Hydrogen Sulfide and Carnosine: Modulation of Oxidative Stress and Inflammation in Kidney and Brain Axis

Emerging evidence indicates that the dysregulation of cellular redox homeostasis and chronic inflammatory processes are implicated in the pathogenesis of kidney and brain disorders. In this light, endogenous dipeptide carnosine (β-alanyl-L-histidine) and hydrogen sulfide (H2S) exert cytoprotective actions through the modulation of redox-dependent resilience pathways during oxidative stress and inflammation. Several recent studies have elucidated a functional crosstalk occurring between kidney and the brain. The pathophysiological link of this crosstalk is represented by oxidative stress and inflammatory processes which contribute to the high prevalence of neuropsychiatric disorders, cognitive impairment, and dementia during the natural history of chronic kidney disease. Herein, we provide an overview of the main pathophysiological mechanisms related to high levels of pro-inflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and neurotoxins, which play a critical role in the kidney–brain crosstalk. The present paper also explores the respective role of H2S and carnosine in the modulation of oxidative stress and inflammation in the kidney–brain axis. It suggests that these activities are likely mediated, at least in part, via hormetic processes, involving Nrf2 (Nuclear factor-like 2), Hsp 70 (heat shock protein 70), SIRT-1 (Sirtuin-1), Trx (Thioredoxin), and the glutathione system. Metabolic interactions at the kidney and brain axis level operate in controlling and reducing oxidant-induced inflammatory damage and therefore, can be a promising potential therapeutic target to reduce the severity of renal and brain injuries in humans.


Introduction
The dysregulation of cellular redox homeostasis and chronic inflammatory processes represent interdependent factors implicated in the pathogenesis of multiple diseases, including atherosclerosis and cardiovascular diseases, neurodegenerative diseases, chronic kidney disease, diabetes, cancer,

Hydrogen Sulfide Signaling
Hydrogen sulfide (H2S) is a small gaseous molecule with profound biological effects within living organisms. It exerts key roles in cytoprotection, inflammation, vascular function, neurological systems, mitochondrial function, energy metabolism, and ageing [74,75]. However, H2S was originally known for its deleterious effects on health and the environment. First, in 1700, Italian physician Bernardino Ramazzini [76] described a severe ocular irritation and inflammation in sewer workers, caused by an unspecified volatile acid. Later, the chemical composition of H2S was elucidated and its association with ocular adverse effects and intoxication in sewer workers was recognized. For over a century, studies focused on its major toxic effects-e.g., inhibition of cytochrome c oxidase, carbonic anhydrase, monoamine oxidase, and sodium/potassium-ATPase (NaC/KC ATPase) [77]. The image of H2S was revolutionized when Kimura, in 1996, revealed its role as an endogenous neuromodulator [78]. Recently, H2S was classified as the third gasotransmitter along with NO and carbon monoxide (CO). H2S is enzymatically released in our body and it has a highly regulated metabolism. It is freely permeable to membranes and exerts specific physiological functions in several systems that can be mimicked by H2S donors applied exogenously [79]. In mammals, hydrogen sulfide (H2S) is primarily produced by two cytosolic pyridoxal-5′-phosphatedependent enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), which use the substrates homocysteine and L-cysteine ( Figure 1).   A third enzyme, 3-mercaptopyruvate sulfurtransferase , catalyzes H 2 S production mainly in mitochondria by the conversion of 3-mercaptopyruvate to pyruvate and H 2 S [80]. Recently, an additional biosynthetic pathway has been described for the production of hydrogen sulfide from D-cysteine involving 3-mercaptopyruvate sulfurtransferase and D-amino acid oxidase that operates predominantly in the cerebellum and the kidney, i.e., within the KB axis [81]. D-cysteine is mainly adsorbed with food and derives from L-cysteine racemization during food processing. This novel pathway is of particular interest since supplementation with D-cysteine showed to protect renal cortex cells and cerebellum cells (i.e., KB axis) more efficiently than L-cysteine [81]. Furthermore, it was reported that gut microbiota would be another source of H 2 S that might influence health and function [81]. The contribution of each of these enzymes to net H 2 S production is dictated by its presence and relative tissue concentration, which varies in a cell-specific manner [81][82][83]. CBS is the major H 2 S-producing enzyme in the brain, while CSE is significantly expressed in the mammalian cardiovascular system and respiratory system and it seems to be the main H 2 S-forming enzyme in the liver, kidney, and pancreas [84].
Oxidative stress seems to influence CSE and CBS in a different manner. Reactive oxygen species appear to induce CSE expression, whereas they clearly suppress the transcription of the human CBS gene [85]. Eventually, H 2 S is metabolized to sulfite in the mitochondria by thiosulfate reductase, and then, is oxidized to thiosulfate and sulfate by sulfite oxidase. The sulfates are excreted in the urine [80]. Interestingly, urinary sulfates have been used as markers of H 2 S plasma levels and increased urinary sulfate concentrations have been correlated with a decreased risk of renal events in type 2 diabetic patients with nephropathy [86]. However, urinary sulfate and thiosulfate are not specific markers for endogenous H 2 S formation, and can also be the products of exogenous H 2 S production by sulfate-reducing bacteria in the gut [86]. H 2 S acts independently of any specific transporters by mechanisms not fully understood. S-sulfhydration, a novel posttranslational modification, is emerging as a mechanism responsible for many biological effects mediated by H 2 S [75,87]. H 2 S sulfhydrates protein thiol groups by transferring its sulfhydryl group to the cysteine residue of targeted proteins. Furthermore, H 2 S is oxidized in biological systems to polysulfides, which are now increasingly recognized as effectors of the H 2 S signaling mechanism [88]. H 2 S S-sulfhydration of enzymes, transcription factors, and ion channels has been described accounting for several protective effects of H 2 S, ranging from response to inflammation to cytoprotection [82,89]. For instance, H 2 S attenuates inflammation through the S-sulfhydration of Nuclear Factor-kappa B (NF-κB) [90]. H 2 S increases the antioxidative properties of cells by sulfhydration of Kelch-like ECH-associated protein 1 (Keap1), leading to its dissociation from Nrf2, which translocates in the nucleus and binds to the antioxidant response element (ARE) promoting antioxidant gene transcription, such as GCLM, GCLC, and glutathione reductase [91]. S-sulfhydration may be involved in the augment of the life span of Caenorhabditis elegans induced by H 2 S through sirtuins [92]. H 2 S may also inhibit mitochondria ROS production through sulfhydration of p66Shc [91]. In this regard, gut microbiota has attracted considerable interest for its role in microbial-mediated ROS generation, which might influence many signaling and homeostatic processes. Notably, lactobacilli ROS-mediated signaling has been described to induce Nrf2, opening the prospect that probiotic bacteria may elicit beneficial effects on disease states that involve Nrf2, including diabetes and neurodegenerative diseases [93]. In particular, in the (nod-like receptor) NLR family, the nod-like receptor pyrin domain-containing 3 (NLRP3) inflammasome has been reported to play a pathogenic role in the initiation and progression of metabolic and neurodegenerative diseases [94,95]. Recently, in vitro and in vivo studies showed that H 2 S mitigated lipopolysaccharide (LPS)-induced sepsis against oxidative stress and inflammation damage mediated by the NADPH oxidase 4 (Nox4) pathway [96], inhibiting the vicious cycle of NLRP3 inflammasome and oxidative stress in human retinal pigment epithelial cells [97] and in hypertensive rats [98]. In addition, H 2 S mediated effects on neuroinflammation and Aβ 1-42 production by suppressing the activation of STAT3 and cathepsin S [99]. H 2 S suppresses oxidative stress-induced mtROS production and NLRP3 inflammasome activation via S-sulfhydrating c-Jun at cysteine 269 in macrophages [100].

H 2 S as a Signaling Mediator in the Kidney-Brain Axis
Over the past few years, a crosstalk between kidney and brain emerged, as elucidated through the modulation of H 2 S for neuroprotection, particularly to unveil the pathophysiological mechanisms of oxidative and inflammatory stress. Consistent with this notion H 2 S, as a biological signaling mediator, is involved in various functions, including antioxidant, neuromodulatory, regulation of vascular tone, cytoprotective, anti-inflammatory, modulator of immune response, oxygen sensing, angiogenesis, mitochondrial bioenergetics, and blood-brain barrier permeability [101]. The kidney and brain are vital organs exposed to high-volume blood flow and thus, are highly susceptible to vascular damage [102]. Normal renal function regulates whole-body homeostasis, including neuronal homeostasis [103]. Therefore, it was well recognized that oxidative stress and inflammation are common outcomes strongly associated with both kidney and brain dysfunctions, leading to the development and progression of several diseases, including acute kidney disease (AKD), chronic kidney disease (CKD), neuropsychiatric disorders, and cognitive dementia [104,105]. Recent data have demonstrated that patients with CKD, especially at advanced stages, are highly susceptible to developing neuropsychiatric disorders (i.e., depression and anxiety) and cognitive impairment (i.e., Alzheimer and Parkinson diseases) [31]. In the kidney, H 2 S induces important diuretic, natriuretic, and kaliuretic effects by raising glomerular filtration rate and inhibiting tubular sodium re-absorption [106,107]. Notably, under hypoxic conditions, H 2 S functions as an oxygen sensor in the renal medulla that restores oxygen balance by enhancing medullary blood flow, decreasing energy requirements for tubular transport and directly suppressing mitochondrial respiration [107]. Since medullary hypoxia is a common feature of CKD, H 2 S deficiency can lead to progression of CKD by limiting this significant adaptive mechanism [108].

Anti-Inflammatory Role of H 2 S
Inflammation is a common feature in brain and kidney injuries, and it is quite reasonable to assume that inflammatory mediators may amplify the kidney-brain crosstalk. Particularly, the nucleotide-binding oligomerization domain NLRP3 inflammasome, a multiprotein complex, is involved in the pathogenesis of inflammation in chronic kidney and brain diseases [109,110]. NLRP3 induces the production of many proinflammatory cytokines and chemokines such as IL-1β, IL-6, TNF-α, transforming growth factor-β (TGF-β), and NF-κB, which are frequently associated with the pathogenesis of CKD [111]. These cytokines then initiate or amplify different downstream signaling pathways and drive proinflammatory factors [112], leading to cellular damage, such as autophagy dysfunction and ROS production [113]. In the kidney, generation and activation of the NLRP3 inflammasome have been reported to occur not only in immune cells like dendritic cells [114] and infiltrating macrophages [115], but also in other renal cells such as tubular epithelial cells [116] and podocytes [117]. Several human studies reported that the inflammatory response triggered by CKD may contribute toward increasing proinflammatory cytokines that can cross the blood-brain barrier (BBB) to reach the CNS and lead to neuroinflammation such as reported with neuropsychiatric and neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, major depressive disorder, and schizophrenia. This reinforces the concept of kidney-brain inflammatory crosstalk [118][119][120]. Accordingly, these pro-inflammatory molecules operate in the brain to induce common symptoms of sickness, including loss of appetite, sleepiness, withdrawal from normal social activities, fever, aching joints, fatigue, embezzlement of cognition, malaise, inattention, and depression [121]. In this context, H 2 S possess powerful anti-inflammatory, antioxidant, and anti-apoptotic effects [122]. On the other hand, H 2 S depletion may contribute to the progression of CKD and cognitive disorders [123,124]. Moreover, accumulating evidence reported that H 2 S appears to play a crucial role in the modulation of the immune response by regulating posttranslational modification of the NF-kB pathway in vitro and in vivo [125,126]. In the brain, H 2 S protects neurons from apoptosis and degeneration by inducing anti-inflammatory effects and upregulating antioxidant enzymes [127]. Additionally, treatment of H 2 S in the APP/PS1 (human amyloid precursor protein and presenilin 1) mouse model of AD reduced cognitive impairment and mitigated oxidative stress [128]. Moreover, H 2 S mediated the process of altered blood pressure in response to changes in serum homocysteine levels by blocking the activation of extracellular signal-regulated kinase 1/2 (ERK1/2)-STAT3 signaling pathway [129], exerted antidepressant effects by the induction of the mTORC1-TrkB-AMPA receptor pathway [130,131] as well as prevented cisplatin-induced nephrotoxicity [132]. Furthermore, NaHS, an exogenous H 2 S donor, also inhibits macrophage pro-inflammatory cytokine production, as well as cyclooxygenase-2 and nitric oxide production, and decreases macrophage motility. In addition, recent in vivo studies reported that NaHS ameliorates CKD-mediated brain dysfunctions through interaction with NO signaling in the hippocampus [133], as well as improves apoptosis, inflammation, and autophagy, exerting nephroprotective effects against CKD in rats [134,135].

Antioxidant Role of H 2 S
During pathological conditions, there is a mutual upregulation between factors promoting inflammation and oxidative stress, which sustains CDK progression and neurological disorders [136]. Under physiological conditions, ROS provides beneficial effects, regulating cellular stress responses by redox-sensitive signaling pathways. Indeed, ROS control cellular growth, differentiation, and migration; regulate vascular tone and cellular adhesion, leading to the production of iNOS at the transcriptional and posttranscriptional level by redox-dependent NF-κB or mitogen-activated protein kinases (MAPKs); modulate immune response; and control angiogenesis and apoptosis [137]. When ROS generation is prolonged or excessive, harmful consequences are observed with peculiar changes in cellular proteins, lipids, and ribonucleic acids, leading to cell dysfunction or death. Numerous enzymes with antioxidant activity are involved in neutralizing ROS, including superoxide dismutase (SOD), γ-glutamyltransferase (GGT), glutathione (GSH), glutathione reductase (GSSG-Rd), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), catalase (CAT), and nuclear factor erythroid 2-related factor 2 (Nrf2) [138]. All these antioxidant pathways are differently expressed in various cells and organs including kidney and brain tissues [139,140]. Intriguingly, recent evidence highlighted the complex role of H 2 S as a direct antioxidant scavenger to inhibit ROS generation and the inflammatory process in the pathogenesis of kidney and brain injuries [141,142]. Additionally, the formation of free radicals (i.e., ROS and RNS) as signaling molecules to promote inflammation induces the activation of the transcription of NF-κB by upregulating the production of pro-inflammatory cytokines and chemokines, leading to the recruitment and activation of leukocytes and resident cells [143]. The "oxidative" linkage between CKD and its complications is achieved through several pathophysiological mechanisms, including mitochondrial dysfunction [144], uremic neurotoxin-induced endothelial nitric oxide synthase (eNOS) uncoupling [145], and increased nicotinamide adenine dinucleotide phosphate-oxidases (NADPH oxidases (NOX)) activity [146], myeloperoxidase (MPO) [147], but also antioxidant enzymes depletion due to dietary restriction, and/or decreased intestinal absorption [148]. In particular, preclinical and clinical studies confirmed beneficial effects by upregulation of the Nrf2-HO1 antioxidant pathway during acute and chronic kidney diseases [149,150]. In addition, activation of the glutathione peroxidase pathway reduced not only the oxidative stress but also the inflammatory process induced by the uremic neurotoxins on the endothelium during CKD [151]. On the other hand, glutathione peroxidase deficiency in kidney diseases contributed to the development of cardiac diseases risk due to an increase in ROS generation and inflammatory pathways [152]. Furthermore, CKD patients also display hypovitaminosis D [153] as well as hypoalbuminemia [154] and zinc deficiency [155].
Fascinatingly evidence demonstrated that NaHS suppresses the expression of the ROS-generating enzyme, NADPH oxidase (NOX) and its essential subunit, Rac-1, in cultured vascular smooth muscle cells [156]. Likewise, NaHS has been shown to lower NOX-4 expression and potentiates the antioxidant effects of apocyanin, N-acetyl-L-cysteine, catalase, superoxide dismutase, and GSH, in the brain endothelial cells [157]. In addition, H 2 S can scavenge and/or degrade lipid peroxides [158] and increases the production of GSH [148]. Moreover, intraperitoneal application of NaHS to pregnant rats protects fetal brains from ischemia-reperfusion injury by restoring the glutathione levels reduced by ischemia-reperfusion [156]. However, experimental evidence demonstrated the scavenging effect of H 2 S in the presence of intracellular glutathione concentration between 1 and 10 mM, and 1 and 100 µM of cysteine in neurons [159]. Thus, the suppression of oxidative stress by increased levels of glutathione should be more effective than ROS scavenging by H 2 S itself.
GSH is a ubiquitous thiol tripeptide composed of cysteine, glycine, and glutamate, existing often as a reduced form, and it is synthesized from cysteine. Moreover, H 2 S also facilitates the transport of cysteine into cells. Glutathione is produced by two enzymes, glutamate cysteine ligase (GCL) (γ-glutamylcysteine synthase (γ-GCS)), which is a rate-limiting enzyme in the production of γ-glutamyl cysteine from glutamate and cysteine, and glutathione synthetase (GS), which produces glutathione by adding glycine to γ-glutamyl cysteine. GSH reduces disulfide bonds formed within cytoplasmic proteins to cysteines by serving as an electron donor. In this process, GSH is converted to its oxidized form, glutathione disulfide (GSSG). Intracellular cysteine plays an important role in cellular homeostasis as a precursor for protein synthesis, and for the production of GSH, hydrogen sulfide (H 2 S), and taurine. Cysteine exists as two unstable redox forms in the body: the oxidized form cystine and the reduced form cysteine. The extracellular cystine form is carried into cells through the cystine/glutamate antiporter system, after which cysteine is reduced and ready for GSH synthesis. The release of H 2 S into the extracellular space promotes the reduction of cystine to cysteine, increasing the amount of cysteine available as a substrate for GSH synthesis, and improving cystine transport [156,160,161]. Another significant mechanism for the effect of H 2 S on GSH may be by enhancing glutamate uptake [162]. Interestingly, kidney and brain tissues contain significant amounts of γ-glutamyl transpeptidase and γ-glutamyl cyclotransferase and nevertheless, maintain an appreciable concentration of glutathione. This suggested to us that these organs might also possess the enzymatic equipment needed for the synthesis of glutathione. Many in vivo studies have demonstrated that the kidney-brain axis contains a high concentration of both γ-glutamylcysteine synthetase and glutathione synthetase [163,164]. The presence of these enzymes in kidney and brain enables a series of catalytic events involving the synthesis and degradation of glutathione, and the coupled uptake and release of free amino acids from γ-glutamyl linkage. These reactions are steps in a cyclical process referred to as the "γ-glutamylcycle" (Figure 2). Antioxidants 2020, 9, x FOR PEER REVIEW 9 of 35 between 1 and 10 mM, and 1 and 100 μM of cysteine in neurons [159]. Thus, the suppression of oxidative stress by increased levels of glutathione should be more effective than ROS scavenging by H2S itself. GSH is a ubiquitous thiol tripeptide composed of cysteine, glycine, and glutamate, existing often as a reduced form, and it is synthesized from cysteine. Moreover, H2S also facilitates the transport of cysteine into cells. Glutathione is produced by two enzymes, glutamate cysteine ligase (GCL) (γglutamylcysteine synthase (γ-GCS)), which is a rate-limiting enzyme in the production of γ-glutamyl cysteine from glutamate and cysteine, and glutathione synthetase (GS), which produces glutathione by adding glycine to γ-glutamyl cysteine. GSH reduces disulfide bonds formed within cytoplasmic proteins to cysteines by serving as an electron donor. In this process, GSH is converted to its oxidized form, glutathione disulfide (GSSG). Intracellular cysteine plays an important role in cellular homeostasis as a precursor for protein synthesis, and for the production of GSH, hydrogen sulfide (H2S), and taurine. Cysteine exists as two unstable redox forms in the body: the oxidized form cystine and the reduced form cysteine. The extracellular cystine form is carried into cells through the cystine/glutamate antiporter system, after which cysteine is reduced and ready for GSH synthesis. The release of H2S into the extracellular space promotes the reduction of cystine to cysteine, increasing the amount of cysteine available as a substrate for GSH synthesis, and improving cystine transport [156,160,161]. Another significant mechanism for the effect of H2S on GSH may be by enhancing glutamate uptake [162]. Interestingly, kidney and brain tissues contain significant amounts of γ-glutamyl transpeptidase and γ-glutamyl cyclotransferase and nevertheless, maintain an appreciable concentration of glutathione. This suggested to us that these organs might also possess the enzymatic equipment needed for the synthesis of glutathione. Many in vivo studies have demonstrated that the kidney-brain axis contains a high concentration of both γ-glutamylcysteine synthetase and glutathione synthetase [163,164]. The presence of these enzymes in kidney and brain enables a series of catalytic events involving the synthesis and degradation of glutathione, and the coupled uptake and release of free amino acids from γ-glutamyl linkage. These reactions are steps in a cyclical process referred to as the "γ-glutamylcycle" (Figure 2). A great number of accumulated data indicate that oxidative stress and inflammation are major contributing factors of renin-angiotensin system (RAS) imbalance both in peripheral and brain Figure 2. The kidney-brain crosstalk by the H 2 S signaling pathway. γ-glutamyl transpeptidase (γGGT) (3), γ-glutamyl cyclotransferase (4), dipeptidase (5), oxoprolinase (6), γ-glutamyl-cysteine synthase (1), and glutathione synthetase (2) operate in the Meister cycle to generate glutathione (GSH) and internalize amino acids (AA).
A great number of accumulated data indicate that oxidative stress and inflammation are major contributing factors of renin-angiotensin system (RAS) imbalance both in peripheral and brain tissues [165,166]. The potential homeostatic role between kidney-brain crosstalk by RAS to regulate sodium/water balance and maintain normal blood pressure has been purported [167]. The first end-product of this system related to a biological activity is the octapeptide angiotensin (ANG) II, synthesized by the angiotensin converting enzyme (ACE). This pathway may also be involved in neurocognitive performance decline [168]. The role of RAS peptides in the interaction between the kidney and brain is supported by studies showing that treatment with ACE inhibitors and type I receptor (AT1) blockers, besides exerting renoprotection, also have beneficial actions in neurodegenerative disorders [169]. Dysfunctional RAS activation induces angiotensin II release, increasing systemic vascular resistance [170]. It has been established that treatment with captopril reduces oxidative stress and protects dopaminergic neurons in a 6-hydroxydopamine rat model of Parkinson's disease [171]. Analogue results were obtained with the administration of AT1 receptor antagonists in patients and in experimental models of Alzheimer's disease, Parkinson's disease, stroke, traumatic brain injury, and spinal cord injury [172]. Surprisingly, exogeneous H 2 S administration regulates renin release by downregulating the intracellular cAMP level in several cell types [173] and decreases protein expression of AT1R [174], resulting in renoprotection from kidney diseases. Moreover, H 2 S treatment inhibits the upregulation of renin level in renovascular hypertensive rats and reduces the intracellular cAMP level in primary cultures of renin-rich kidney cells [175]. In addition, H 2 S blocks forskolin-induced renin degranulation in mast cells by lowering the intracellular cAMP level, thus protecting against isoproterenol (ISO)-induced heart failure [176]. Likewise, H 2 S therapy protects multiple organs including the heart, kidney, and blood vessels and improves exercise capacity, coupled with inactivation of RAS in a murine model of transverse aortic constriction-induced heart failure [177]. Recent in vivo studies suggested that treatment with NaHS, increases renal H 2 S concentrations, restores NO bioavailability, and blocks RAS in the kidney, thus promoting vasodilatation to prevent the development of hypertension in rats [178]. In addition, H 2 S mitigates the development of diabetic nephropathy through suppressing RAS activity in diabetic rats [179].
As a rationale for neuropsychiatric disorders secondary to kidney damage, known as the "vascular theory", kidney and brain hemodynamic analogies occur, both being low resistance end organs exposed to high-volume blood flow and, consequently, more vulnerable to vascular damage [180]. Accordingly, magnetic resonance imaging (MRI) studies have shown high occurrence of silent brain infarction in patients with CKD, supporting the vascular theory [181]. Consistent with this, approximately 50% of CKD patients present ischemic white matter lesions in the MRI compared with 10% of the general population [182]. The loss of a direct correlation between known vascular risk factors, like diabetes and hypertension with CKD-related cognitive decline [183], the onset of neuropsychiatric comorbidities in pediatric patients with CKD preceding the vascular damage [184], as well as inconsistent findings regarding antihypertensive drugs' beneficial effects in cognition [185], may indicate that other mechanisms underlie CKD-associated brain dysfunctions. Cerebrovascular injuries in CKD have also been associated with the retention of uremic toxins along with electrolyte imbalance, which ultimately leads to neuropsychiatric diseases, especially cognitive impairment and dementia [186]. Importantly, high neurotoxin levels (up to 10-fold higher in CKD patients than in controls) of guanidino compounds were present in brain regions that play a determinant role in cognition, such as the thalamus, the mammillary bodies, and the cerebral cortex [187]. A recent study reported several uremic toxins that potentially mediate the interactions between kidney and brain, which in turn, may influence brain homeostasis. Convincingly, uric acid, indoxyl sulfate, p-cresyl sulfate, IL-1β, IL-6, TNF, and parathyroid hormone have a strong impact on cognition in uremic conditions [188]. Interestingly, uremic neurotoxic effects seem to be also mediated by guanidino compounds, including creatinine, guanidine, guanidinosuccinic acid, and methylguanidine. Indeed, some studies reported that guanidine compounds affect the CNS by inhibition of GABA (A) receptors and concomitant activation of N-methyl-d-aspartate (NMDA) receptors [189,190]. Moreover, other studies showed that uremic mice decrease central dopamine turnover in the striatum, mesencephalon, and hypothalamus, which was correlated with the impairment of motor activity [191].
In this scenario, H 2 S plays a beneficial role by inhibiting uremic toxins release in vitro and in vivo. During uremia, CSE activity and also H 2 S are significantly decreased in blood mononuclear cells from uremic patients on hemodialysis [192]. On the contrary, its metabolic-related compounds such as cystathionine, homolanthionine, and lanthionine are significantly raised [193]. CSE inhibition could be due to a uremic toxin, lanthionine, which is able to decrease H 2 S production in hepatoma cells. Recent in vitro studies suggested that slow-releasing H 2 S donors, such as diallyl disulfide (DADS) and diallyl trisulfide (DATS) enhance levels of alkaline phosphatase, osteopontin, osteocalcin, and collagen type I that are downregulated in human mesenchymal stem cells derived from serum of uremic patients in hemodialysis [194]. Although the potential role of uremic toxins in mediating the kidney-brain crosstalk has become clearer over the past years, the role exerted by H 2 S on these neurotoxic compounds and how it may directly or indirectly influence these compounds and restore the CNS function remain still elusive.
Moreover, hyperhomocysteinemia is a clinical hallmark in patients with CKD or AKI, in the latter setting often caused by ischemia-reperfusion. It causes arteriolar constriction, arterial stiffness, and endothelial damage [195]. H 2 S treatment in rodents attenuates renovascular damage by reducing hyperhomocysteinemia [196]. The pathophysiology of chronic kidney disease and neuropsychiatric disorders has been commonly associated with mechanisms related to the decreased availability of brain-derived neurotropic factor (BDNF) for renal and neuronal failure [197]. BDNF exerts traditional antidepressant actions, and its deletion in the hippocampus weakens antidepressant behavioral responses [198]. Interestingly, epidemiological and experimental studies point to a potential role of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) and BDNF in neuropsychiatric disorders, in particularly in depression [199]. BDNF regulates the growth and maintenance of the neuronal system as well as neuronal plasticity, like long-term potentiation of learning [200], which has been shown to be reduced in major depressive disorders [201]. Several preclinical and clinical data reported that an increase in ADMA infusion alone causes a marked reduction in serum BDNF levels leading to behavioral changes and depression of CKD in 11 hemodialyzed patients as well as in nephrectomized rats. Thus, ADMA is considered a uremic toxin that acts as an endogenous inhibitor of NO [197]. Recently, it has been reported that BDNF and tropomyosin receptor kinase B (TrkB) are expressed in podocytes and in the zebrafish model, being essential for actin polymerization and cell survival [202]. Moreover, a recent study demonstrated that BDNF is required for glomerular development, morphology, and function, and the expression of BDNF and KIM-1 is highly correlated in urine cells of CKD patients. Therefore, BDNF mRNA in urine cells could serve as a potential prognostic biomarker for CKD [203]. Some studies demonstrated that H 2 S reversed the decrease in TrKB receptors against chronic unpredictable mild stress (CUMS)-induced hippocampal oxidative stress, demonstrating the critical role of neurotrophic signaling in the antidepressant effects mediated by H 2 S [204]. These outcomes are consistent with premises that H 2 S exerted neuroprotective effects against formaldehyde-induced toxicity in PC12 cells [205] as well as against homocysteine-induced ER stress and neuronal apoptosis in the hippocampus of rat via the BDNF-TrKB pathway [206]. Taken together, the data indicate that the interactions between kidney and brain are complex and multifaceted, thus justifying the significant neuropsychiatric comorbidity observed in patients with CKD. Despite much research efforts, to date, kidney-brain crosstalk is still an area with excitingly few publications, especially as regards the molecular mechanisms underlying CDK and brain damage. Consistent with this, H 2 S signaling could represent a novel direct link between kidney and brain diseases, conferring protection, and limiting neuropsychiatric disorder occurrence in CKD patients.

H 2 S and Diabetes
H 2 S is abundant in kidney and is generated mainly by CBS and CSE. In the glomeruli, CSE is the main H 2 S-producing enzyme expressed by endothelial cells, mesangial cells, and podocytes [207], while both CBS and CSE have been reported to be expressed on renal proximal tubules [78]. H 2 S is important in kidney for its role in homocysteine metabolism, regulation of GFR and of urinary sodium and potassium excretion [76]. In a murine model of renovascular hypertension, NaHS, a donor of H 2 S, suppressed the upregulation of renin mRNA by downregulating cAMP levels [208]. Emerging evidence indicates an active role of H 2 S in diabetes and in diabetic nephropathy (DN). CSE expression is upregulated in liver and pancreas in streptozotocin-induced diabetic rats, a model for type-1 diabetes. Insulin treatment reduced CSE expression [81]. CSE is also overexpressed in pancreatic β-cells in Zucker diabetic fatty rats, a model for type-2 diabetes [209]. On the other hand, renal expression of CBS and CSE enzymes which synthetize H 2 S, has been reported downregulated both in patients and in animal models of diabetes [76]. Hyperglycemia impairs cell redox homeostasis increasing ROS generation, reducing the activities of endogenous antioxidants such as GSH and SOD and downregulating Nrf-2, which control the expression of protective enzymes [207]. In addition, hyperglycemia-induced oxidative stress reduced CBS and CSE expression by upregulating MMP-9. These effects have been reported in cultured mesangial cells [210] and in diabetic rats [211] treated with high glucose and were reversed by treatment with NaHS [78]. H 2 S have been also reported to attenuate renovascular remodeling in DN through its regulatory action on MPP-9 and NADPH oxidase 4 (NOX4) [212,213]. In renal epithelial cells, NaHS reversed the inactivation of AMPK, responsible for the cascade that leads to hyperactivation of mTOR and subsequent matrix deposition [214]. Later, it was demonstrated that H 2 S inhibits NOX4 expression and matrix deposition by activation of iNOS and NO production [94]. Interestingly NO in turn is able to induce CSE and H 2 S production, suggesting a crosstalk interaction between the two gasotransmitters [215]. H 2 S have demonstrated an antifibrotic effect in renal tubular epithelial cells by attenuating the TGF-beta1-induced epithelial-to-mesenchymal transition (EMT) through both ERK-dependent and β-catenin-dependent pathways [216]. In addition, H 2 S accelerates wound healing in diabetic rats [217]. H 2 S levels and H 2 S-producing enzymes were found decreased in plasma, urine, and kidney of aging mice. Chronic H 2 S donor (NaHS) treatment could attenuate oxidative stress levels and renal tubular interstitial collagen deposition and restores H 2 S production in aging kidney. These protective effects may refer to Nrf2 activation and transcription of antioxidant proteins, including HO-1, SIRT1, SOD1, and SOD2, which are upregulated in the ageing kidney after NaHS treatment [218].

H 2 S and Brain
In the brain, CBS is the main H 2 S-producing enzyme, present in both neurons and astrocytes [219], and it was expressed mainly in the hippocampus and cerebellum when compared with the cerebral cortex and brain stem [220]. Furthermore, H 2 S production in astrocytes is approximately 10-fold higher than in cultured microglial and neuronal cells, suggesting that astrocytes are the main brain cells producing H 2 S [1]. H 2 S and sulfhydration play significant roles in the optimal functioning of the nervous system. For instance, H 2 S takes part in the regulation of intracellular Ca 2+ in neurons and microglia, maintains pH homeostasis [76], and induces hippocampal long-term potentiation in active synapsis by activating N-methyl-D-aspartate (NMDA) receptors [221]. By regulating the NR2B subunit of NMDA receptors, NaHS treatment improved the cognitive dysfunction associated with hepatic ischemia/reperfusion in hippocampus of rats [222]. Various studies demonstrated the protective effects of H 2 S on neurons against oxidative stress [223]. H 2 S is unlikely to scavenge oxidants by itself due to its low endogenous concentration [83], whereas it seems to exert potent antioxidant effects indirectly regulating GSH production through the upregulation of the Keap1/Nrf2/ARE pathway. In addition, in vitro and in vivo models demonstrated that activation of Nrf2 in astrocytes provides protection also in neurons, prompting the hypothesis that this effect is the primary factor leading to neuroprotection of both cortical and motor neurons [1]. Notably, both D-Cys and L-Cys, substrates for H 2 S synthesis showed to protect the neurons of the cerebellum from hydrogen peroxide (H 2 O 2 )-induced oxidative stress [81]. In human cultured neuron cells, H 2 S inhibits peroxynitrite, acting as an endogenous peroxynitrite scavenger [1]. Dysregulation of H 2 S metabolism has been described in different neurological diseases.
Hu et al. showed reduced levels of H 2 S in striatum and substantia nigra in two different murine models of Parkinson's disease [224]. NaHS treatment attenuated neuronal loss, lipid oxidation, accumulation of inflammatory markers, and improved movement dysfunction [224]. In addition, H 2 S sulfhydration plays a relevant role in Parkinson's disease by activating the neuroprotective ubiquitin E3 ligase, parkin, responsible for clearance of toxic, misfolded proteins [82]. Parkin sulfhydration was found markedly depleted in the brains of patients with Parkinson's disease, suggesting that this loss may be pathologic and hydrogen sulfide donors may be therapeutic [225]. Likewise, H 2 S levels were found reduced in the plasma of patients with Alzheimer disease (AD) compared to controls, and H 2 S levels correlated negatively with the severity of the disease [226]. Depletion of H 2 S and concomitant increase in homocysteine levels in AD have been attributed to a lack of S-adenosylmethionine (SAM), an allosteric activator of CBS. Moreover, NaHS treatments in murine models of AD reduced oxidative stress through Nrf-2 and provided protection against homocysteine-induced cognitive dysfunction [227]. In addition, recent studies have shown that inhalation of H 2 S protected against PD-induced movement dysfunction and prevented neuronal apoptosis and microglia activation in the nigrostriatal region. CBS overexpression has also been reported to protect against the 6-hydroxydopamine-induced model of PD [228]. On the other hand, H 2 S production by CBS has been found elevated in amyotrophic lateral sclerosis (ALS) both in mice tissues and cerebrospinal fluid of ALS patients. Similarly, high H 2 S levels have been observed in trisomy of chromosome 21 trisomy (Down syndrome), on which the CBS gene is located [82]. It is well documented in the literature that H 2 S exerts either antioxidant and anti-inflammatory effects or prooxidant and pro-inflammatory effects depending on its local concentration. Following a bell-shaped dose-response curve, H 2 S produces protective effects at a lower concentration and a variety of deleterious/cytotoxic effects at higher concentrations [83,91]. The varying effects of H 2 S reported in several lines of evidence could be due to the dual effects of H 2 S [82]. This has been widely suggested in investigations concerning various types of inflammatory processes, vasorelaxation/tension in aortic tissue, cell proliferation/apoptosis, as well as in the case of tumor promotion and inhibition. Since the concentration of H 2 S can vary considerably both within and between tissues and under differing conditions, it is likely that H 2 S could affect a complex array of biological responses that may challenge the capacity to make definitive biomedical and/or clinical interpretations or predictions. Li and Moore [229] closed their recent insightful review of the role of H 2 S in health and disease with a statement probing their articulated biological conundrum of how does one molecule display such widely differing effects and could it be due to different effects at differing concentrations. The answer to this seminal question is found within the framework of a hormetic dose-response interaction that is independent of biological model, tissue, and endpoint [1].

H 2 S Redox Signaling and Resilience
Emerging evidence has highlighted the crucial role of H 2 S in maintaining redox homeostasis, which occurs by modulating levels of cellular antioxidant enzymes and increasing the expression of the transcription factor nuclear erythroid-related factor 2 (Nrf2) during oxidative stress. The latter is an intracellular excess of reactive oxygen species (ROS) relative to depletion of antioxidant capacity of the cell [230]. Interestingly, Nrf2 is a master regulator of redox cellular stress response in various pathological states [1,231]. Under physiological conditions, Nrf2 is localized in the cytosol and regulated by its inhibitor Kelch-like ECH-associated protein 1 (Keap1). Recently, much evidence has demonstrated that H 2 S induces cytoprotection against oxidative stress by the stimulation of Nrf2, which accumulates and translocates into the nucleus where it binds to the antioxidant response element (ARE) inducing the transcription of multiple target genes, including phase II detoxification enzymes such as NAD(P)H: quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1), thioredoxin, γ-glutamylcysteine synthetase, and glutathione S-transferase (GST) [232]. In line with this observation, Nrf2 encodes the vitagene antioxidant pathway which exists to counteract different forms of stress (e.g., oxidative, environmental, and mitochondrial stress). Vitagenes include heat shock protein 70 (Hsp70), heme oxygenase 1 (HO-1), γ-glutamylcysteine synthetase (γ-GCs), thioredoxin (Trx), and sirtuins (SIRTs) (Figure 3) [1,[233][234][235] as biomarkers for stress adaptation, cross-tolerance, and resilience underlying hormesis or preconditioning [236] (Figure 3). For instance, the benefits of hormetic stimuli are well documented, such as preconditioning, dietary restriction as well as intermittent fasting in regulating endogenous H2S production by the conserved trans-sulfuration pathway (TSP) in a dose-response manner during ischemia reperfusion (I/R) injury [237][238][239]. Moreover, H2S can also act as a mimetic of dietary restriction and extend lifespan in yeast and worms [240]. Intriguingly, a growing number of studies have demonstrated that H2S decreases the ROS level in cardiomyocytes under ischemia/reperfusion injury in the setting of diabetes by relieving oxidative stress, and the ability of H2S to upregulate cellular antioxidants in the heart in a Nrf2-dependent manner [241,242]. Especially H2S-mediated cardioprotection increases in antioxidant (i.e., HO-1, Trx, Hsp70, and Hsp90) and anti-apoptotic (i.e., Bcl-2, Bcl-xL, and COX-2) signaling in a mouse model of pharmacological preconditioning [241]. These findings are consistent with the integrated view that cellular resilience is mediated by many distinct pathways that converge upon mitochondria to supply the extra energy for building resilience. Accordingly, several research groups have characterized the effect of H2S on mitochondrial activity and cellular bioenergetics [242][243][244]. These studies mentioned above demonstrated that H2S induces a U-shaped biological doseresponse concentration typical of hormetic compounds in in vitro and in vivo models, with activation at lower concentrations and inhibition at higher concentrations [245]. Thus, the toxic and therapeutic effects of H2S depend on its endogenous concentration. Specifically, H2S is synthesized endogenously and mainly metabolized by a mitochondrial sulfide-oxidizing pathway including sulfide quinone oxidoreductase (SQR) and utilizes GSH as a thiophilic acceptor to produce GSSH [246]. Therefore, H2S produced in the mitochondria scavenges ROS [156]. In principle, H2S is a potent inhibitor of oxidative phosphorylation (OXPHOS) by its well-known ability to inhibit cytochrome c oxidase (COX) [247]. On the other hand, H2S therapy has been observed to preserve mitochondrial function in the heart muscle of rodents after I/R injury [238,248]. It has been well established that a low level of H2S administration increases the phosphorylation of protein serine/threonine kinase B (Akt) and For instance, the benefits of hormetic stimuli are well documented, such as preconditioning, dietary restriction as well as intermittent fasting in regulating endogenous H 2 S production by the conserved trans-sulfuration pathway (TSP) in a dose-response manner during ischemia reperfusion (I/R) injury [237][238][239]. Moreover, H 2 S can also act as a mimetic of dietary restriction and extend lifespan in yeast and worms [240]. Intriguingly, a growing number of studies have demonstrated that H 2 S decreases the ROS level in cardiomyocytes under ischemia/reperfusion injury in the setting of diabetes by relieving oxidative stress, and the ability of H 2 S to upregulate cellular antioxidants in the heart in a Nrf2-dependent manner [241,242]. Especially H 2 S-mediated cardioprotection increases in antioxidant (i.e., HO-1, Trx, Hsp70, and Hsp90) and anti-apoptotic (i.e., Bcl-2, Bcl-xL, and COX-2) signaling in a mouse model of pharmacological preconditioning [241]. These findings are consistent with the integrated view that cellular resilience is mediated by many distinct pathways that converge upon mitochondria to supply the extra energy for building resilience. Accordingly, several research groups have characterized the effect of H 2 S on mitochondrial activity and cellular bioenergetics [242][243][244]. These studies mentioned above demonstrated that H 2 S induces a U-shaped biological dose-response concentration typical of hormetic compounds in in vitro and in vivo models, with activation at lower concentrations and inhibition at higher concentrations [245]. Thus, the toxic and therapeutic effects of H 2 S depend on its endogenous concentration. Specifically, H 2 S is synthesized endogenously and mainly metabolized by a mitochondrial sulfide-oxidizing pathway including sulfide quinone oxidoreductase (SQR) and utilizes GSH as a thiophilic acceptor to produce GSSH [246]. Therefore, H 2 S produced in the mitochondria scavenges ROS [156]. In principle, H 2 S is a potent inhibitor of oxidative phosphorylation (OXPHOS) by its well-known ability to inhibit cytochrome c oxidase (COX) [247]. On the other hand, H 2 S therapy has been observed to preserve mitochondrial function in the heart muscle of rodents after I/R injury [238,248]. It has been well established that a low level of H 2 S administration increases the phosphorylation of protein serine/threonine kinase B (Akt) and enhances the nuclear localization of two transcription factors, nuclear respiratory factors-1 and -2, which are involved in increasing the levels of endogenous antioxidants, attenuating apoptosis, increasing mitochondrial biogenesis, and confer resilience against cellular stress [249]. Recently, the modulation of vitagenes elicited through H 2 S antioxidant molecule has taken on a considerable importance in preserving redox homeostasis, mitochondrial stress, protein quality control, and enhancing resilience in a hormetic dose-response manner during metabolic diseases such as diabetes and its complications. In this context, H 2 S provides antioxidant function by enhancing γ-glutamyl synthetase activity and upregulation of cysteine transport, restoring the levels of GSH in glutamate-mediated oxidative stress [148]. Some experimental studies suggested that administration of H 2 S attenuates high glucose-induced elevation in ROS production in renal mesangial cells and diabetic rat kidneys [250]. In addition, H 2 S treatment exerts protective effects in the kidney of type 1 diabetic rats, related to the suppression of oxidative stress through upregulation of superoxide dismutase (SOD) activity [251]. Interestingly, H 2 S mitigates diabetic renal damage by blocking mitochondrial Ca 2+ permeability through the N-methyl-d-aspartate receptor-R1 (NMDA-R1) pathway [252]. Based on the concept of hormesis [253], in which low levels of H 2 S confer cytoprotective actions against oxidative stress, it can also cause toxicity when present in high concentrations [1,254]. Under oxidative stress, reactive sulfur species (RSS) are generated that can act as aggressive oxidizing agents. This phenomenon is mainly due to the high concentration of the H 2 S signaling molecule [255]. Thus, efforts have been made to identify suitable exogenous H 2 S donors. Indeed, some in vitro studies indicate that a high concentration of 0.1 and 1 mM NaHS induces pro-oxidant effects such as lipid peroxidation and protein carbonylation in human plasma [256]. In addition, in vivo studies demonstrate that NaHS improves diabetic state-induced muscle atrophy by increasing Akt/mTOR signaling and decreasing the expression of myostatin and the FoxO1/MuRF1/atrogin-dependent pathway [257]. Recent studies revealed a novel H 2 S-induced posttranslational modification, termed protein S-sulfhydration (also known as S-persulfidation) [87,258]. During this process, the -SH group of cysteine residues becomes covalently converted to a -SSH group, which can result in changes in the activity of the protein. This process importantly contributes to physiological and pathophysiological H 2 S-signaling. In this context, the S-sulfhydration reaction with H 2 S or hydrogen polysulfide (H 2 Sn) participation plays a crucial regulatory role in the biogenesis of RSS from endogenous and exogenous precursors and on regulatory properties of this reaction. On the other hand, these reversibly oxidized -SH groups are under the control of intracellular antioxidant pathways such as glutathione (GSH), cysteine (Cys), and thioredoxin (Trx) that actively participate to restore redox H 2 S homeostasis [259]. It is noteworthy that excessive levels of H 2 S can be countered by activation of the sulfide oxidation pathway, which involves the enzyme sulfide quinone oxidoreductase that oxidizes H 2 S and reduces coenzyme Q [260]. Several studies have reported that S-sulfhydration is one mechanism where H 2 S interacts directly with the Nrf2 pathway. In this respect, H 2 S has been shown to S-sulfhydrate Keap1 at the cysteine-151 residue, leading to Nrf2 dissociation and increased nuclear translocation and expression of antioxidant genes through binding to promoters' ARE sites [232]. Furthermore, H 2 S can S-sulfhydrate Keap1 at the cysteine-226 and cysteine-613 residues, leading to Keap1 inactivation, Nrf2 release, and activation of Nrf2-dependent gene expression [261]. Additionally, NaHS upregulated Nrf2 nuclear translocation, and the transcription of the two key downstream antioxidant genes peroxiredoxin-1 and NAD(P)H dehydrogenase quinone 1 [262]. Most studies indicate that insufficient endogenous H 2 S concentration is implicated to increase oxidative stress leading to type 2 diabetes in mice and in humans [263,264]. Conversely, exogenous H 2 S promoted antioxidant effects by increasing Keap-1 and suppressing its ubiquitination, facilitating ubiquitin aggregate clearance via autophagy in the hearts of db/db mice [265]. In addition, recent studies demonstrated that H 2 S, in the form of a NaHS donor, may reduce high glucose-induced oxidative stress, inflammation, and apoptosis by activating the Nrf2/ARE pathway and may exert anti-apoptotic effects in diabetic myocardium by inhibiting c-Jun N-terminal kinase (JNK) and p38 MAPK pathways and activating PI3K/Akt signaling in vitro and in vivo [266]. Nevertheless, other recent studies showed that slow-releasing H 2 S donor, GYY4137, induces cardioprotection against myocardial ischemia and reperfusion injury by attenuating oxidative stress and apoptosis via activation of the PHLPP-1/Akt/Nrf2 pathway in mice [267], inhibition of the STAT3/HIF-1α signaling pathway [268], and activation of the AMPK (5 AMP-activated protein kinase)/mTOR signal pathway in high glucose-induced H9c2 cardiomyocyte damage [269]. Moreover, the GYY4137 donor exerts anti-inflammatory effects by suppression of the NF-kB and MAPK signaling pathway in Coxsackie virus B3-infected rat cardiomyocytes [270] as well as attenuating the development of diabetic cardiomyopathy via FoxO1 signaling pathway in vitro and in vivo [271]. Recent findings reported that the H 2 S-Nrf2-antioxidant proteins axis protects renal tubular epithelial cells and rescues cells from the native hibernator and from lipid peroxidation-mediated cell death under reoxygenation conditions [272]. Recently, Kimura et al. provided novel insights into generated potential redox regulators cysteine-and glutathione-persulfide species (Cys-SSH, GSSH) as well as signaling molecules such as H 2 S 2 and H 2 S 3 produced by 3MST in the presence of physiological concentrations of cysteine and glutathione, to maintain neuronal transmission, vascular tone, cytoprotection, inflammation, and oxygen-sensing [273]. In this scenario, it has been shown that H 2 S 2 along with H 2 S 3 shields neuronal cells from oxidative as well as carbonyl stress through exerting reduced synthesis of glutathione [101]. Additional studies suggested that H 2 Sn rather that H 2 S regulates the activity of the tumor suppressor phosphatase and tensin homolog (PTEN) and reduces blood pressure by dilating vascular smooth muscle [274] through the modulation of the Nrf2 pathway [275]. Interestingly, H 2 Sn activates transient receptor potential ankyrin 1 (TRPA1) channels by sulfhydrating two cysteine residues at the amino terminus of the channels [276] and the species also facilitates the translocation of Nrf2 to the nucleus to upregulate antioxidant genes by sulfhydrating its binding partner Keap1 to release Nrf2 [275]. It is well known that decreased endogenous H 2 S levels, redox imbalance, and oxidative damage are closely correlated with disease severity and progression in cardiac, neurological, pulmonary, gastric, nephrological, hepatic diseases, as well as in aging. Notably, Xie and coworkers detect significantly lower levels of plasma H 2 S in diabetic mice that is corrected by administration of exogenous H 2 S donor GYY4137 [277]. Intriguingly, GYY4137 exerts anti-atherogenic [277] and inflammatory effects [278] via the Nrf2/HO-1 pathway in mice. In addition, H 2 S induced NRF2 activity, the upregulation of antioxidant genes HO-1, Trx-1, and GSH, and the reduction in inflammation response by suppressing the NF-κB pathway [279] as well as apoptosis in rat models [280]. In these conditions of inflammation, activation of the cellular stress response represents an essential system that requires upregulation of the antioxidant vitagene pathway to preserve resilience and redox homeostasis of the cell in various pathological conditions [1,[281][282][283]. Within this context, silent mating type information regulator 2 homolog 1 (SIRT1) is a NAD+-dependent deacetylase of lysine residue of the target protein. Mammals have seven different sirtuins, SIRT1-SIRT7 [284]. SIRT1 extends lifespan [285] and improves cell tolerance to inhibit environmental stress [286]. Recently, it has been shown that H 2 S is a novel SIRT1 activator by direct sulfhydration. Numerous experimental studies in vitro and in vivo indicate protective effects of H 2 S signaling through activation of Sirtuin's family in different pathologies and, in particular, in type 2 diabetes and related complications. In this regard, NaHS increased SIRT1 and reversed biochemical, apoptotic, oxidant, and pathologic parameters characteristic of diabetic nephropathy, at a dose of 100 µmol/kg/day [287]. In vivo studies reported that hydrogen sulfide ameliorated reperfusion-induced oxidative stress and mitochondrial dysfunction via activation of Sirtuin3 signaling, thereby decreasing lung ischemia-reperfusion damage in rats with a model of type II diabetes [288]. Endogenous CSE/H 2 S directly sulfhydrated SIRT1, enhanced SIRT1 binding to zinc ion, then promoted its deacetylation activity, and increased SIRT1 stability, thus reducing atherosclerotic plaque formation [289]. In vitro studies suggested that H 2 S attenuates CSE-induced cellular senescence and apoptosis by improving mitochondrial function and reducing oxidative stress in alveolar epithelial cells [290] as well as protects against hyperglycemia-induced neuronal senescence and neurotoxicity in the mouse hippocampal cell line [291] via upregulation of the SIRT1 pathway. Compelling evidence reported that H 2 S plays a peculiar role in protecting the ageing kidney from antifibrosis and anti-apoptosis through the regulation of redox homeostasis. Consistent with this, SIRT1, which emerges as a major lifespan regulator, has been widely investigated in the cardiovascular system and nervous system, but it is rare in the urinary system. Recent studies have demonstrated that a low concentration of NaHS (25 µmol/L) could directly induce SIRT1 activation in a cell-free system, whereas chronic treatment of NaHS (50 µmol/kg/day) selectively improves the expression but not the activity of SIRT1 in the ageing kidney. SIRT1 also regulates lipid metabolism by modulating a great variety of signaling pathways such as PPAR-α, LXR, FXR, and SREBP signals [292]. It is noteworthy that SIRT3 is a crucial regulator of mitochondrial function. SIRT3 catalyzes the deacetylation of mitochondrial proteins, which in turn affects mitochondrial energy metabolism. SIRT3 is regulated by nutritional status and metabolic stress. In this context, a recent in vivo study has demonstrated that exogenous H 2 S supplement attenuated isoproterenol-(ISO-)-induced myocardial hypertrophy through SIRT3 protein in mice [293]. It is well observed that chronic NaHS treatment modulates Nrf2 downstream genes such as HO-1, SOD1, and SOD2, consequently enhancing resistance to oxidative stress in the ageing kidney. Chronic exogenous H 2 S treatment could protect the ageing kidney by reducing oxidative stress, decreasing collagen deposition, and enhancing Nrf2 nuclear translocation, as well as increasing endogenous H 2 S production. Reactive oxygen species are constantly generated during metabolic diseases such diabetes. Among various signals activated by ROS, the thioredoxin pathway is among the extensively investigated signaling cascades that mediate oxidative cell proliferation, inflammation, senescence, and survival [294,295]. Trx represent the primary defense mechanism against oxidative stress. It contains two redox-active cysteine residues that protect protein against unwarranted oxidant-mediated inter-or intra-molecular disulfide bond formation. Some studies have demonstrated that H 2 S exerts antioxidative effects on the cells through the regulation of the redox state of Trx and interference with the ASK1/P38 signaling pathway [296]. Importantly, H 2 S modulates cellular redox signaling via direct S-sulfhydration of the Nrf2-Trx pathway. Consistent with this notion, Trx is the major regulator enzyme of intracellular persulfidation that cleaves disulfides in proteins and acts as an S-denitrosylase [297]. Especially, cysteine-32 within Trx is responsible for the direct interaction of Trx and S-sulfhydrated proteins following the breakage of the hydropersulfide group [298]. Likewise, Trx can desulfhydrate the protein tyrosine phosphatases 1B (PTP1B), restoring endoplasmic reticulum (ER) stress response in an in vitro setting [299]. Several studies revealed that H 2 S preconditioning could protect mice against cerebral I/R injury by the induction of HSP70 and the PI3K/Akt/Nrf2 pathway [300]. The heme oxygenase 1 (HO-1), also referred to as Hsp32, is induced by various oxidative stimuli, including ROS, RNS, and RSS. Accordingly, HO-1 has been recognized also as dynamic sensors of cellular oxidative stress and modulators of redox homeostasis throughout the phylogenetic spectrum. In this light, H 2 S exerts a protective role by upregulating the antioxidant enzyme HO-1 expression in human kidney cells [301] (Figure 4).
In addition, recent in vivo studies reveal the nephroprotective effects of H 2 S on renal tissue through upregulation of antioxidant proteins and anti-inflammatory cytokines, as well as the expression of eNOS and iNOS via induction of the Nrf2/HO-1 pathway in renal injury [302] and in the spinal cord of rats [303]. Moreover, H 2 S could attenuate high glucose-induced myocardial injury in rat cardiomyocytes by suppressing the Wnt/β-catenin pathway and upregulating the expression of HO-1 and NQO1 [304]. In addition, H 2 S protected renal tissue against ischemia-reperfusion injury-induced lipid peroxidation, inflammation, and apoptosis, which may be attributed to the upregulation of HSP 70, HO-1, and HSP 27 [305]. H 2 S reduces myocardial fibrosis in diabetic rats, which is related to the inhibition of protein kinase Cα (PKCα), upregulation of HSP70 expression [306], and downregulation of the JAK/STAT signaling pathway [307]. Finally, H 2 S protects cells from oxidative stress [308] and delays programmed cell death by increasing the levels of antioxidant glutathione and HO1 expression [309]. Taken together, the data above convincingly indicate the crucial role of H 2 S as a potent antioxidant molecule that, at low concentrations, induces protective actions exploited through the redox modulation of the Nrf2 vitagene signaling pathway which may provide a novel potential therapeutic approach to confer resilience against oxidative stress, inflammation as well as apoptosis during pathological conditions such as diabetes and related complications.
Especially, cysteine-32 within Trx is responsible for the direct interaction of Trx and S-sulfhydrated proteins following the breakage of the hydropersulfide group [298]. Likewise, Trx can desulfhydrate the protein tyrosine phosphatases 1B (PTP1B), restoring endoplasmic reticulum (ER) stress response in an in vitro setting [299]. Several studies revealed that H2S preconditioning could protect mice against cerebral I/R injury by the induction of HSP70 and the PI3K/Akt/Nrf2 pathway [300]. The heme oxygenase 1 (HO-1), also referred to as Hsp32, is induced by various oxidative stimuli, including ROS, RNS, and RSS. Accordingly, HO-1 has been recognized also as dynamic sensors of cellular oxidative stress and modulators of redox homeostasis throughout the phylogenetic spectrum. In this light, H2S exerts a protective role by upregulating the antioxidant enzyme HO-1 expression in human kidney cells [301] (Figure 4).   (4), dipeptidase (5), oxoprolinase (6), γ-glutamyl-cysteine synthase (1), and glutathione synthetase (2) operate in the Meister cycle to generate glutathione (GSH) and internalize amino acids (AA). GSH interacts with Cystathionine-β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3MST) to produce H 2 S.

Analytical Approaches to Quantify H 2 S in Biological Matrices
Hydrogen sulfide, present in mammalian tissues, plays a vital role in physiological and pathophysiological processes. The long identified toxic gas, which has also been confirmed as the third gaseous signaling molecule following NO and CO, plays important roles in various physiological and pathological processes. However, striking differences with orders of magnitude were observed for the detected hydrogen sulfide concentrations in biological matrices among different measurements in the literature, which lead to the uncertainty for examination of the biological relevance of hydrogen sulfide. The monitoring of the quality control of H 2 S donor drugs and the study on the pathophysiology and pharmacology mechanism of H 2 S on experimental animals and even on humans require for detection methods with high sensitivity, selectivity, precision, and accuracy. Various techniques for the analysis of low concentrations of H 2 S have been developed over the past 50 years. Some of the older methods are still used by health departments, but due to their very low sensitivity, they cannot be used in the quantification of very low concentrations of this gas in mammalian tissues useful for scientific research. Also, due to the variety of chemical species with multiple properties, make it difficult accurate and reliable measurements of hydrogen sulfide in biological matrix [310]. Among the various methods that have been established for the measurement of endogenous hydrogen sulfide, the most widely used are: colorimetry [311], gas chromatography [312], electrochemical measurements by electrodes selective for sulfide (ion-selective electrodes, ISEs) [313], polarographic sensors [314], estimation by fluorescent probes [315], and high performance liquid chromatography (HPLC coupled with ultraviolet, fluorescence or electrochemical detection) [316,317]. Currently, fluorescence derivatization is the most used method. Hydrogen sulfide has two possibilities to perform nucleophilic substitution on fluorescence probes to form a fluorescent derivative that can be detected using a fluorimeter coupled with high performance liquid chromatography (HPLC) [318,319]. For example, monobromobimane (MBB) is a common fluorescent reagent that reacts rapidly and completely with thiol groups [320,321]. However, these methods have obvious limitations, such as complex preparation processes, low specificity and sensitivity, and time-consuming procedures [310]. Another analytical technique considered most effective today for the analysis of substances at such low concentrations is MS/MS, the analysis with triple quadrupole mass spectrometry in particular. Due to its low molecular weight and simple chemical structure, hydrogen sulfide is not suitable for direct quantification by triple quadruple mass spectrometry and thus, chemical derivatization was used before detection by LC-MS/MS [322][323][324]. Compared to all other methods, HPLC-MS/MS methods can determine concentrations in the order of 10 −9 -10 −10 molar, the derivatization reactions allow high selectivity, and with the use of deuterated standards, it can calculate the recovery very simply. Therefore, in particular as regards those studies aimed to define the signaling role of H 2 S, HPLC-MS/MS methods, based on derivatization probes, are the most promising methodology to address the need to detect very small amounts of H 2 S in mammalian tissues and in other biological matrices.

Conclusions
This evaluation has demonstrated that both H 2 S and carnosine can modulate oxidative stress and inflammation in the brain and the kidney, suppressing damage interactive synergies between these two organs. Substantial evidence has emerged that these two agents independently activate the Nrf2 transcription factor, which then leads to the formation of acquired resilience following the quantitative features of the hormetic dose response. These processes also may involve the occurrence of transcription factor crosstalk that is both dose-and biological context-sensitive. The process is a general one, and is proposed to account for observations that hormetic effects are independent of biological model, cell type, level of biological organization, inducing agent, and endpoint measured. The emerging findings of H 2 S and carnosine are consistent with similar mechanistic and dose response findings for a wide range of chemopreventive agents such as sulforaphane and EGCG that mediate their protective effects via Nrf2 activation within the context of the hormetic dose response.

Conflicts of Interest:
The authors declare no conflict of interest.