one hour before NaHS and daily for 1 week.In parallel, another series of rats received aminooxy acetic acid (AOAA), CBS inhibitor (100 µg/rat in 4 µl) (Yamamoto et al. 2020) and then CORM3 after half an hour intra-cerebro-ventricularly.24 h after the last injection and at the end of the 11th week, rats were anesthetized for obtaining blood samples from the abdominal aorta to determine blood urea and serum creatinine (sCr) levels and finally the brain tissues were rapidly removed to be stored at -80 ˚C for analysis.The concentration of urea and creatinine was determined spectrophotometrically using assay kits from Pars Azmoon Company, Iran.

Oxidative stress assay
The hippocampus and prefrontal cortex samples were used to evaluate the state of oxidative stress.The SOD activity was measured by using a Nasdox ELISA kit according to the manufacturer's instructions.For 50 mg of hippocampus tissue, 500 µl of lubricating buffer was added and homogenized.The solution was then centrifuged at 12,000 rpm for 5 min at 4 °C and the supernatant was taken to measure SOD activity.Enzyme activity was expressed as a unit per mg of protein.For lipid peroxidation assay, the malondialdehyde (MDA) levels in hippocampus tissue samples were measured by spectrophotometry method with thiobarbituric acid (TBA).MDA reacts with TBA to create a pink pigment with maximum absorption of 532 nm.The values were expressed as µmol/100 mg tissues (Kianian et al. 2022).

Western blot (WB)
CBS protein expression in the hippocampus and prefrontal cortex was measured by western blotting as previously described (Babaei et al. 2018;Jabarpour et al. 2018).For western blotting, tissues were lysed with RIPA buffer.The lysates were removed by centrifugation at 14,000 rpm for 20 min at 4 °C.Protein concentration was determined by the BCA Protein Quantification kit according to the manufacturer's instructions.The cell lysates were mixed with an equal volume of 2X Laemmli sample buffer.Lysates (20 µg) were then subjected to SDS-PAGE after a 5 min boiling and subsequently transferred to a 0.2 μm immune-Blot™ polyvinylidene difluoride (PVDF) membrane (Cat No: 162-017777; Bio-Rad Laboratories, CA, USA).The membranes were then blocked with 5% BSA (Cat No: A-7888; Sigma Aldrich, MO, USA) in 0.1% Tween 20 for 1 h.Then, the membranes were incubated with anti-CBS (Cat No: RP1041, Bosterbio) and anti-beta actin-loading control antibodies (Cat No: ab8227; Abcam) for 1 h at room temperature.Then, membranes were washed thrice with TBST, and incubated with goat anti-rabbit IgG H&L (HRP) secondary antibody.β-actin was used to normalize the protein expression.Densitometry of protein bands was performed using the gel analyzer Version 2010a software (NIH, USA), such that, the percentage area under the curve of each band was divided by the percentage area under the curve of its corresponding actin band, and then calculated values were compared between groups as we described previously (Siavashi et al. 2016).

Histology
The hippocampus and prefrontal cortex samples were placed in 10% buffered formalin, and after regular tissue passage and fixation, a paraffin mold was made from it and after preparing 5-micron slices, it was stained with hematoxylin and eosin acid paints.Tissue analysis was performed by a pathologist after the provision of a proper slide.The hippocampus and prefrontal damage were assessed by counting the injured neurons in the CA1, CA3, Dentate gyrus (DG) and medial prefrontal cortex (mPFC) at a magnification of 200×.The hippocampus and prefrontal damage were assessed by counting the number of injured neurons in the CA1, CA3, Dentate gyrus (DG) and medial prefrontal cortex (mPFC) at a magnification of 200×.The injured cells characterized with condensing nuclei without euchromatin were calculated in 3 sections for each sample using image analysis software (Image J, National Institutes of Health, Bethesda, MD).The number of injured cells of CA1, CA3, DG and mPFC compared with the sham group (P < 0.05).

Statistical analysis
All statistical analyses were carried out by one-way analysis (ANOVA) followed by Tukey's post-hoc and data are expressed as mean ± standard error of mean (SEM).P-values equal to or less than 0.05 were considered as statistically significant.

Effect of CORM3 and NaHS treatment on kidney function in CKD rat
The outcomes showed that as compared to the sham group the levels of BUN (56.8 ± 7.7 vs. 16.2 ± 2.5 mg/dl; p < 0.001) and serum creatinine (0.93 ± 0.1 vs. 0.34 ± 0.03 mg/dl; p < 0.001), 11 weeks after 5/6 nephrectomy were significantly higher in the CKD group.The levels of BUN and serum creatinine were significantly decreased in the CORM3 (38.4 ± 4.5 mg/dl; p < 0.05 and 0.45 ± 0.07 mg/dl; p < 0.001, respectively) and or NaHS treatment groups compared to the CKD group (30.2 ± 10 mg/ dl and 0.44 ± 0.06 p < 0.001, respectively).One-way ANOVA revealed a significant increase in the levels of BUN (p < 0.05) and serum creatinine (p < 0.001) in the groups that received Znpp or AOAA compared to treatment group NaHS and CORM3 respectively (Fig. 1).

Effect of CORM3 treatment on the hippocampus and prefrontal cortex MDA levels
The results of this study revealed that induction of CKD in rats significantly enhanced hippocampus MDA levels (1.49 ± 0.2 vs. 0.09 ± 0.06 µmol/50 mg tissue, p < 0.001) and prefrontal cortex (1.62 ± 0.07 vs. 0.08 ± 0.04 µmol/50 mg tissue, p < 0.001) in comparison with the sham group.although the CORM3 treatment significantly decreased MDA levels in the hippocampus and prefrontal cortex of the CKD rats (p < 0.001).Interestingly, compared to the CORM3 group, the MDA levels were significantly enhanced in the hippocampus and prefrontal cortex (p < 0.001) of the AOAA group (Fig. 2A, B).

Effect of CORM3 treatment on the hippocampus and prefrontal cortex SOD activity
The results of this study showed that CKD caused a significant reduction in the activity of the SOD enzyme in the hippocampus (2.47 ± 0.22 vs. 1.37 ± 0.3 U/mg tissue, p < 0.001) and prefrontal cortex (2.56 ± 0.07 vs. 1.43 ± 0.12 U/mg tissue, p < 0.001) of the CKD rats compared with the sham group.Treatment with the CORM3 significantly enhanced the activity of the SOD enzyme in the hippocampus and prefrontal cortex of the CKD rats (p < 0.001).Prominently compared to the CORM3 group, the activity of the SOD enzyme was significantly decreased in the hippocampus and prefrontal cortex (p < 0.001, p < 0.05 respectively) of the AOAA group (Fig. 2C, D).These results suggest the improving effects of CORM3 on the MDA levels and the activity of the SOD enzyme in the hippocampus are mediated by the H 2 S levels.

Effect of NaHS treatment on the hippocampus and prefrontal cortex MDA levels
The results showed that NaHS treatment significantly decreased MDA levels in the hippocampus and prefrontal cortex of CKD rats (p < 0.001).Interestingly, compared to the NaHS group, the MDA levels were significantly enhanced in the hippocampus and prefrontal cortex (p < 0.001) of the Znpp group (Fig. 2A, B).

Effect of NaHS treatment on the hippocampus and prefrontal cortex SOD activity
NaHS treatment significantly enhanced the activity of the SOD enzyme in the hippocampus and prefrontal cortex of CKD rats (p < 0.001).Importantly compared to the NaHS group, the activity of the SOD enzyme significantly decreased in the hippocampus and prefrontal cortex (p < 0.01) of the Znpp group (Fig. 2C,  D).These results suggest the improving effects of the NaHS on the MDA levels and the activity of the SOD enzyme in the hippocampus and prefrontal cortex are mediated by CO levels.

Effect of CORM3 treatment on the hippocampus and prefrontal cortex CBS protein levels in CKD rats
Based on the results, the CBS protein levels in the hippocampus (0.25 ± 0.04 vs. 1.19 ± 0.09, p < 0.001) (Fig. 3A) and prefrontal (0.39 ± 0.09 vs. 1.99 ± 0.2, p < 0.001) (Fig. 3B) cells of the CKD group was significantly lower than the sham group.WB analysis showed that administration of the CORM3 significantly increased the CBS protein levels in the hippocampus (0.49 ± 0.9, p < 0.01) and prefrontal (0.92 ± 0.1, p < 0.001) of the CKD rats (Fig. 3A,  B).These results indicate that the CBS protein levels are probably dependent on the CO and HO-l levels.Compared to the CORM3 group, the CBS protein levels significantly decreased in the hippocampus (0.17 ± 0.02, p < 0.01) and prefrontal (0.19 ± 0.09, p < 0.001) of the AOAA group.

Effect of NaHS treatment on the hippocampus and prefrontal cortex CBS protein levels in CKD rats
WB analysis showed that NaHS treatment significantly increased CBS protein levels in the hippocampus (0.55 ± 0.1, p < 0.01) (Fig. 3A) and prefrontal (1.01 ± 0.2, p < 0.001) (Fig. 3B) of CKD rats.Notably, Znpp administration along with NaHS significantly reduced its enhancing effects on CBS protein levels in the hippocampus (0.37 ± 0.02, p < 0.01) and prefrontal (0.22 ± 0.08, p < 0.001) of CKD rats (Fig. 3A, B).These results suggest that the level of CBS protein is dependent on the level of HO-1 and probably many of its effects are mediated by CO.
Fig. 3 Effects of CORM3 or NaHS on CBS protein levels in hippocampus (A) and medial prefrontal (B) cells among the groups.Data are expressed as the mean ± SD; *** p < 0.001 in comparison with the sham group; ++ p < 0.001 and +++ p < 0.001 in comparison with the CKD group; ## p < 0.01 and ### p < 0.001 treatment groups in comparison with the groups that revised Znpp or AOAA.CKD: Chronic kidney disease; CORM3: CO-releasing; AOAA: Amino-oxy acetic acid; NaHS: Sodium hydrosulfide; Znpp: Zinc protoporphyrin IX.

Effect of CORM3 treatment on the numbers of the hippocampus and prefrontal cortex intact neurons
Sample sections of hippocampus areas (CA1, CA3 and dentate gyrus) and medial prefrontal cortex (mPFC) are demonstrated in Fig. 4A-F for experimental groups.One-way ANOVA and Tukey's post-test showed that the number of neurons in the dentate gyrus (DG), CA1 and CA3 of the hippocampus and mPFC in the CKD rats were significantly fewer compared with corresponding areas of the sham group (p < 0.001 for CA1; CA3; mPFC and p < 0.05 for DG, Fig. 5).Treatment with the CORM3 significantly decreased counted number of neurons in all three areas of the hippocampus and mPFC in the CKD rats (p < 0.001 for CA1; CA3; DG and mPFC, Fig. 5).Moreover, CBS inhibition significantly decreased the protective effects of the CORM3 on the hippocampus and mPFC damage (p < 0.001 for CA1; CA3 and mPFC; p < 0.05 for DG, Fig. 5).

Effect of NaHS treatment on the numbers of hippocampus and prefrontal cortex intact neurons
Based on the results, treatment with NaHS significantly increased neural cells count in all three areas of the hippocampus and mPFC compared with the CKD group (p < 0.001 for CA1; CA3; mPFC and p < 0.01 for DG).In addition, inhibition of HO-1 by Znpp significantly decreased the therapeutic effects of NaHS on reducing neuronal loss in the hippocampus and mPFC (p < 0.001 for CA1; CA3; mPFC and p < 0.05 for DG, Fig. 5).

Discussion
The data of this study showed that treatment of NaHS or CORM3 ameliorated oxidative stress, neural loss and increased CBS protein levels in the hippocampus tissues and medial prefrontal cortex of CKD rats.In addition, improved kidney functions in CKD rats.Remarkably, it was observed that the therapeutic effects of CORM3 on SOD and MDA levels and neural loss in the hippocampus and prefrontal cortex and renal injury markers were reduced by inhibiting CBS enzyme activity, confirming that CO acts by H 2 S production.Moreover, CO-producing enzyme inhibitors (Znpp) prevented the ameliorating effects of NaHS on SOD, MDA levels and CBS protein expression in the hippocampus and prefrontal cortex and kidney functions.Notably, since many studies have investigated the effects of AOAA and Znpp in oxidative stress conditions in the CNS, the effects of these two inhibitors alone were not examined in the present study (Pérez-de-Puig et al. 2013;Hadadha et al. 2015;Liu et al. 2017;Wu et al. 2022).Previous studies have shown that CO and H 2 S both reduce inflammation and oxidative stress in various pathological conditions such as CKD-induced neurotoxicity, lipopolysaccharide-induced cognitive impairment and ischemic brain injury (Bani-Hani et al. 2006;Yabluchanskiy et al. 2012;Askari et al. 2018;Kshirsagar et al. 2021).
In agreement with previous studies (Kujal and Vernerová 2008), our results demonstrated that induction of 5/6 nephrectomy leads to renal dysfunction (increased concentration of BUN and sCr), elevated MDA levels and neural loss and decreased SOD and CBS expression in the hippocampus tissues and medial prefrontal cortex.The accumulation of uremic toxins (especially neurotoxins) following CKD leads to the release of proinflammatory cytokines, reactive oxygen species, and lipid peroxidation in neurons of various brain regions, such as the frontal cortex and hippocampus (Small et al. 2012;Watanabe et al. 2014).
In addition, several studies have shown that the inhibition of CBS expression aggravates the processes of oxidative stress and reduces the activity of antioxidant enzymes (Liu et al. 2017).The enhancement of CBS activity and the level of H 2 S derived from it can promote the activity of the antioxidant enzyme catalase at the post-translational level (Ma et al. 2021).In the neonatal hypoxic-ischemic (HI) injury rat model, L-Cysteine exerts neuroprotection in neonates through the CBS/H 2 S pathway, which is partially mediated by anti-apoptotic effects and reduction of oxidative stress and ER stress.Furthermore, the results of this study showed that pre-treatment with a CBS inhibitor significantly reduced the neuroprotection of L-Cysteine (Liu et al. 2017).In our previous study, a rat model of chronic kidney diseaseinduced cognitive impairment, CORM3 treatment of CKD rats reduced neuronal loss and memory and learning deficits by enhancing the Nrf2/HO-1 signaling pathway.Interestingly, the results of this study showed that Inhibition of CBS significantly reduced the therapeutic effects of CORM3 (Hamidizad et al. 2022).
The results of this study showed that the intracerebroventricular injection of the CORM3 and NaHS reduced the renal injury induced by 5/6 nephrectomy.Previous studies have shown that H 2 S and CO have renal protective effects that can be exerted through their effects on renal tissue antioxidants, and proinflammatory and anti-inflammatory cytokines (Wesseling et al. 2015;Uddin et al. 2018).H 2 S and CO are high lipid solubility, the molecules can pass easily through the blood-brain barrier and have beneficial effects on multiple organs and tissues (Wang 2004;Kajimura et al. 2010).Notably, unlike other gasotransmitters, CO has biologically stability and is capable of causing effects far from the site of production (Farrugia and Szurszewski 2014).Several previous studies have reported the therapeutic and cytoprotective effects of the central administration of H 2 S. In the angiotensin II-induced hypertension (HTN) rat model chronic intracerebroventricular infusion of NaHS can improve HTN and autonomic function (Donertas Ayaz et al. 2021).In the preeclampsia (PE) rat model the intracerebroventricular injection of NaHS reduced microglia-induced inflammation in the rostral ventrolateral medulla (RVLM), renal sympathetic nerve activity, and PE symptoms in both PE and lipopolysaccharide (LPS)-treated pregnant rats (Du et al. 2022).
In addition, the results of this study showed that treatment of NaHS or CORM3 improved oxidative stress markers (SOD and MDA levels), neural loss and increased CBS protein levels in the hippocampus tissues and medial prefrontal cortex of CKD rats.Previous studies showed that CORM3 and NaHS possess antioxidation, antiapoptotic, and antiinflammatory properties.In a mouse model of transient middle cerebral artery occlusion, CORM3 treatment reduces ischemia-reperfusion-induced brain injury by suppressing neuroinflammation and improving endogenous antioxidant enzymatic activities (Wang et al. 2018).In human plasma and blood platelets in vitro, CORM3 administration protected various molecules in plasma as an antioxidant and showed stronger antioxidant properties compared to CORM-A1 (Adach and Olas 2020).In cognitive impairment and oxidative stress changes induced by high-fat diet (HFD) consumption, NaHS administration significantly improved HFD-induced spatial and passive learning and memory impairment and anxiety-like behavior, somewhat, through its antioxidant activities (Habibitabar et al. 2020).In the myocardial infarction (MI), mice model NaHS treatment protected cardiomyocytes from hypoxic injury and increased CBS levels in a concentration-dependent manner (Li et al. 2016).Furthermore, the neuroprotective effects of CO and H2S against CKD induced oxidative stress in the hippocampus tissues and medial prefrontal cortex of CKD rats may be due to the ameliorating effect of these agents on kidney function.One previous study has shown that the level of endoplasmic reticulum stress as well as oxidative stress in the hippocampus of CKD mice is positively correlated with the serum concentrations of BUN and creatinine.(Kosuge et al. 2018).In a rat model of CKD, NaHS administration improved renal dysfunction, memory loss and enhanced antioxidant/anti-inflammatory responses (Askari et al. 2018).
Our results demonstrated that the protective effects of NaHS on oxidative stress, neural loss, CBS expression and kidney function in CKD rats were prevented by Znpp (which inhibits the production of endogenous CO).Many studies have reported that gasotransmitters interact with each other and their physiological effects are mutually dependent (Huang et al. 2021).In the acetic acid-induced gastric ulcers model rat gastroprotection effects of NaHS treatment were prevented by Znpp (Magierowski et al. 2016).Moreover, in a recurrent febrile seizures rat model, administration of hemin (which increases the production of endogenous CO) increased CBS expression, while administration of Znpp reduced CBS expression, these findings suggest that CO can regulate the H 2 S/CBS system (Han et al. 2006).In CKD-induced brain dysfunction, H 2 S improved behavioral deficits and renal dysfunction, and accelerated anti-oxidant/anti-inflammatory responses, through interaction with NO signaling in the hippocampus (Askari et al. 2018).However, our study suggests the neuroprotective and antioxidant effects of H 2 S may be partially mediated by CO in the hippocampus and prefrontal cortex of CKD rats.Our data indicated that the therapeutic effects of CORM3 on oxidative stress, neural loss, CBS expression and renal injury markers in CKD rats were prevented by AOAA (CBS inhibitor).The bile excretion regulated by CBS in the liver is dependent on the CO levels (Shintani et al. 2009) while in the carotid body, the H 2 S generation, induced by hypoxia was enhanced by CO inhibitor (Prabhakar 2012).In the gentamicin induced renal injury rat model NaHS administration improved kidney functions which can be partially dependent on CO pathway via induction of the HO-1 enzyme (Aziz et al. 2020).In addition, our previous study showed that the biological effects of CO on the Nrf2/ HO-1 signaling pathway are reciprocally mediated by H 2 S in CKD rats (Hamidizad et al. 2022).although studies are showing the protective effects of CO are independent of H 2 S (Magierowski et al. 2018).However, our study suggests a positive interaction between CO and H 2 S on oxidative stress and CBS expression in CKD rats.
There are two limitations to this study.First, we used AOAA to block CBS activity and CORM3 function and investigative the interaction of CO and H 2 S in the brain injury induced by CKD, but the CNS has other enzymes to produce H 2 S, such as 3-mercaptopyruvate sulfur transferase (3-MST), which remain to be investigated in future studies.Secondly, the effect of CORM3 and NaHS treatments on cystathionine γ-lyase (CSE) and 3-MST have not been evaluated.

Conclusion
In summary, this study suggests that H 2 S or CO improved oxidative stress in CKD rats possibly by enhancing the CBS expression.The findings of our study emphasized that the protective effects of CORM3 and NaHS on oxidative stress and CBS expression in CKD rats are reciprocally interdependent.These interactions should be further investigated to elucidate the therapeutic effects of CO and H 2 S releasing substances.

Fig. 2
Fig. 2 Effects of CORM3 or NaHS on MDA levels in hippocampus (A) and medial prefrontal cortex (B) and SOD activity in hippocampus (C) and medial prefrontal cortex (D) among the groups.Results are expressed as mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001 in comparison with the sham group; +++ p < 0.001 in compari-

Fig. 5
Fig. 5 Effects of CORM3 or NaHS on the number of neurons in the CA1, CA3 and dentate gyrus areas of hippocampus and medial prefrontal cortex (mPFC) among the groups.Data are expressed as the mean ± SD; * p < 0.05 and *** p < 0.001 in comparison with the sham group;++ p < 0.01 and +++ p < 0.001 in comparison with the