THE ROLE OF HYDROGEN SULFIDE IN PATHOLOGIES OF THE VITAL ORGANS AND ITS CLINICAL APPLICATION

Hydrogen sulfide (H2S) is a colourless gas with a strong odor of rotten eggs (1). It can be oxidised into sulfur, sulfur oxide as well as sulfates, best known for its toxic effects which range from eye irritation to causing rapid unconsciousness and cardiac arrest (2). However, it has been discovered that H2S is also produced in living organisms and possesses roles beyond its toxic properties as an environmental pollutant.

Similarities have been drawn between H2S and the other gaseous transmitters, such as nitric oxide (NO), carbon monoxide (CO) and ammonium, where they have been demonstrated to be important gaseous mediators in inflammation and sepsis (3-5). They serve as important autocrine and paracrine messengers. NO and CO with the addition of hydrogen cyanide, have been reported to inhibit mitochondrial cytochrome oxidase (6). It was found that H2S could both induce and inhibit NO production, depending on its concentration (7, 8). The interaction between these gaseous transmitters is beyond the scope of this paper and has been explored in recent reviews (5, 9, 10).

Several studies have indicated the roles of H2S in regulating vascular tone and its possible associations with inflammation and disease progression. However, research of H2S is still at its infancy, and the understanding of the mechanisms behind its role still limited. Nevertheless, many researchers have proposed the use of H2S donors and inhibitors as possible therapeutic agents in different disease conditions.

Due to the breadth of the studies available concerning the pathophysiological effect of H2S, it is not possible to include and critically appraise all the information available.

In this article, we will summarise the pathophysiological effects of the H2S on the central nervous system, cardiovascular system, lungs, liver and kidneys, focusing on conditions where H2S demonstrates therapeutic potential.

HYDROGEN SULFIDE SYNTHESIS (Fig. 1)

The production of H2S involves the conversion of L-cysteine to H2S via the action of two enzymes, cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) (11). H2S can also be produced from cysteine through a third enzyme, 3-mercaptopyruvate sulphurtransferase (3-MST). This particular pathway involves another enzyme, cysteine aminotransferase, which generates 3-mercaptopyruvate from cysteine and α-ketoglutarate. 3-MST then produces H2S from 3-mercaptopyruvate. 3-mercaptopyruvate contributes sulfur to the active-site cysteine residue of 3-MST to form persulfide, which releases H2S in the presence of dithiothreitol (12). However, dithiothreitol is not physiologically freely available. A recent study by Yoshinori et al. demonstrated the effects of 2 physiological reducing disulfides, thioredoxin and dihydrolipoic acid in releasing H2S from persulfide at the active site of 3-MST (13).

Fig. 1. Pathway of hydrogen sulfide synthesis. 3-MST, 3-mercaptopyruvate sulphurtransferase; CAT, catalase; CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase; H2S, hydrogen sulfide.

Hydrolisation of H2S yields hydrosulfide and sulfide ions in the following reactions: H2S ⇔ H+ + HS- ⇔ 2H+ + S2–. At pH 7.4 in an aqueous solution, approximately one third of H2S remains undissociated and is permeable to plasma membrane (1). The reports of different concentration levels of H2S in mammals are dependent on the methods used to determine its concentration. These methods include spectophotometric measurement of methylene blue formed from the reaction of N-dimethyl-p-phenylenediamine with sulfide, or alternatively, sulfide anion measurement with an ion-selective electrode. Olson et al. (14) discussed these methods in detail and their respective drawbacks. In mammals, it has been proposed that H2S has a circulating concentration of between 1 and 300 µM (15-17) in blood, and concentrations in the 50–160 µM range (18) in the brain, although the true concentrations are likely to be lower than the values mentioned above. H2S is also present in the lumen of human large intestine at millimolar concentrations, but the level of unbound sulfide is in the micromolar range due to the binding of sulfide to faecal components (19). Even after systemic administration of H2S donors (Table 1) at doses that produce pharmacological effects, plasma H2S concentrations rarely rise above the normal range, or only for brief periods of time (20).

Table 1. List of hydrogen sulfide donors, hydrogen sulfide producing enzymes and their inhibitors (29, 36, 42, 43, 46).

GENERAL FUNCTIONS OF HYDROGEN SULFIDE

Physiologically, H2S is an endogenous gaseous transmitter and an important signaling molecule involved in inflammation (21-24). H2S is able to stimulate and inhibit the mitochondrial electron transport, depending on its concentrations. It has been reported to act as both substrate, at low concentration (<5 µM)(25); and inhibitor, at high concentration (>20 µM) (6) for the cytochrome oxidase system. H2S has also been demonstrated to regulate L and T type calcium channels and open KATP channels.(26-29). H2S also possesses anti-oxidative properties, increasing superoxide dismutase (SOD) activity, glutathione (GSH) turnover, decreasing reactive oxygen species (ROS) production; and anti-apoptotic properties by inhibiting nuclear factor kappa beta (NF-κB) pathways, mitogen-activated protein kinase p38 (MAPK p38), C-Jun N-terminal kinase (JNK), Bcl-2 associated X protein (BAX) and caspase 3, as well as up regulating Bcl (30, 31). Interestingly studies have also demonstrated that H2S can mediate its cytoprotective excitation of the sensory nerve supplying the tissue (29).

Previous studies exploring the roles of H2S have used its donors and inhibitors of its producing enzymes to investigate the effects of H2S as well as its inhibition. For instance, DL-propargyl glycine (PAG) is an irreversible inhibitor of CSE and, when administered to rodents, produces an almost complete inhibition of the activity of this enzyme (32). The examples of H2S donor, H2S enzymes and their inhibitors are listed in Table 1.

Increase in H2S level has been associated with reduced inflammation in various organ systems, such as in inflammatory bowel disease, particle induced airway irritation and neuroinflammation (33-35). It has demonstrated the ability to reduce oedema formation and leukocyte adherence to the vascular endothelium, and to inhibit pro-inflammatory cytokine synthesis (20). Some studies even demonstrate the organ protective effects of H2S against lipopolysaccharides (LPS) exposure (36, 37). However numerous studies have found that in sepsis, H2S administration significantly worsens the inflammation (38, 39). This is associated with significantly increased histological deterioration in various organs and worse disease outcome (40, 41). Inhibition of H2S production significantly reduces pathology severity and overall survival, which would implicate theraputic benefit of H2S production inhibition in sepsis (42, 43). The effects of H2S in endotoxaemia and sepsis related inflammation have been discussed in great detail in other reviews (20, 22, 24, 38, 39, 42, 44). As such we will not attempt to discuss this topic in detail.

The molecular targets of H2S are summarised in Fig. 2.

Fig. 2. General ‘pharmacological’ functions of hydrogen sulfide.
BAX, Bcl-2 associated X protein; CO, carbon monoxide; ERK, extracellular-signal-regulated kinase; GSH, glutathione; GSK3b, glycogen synthase kinase 3 beta; H2S, hydrogen sulfide; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; NF-κB, nuclear factor kappa beta; Nrf2, NF-E2 p45-related factor 2; ROS, reactive oxygen species; SOD, superoxide dismutase; TRPV-1, transient receptor potential cation channel subfamily V member 1.

CARDIOVASCULAR SYSTEM

Hydrogen sulfide is synthesised in the cardiovascular system by CSE and 3-MST, but not by CBS which are found in various blood vessels including the aorta and portal vein (45-47).

Hydrogen sulfide is involved in a number of physiological and pathological processes in the cardiovascular system (28, 48). At low concentrations, exogenous H2S acts as a vasodilator and antihypertensive. Zhao et al. showed that NaHS given at 2.8 µM/kg is enough to cause a measurable reduction in the systolic blood pressure and significant vasodilation (49), while the knockout of the CSE gene increases blood pressure (50). A number of studies have looked into the possible benefit of H2S in hypertension and found that H2S donor administration significantly reduced blood pressure as well as the associated renal injury (51-53). At higher doses, H2S significantly reduced the rate and amplitude of cardiomyocyte action potential, while increasing the rate of repolarisation and significantly reduces pacemaker firing. These studies found significantly reduced potassium and calcium currents across cardiomyocytes, and suggested that the electrophysiological changes are probably due to H2S interaction with the KATP and L type Ca2+ channels (54-56).

At low doses, H2S also promotes endothelial proliferation and angiogenesis. Cai et al. showed that with 10–20 µM NaHS, endothelial cells showed increased proliferation, migration, branching and endothelial tube formation (57). The therapeutic potential of this property has been demonstrated in diabetic wound healing and in heart failure. Liu et al. showed that in diabetic mice administration of H2S donor increased the rate of angiogenesis and the rate of wound closure, and administration of PAG resulted in the opposite (47). Givvimani et al. demonstrated that H2S administration in heart failure mouse model increases angiogenesis and results in better overall ventricular function. Possible mechanisms include Akt phosphorylation, survivin, angiopoietin-1 (ANG1), vascular endothelial growth factor (VEGF) expression and inhibition of angiostatin, endostatin and matrix metalloproteinases (MMP) (58).

However, the pro-proliferative effect is completely abolished with NaHS doses of 100 uM or higher. Indeed, at a higher concentration, H2S exerts an anti-proliferative and pro-apoptotic effect on cardiomyocytes, vascular smooth muscles and endothelial tissue. In CSE over-expressing tissue, proliferation rate and viability are significantly lower, and apoptotic rate is significantly higher (59). Similar effects have been found with regard to aortic smooth muscle cells, vascular endothelium and atrial cardiomyocytes that were exposed to high levels of NaHS (54, 60, 61). These findings were associated with higher activation of extracellular-signal-regulated kinase (ERK), and caspase 3 activities, increased p38 MAPK phosphorylation and reduced cyclin D activity. These perhaps reflect on the ability of H2S to inhibit cardiac remodeling in response to pathology.

It has been suggested that H2S over-expression may play a role in diseases with undesirable angiogenesis, and lowering H2S level may prove therapeutic. Ran et al. showed that patients with proliferative diabetic retinopathy have significantly higher H2S levels than those that have non-proliferative retinopathy (62). Szabo et al. showed that colon cancer cells over-produce H2S, and inhibiting CBS activity decreases angiogenesis and tumor growth (63).

Hydrogen sulfide is also thought to exert protective effects on ischaemia related insults. NaHS administrated at a moderate level of 20–50 µM, before hemorrhagic shock, is enough to significantly improve histological appearance of the heart and haemodynamic stability (64-66). In ischaemic-reperfusion injury, pre-treatment with H2S either immediately or 20 hours before the injury results in less cell damage and apoptosis, histological change and better contractility (67-69). This is associated with reduced BAX and caspase 9 activation, reduced cytokine release and reduced reactive oxygen species (ROS) generation. In addition to that, it is thought that the anti-apoptotic effect is due to the upstream effect of H2S on protein kinase C (PKC) and ATP-sensitive potassium channels (KATP), which causes the phosphorylation of ERK1/2, as well as the phosphorylation of glycogen synthase kinase 3 beta (GSK3β) and the H2S-NO synthase interaction (69). H2S is also thought to contribute to the ischaemic preconditioning (IP) phenomenon - Huang et al. showed that H2S level rises significantly with IP and ischaemic postcoditioning (IPO), while inhibition of H2S synthesis with PAG in heart tissue abolishes the effect of IP and IPO (70). It is suggested that H2S exerts the protective effect via ERK 1/2 activation (68).

Hydrogen sulfide has also demonstrated anti-atherosclerotic properties. Mani et al. showed that CSE knockout mice developed more marked dyslipidemia, atherosclerotic change, increased oxidative stress and adhesion molecule expression, this was reversed by administering NaHS supplement (71). NaSH administration reduces the size of atherosclerotic plaque formation, whereas PAG increases formation (72). S-aspirin has also demonstrated anti-atherosclerotic benefit (73). In a rabbit model of atherosclerosis, restenosis after percutaneous angioplasty was significantly lower with NaHS administration and higher with PAG administration (74). This is the result of decreased oxidative low density lipoproteins (LDL) modification (75), reduced tumor necrosis factor alpha (TNF-α) and intercellular adhesion molecule (ICAM) expression by macrophage (76) and decreased foam cell formation (77).

A number of studies looked into the use of H2S as a marker of cardiovascular disease in humans. Jiang et al. demonstrated that patients with angina have significantly lower levels of plasma H2S compared to a healthy population, and unstable angina patients have even lower H2S levels than stable angina patients (78). In addition to this, multiple vessel pathology, complete occlusion, co-existing hypertension and smoking are all factors associated with lower H2S levels. Kovacic et al. demonstrated that in patients with congestive heart failure, plasma H2S level is closely correlated with the staging of the disease, and its lower level is also a good predictor of mortality and hospitalisation (79).

RESPIRATORY SYSTEM

In the respiratory system, CSE and CBS are expressed in the airway and vascular smooth muscle cells (2).

Hydrogen sulfide administration in airway tissue is associated with anti-proliferative effects. Administration of H2S donor inhibits the proliferation and migration of both smooth muscle cells and fibroblasts, and inhibits the transformation of fibroblasts into myofibroblasts. This is thought to be mediated by the inhibition of ERK expression and phosphorylation, and reduced IL-8 production (80, 81). H2S prevents TGF-β induced epithelial mesenchymal transition, a process which is thought to be implicated in the development of pulmonary fibrosis (82). This finding may have applicability in counteracting drug-induced fibrosis, for example, treatment with H2S would limit bleomycin induced fibrosis through the reduction of inflammatory cell infiltration and the inhibition of NF-κB (81).

Hydrogen sulfide also has pro-apoptotic effects on the airway cells, H2S exposure at levels of around 70 µM is associated with reduced cell viability; increased apoptotic pathway activation and relevant histological changes (83). One possible application of this is in cancer. Indeed, lung adenocarcinoma cells exposed to ACS2 and ACS33 underwent apoptosis, seeming to have a synergistic effect with the conventional chemotherapeutic agent such as cisplatin (84).

Hydrogen sulfide can act as a bronchodilator, as it causes significant airway smooth muscle relaxation and increases airway luminal area. H2S inhibits InsP3-evoked Ca2+ release, through the reduction of disulphide bonds in InsP3 receptors (85). It also activates the calcium activated BK potassium channel, which hyperpolarizes the smooth muscle cell. Guanylyl cyclase and protein kinase G pathway on the other hand does not mediate the effect of H2S (86). The bronchodilatory along with the anti-proliferative properties make H2S a possible therapeutic option for asthma and other airway hyper-responsive conditions. Indeed, it has been found that serum H2S in children with asthma is significantly lower than healthy control, and that H2S level positively correlates with lung function in asthmatics (87). Animal studies also support the role of H2S in asthma. Studies with CSE knockout mice and PAG administration demonstrates that H2S deficiency is associated with higher airway responsiveness, increased airway resistance, goblet cell hyperplasia, increased cellular infiltrates and increased cytokines such as IL4, 5 and 13. H2S exposure reverses the effect of CSE knockout, reduces airway hyper-responsiveness, limits inflammation, airway remodeling and collagen deposition (88-90).

Hydrogen sulfide has also been shown to play a role in the pathology and treatment of chronic obstructive pulmonary disease (COPD). In COPD patients, the serum H2S level is negatively correlated with the disease staging, with late stage COPD patients having significantly lower H2S levels and H2S levels showing a positive correlation with the forced expiratory volume in the first second (FEV1) value of the patient (91). A recent study also found that the H2S sputum-to-serum ratio is significantly higher in COPD patients, especially those with acute exacerbation (92). In animal models of tobacco smoke induced COPD, NaHS significantly reduced airway remodeling and emphysematous change, and demonstrated reduced cytokine release and cell infiltrate (93).

The effect of H2S on pulmonary vasculature is somewhat controversial, as H2S has been shownin vitro to cause significant reduction in pre-constricted human pulmonary artery pressure, and inhibition of CSE with PAG increases the pulmonary arterial pressure (94). On the other hand, H2S administration caused transient vasoconstriction in relaxed pulmonary vasculature, and hypoxia caused an increase H2S generation. It has been suggested that H2S is involved in oxygen sensing in pulmonary vasculature and the hypoxic pulmonary vasoconstriction (HPV) phenomenon (95). This is demonstrated where H2S precursors potentiate HPV, while H2S inhibitors reduce HPV magnitude (96). However, this effect is not seen in chronic hypoxia where H2S administration is associated with clear vasodilatory and anti-hypertensive effects (97). Patients with pulmonary hypertension have significantly lower level of serum H2S than those in healthy controls (98). Similarly, patients with congenital heart disease who develop pulmonary hypertension have significantly lower H2S level (98). This is further supported by animal studies, which show that in animal models of pulmonary hypertension, exposure to sulfur dioxide significantly decreases the pulmonary artery pressure and blood vessel muscularisation (99).

As discussed in the cardiovascular system section, H2S protects tissue from haemodynamic disturbances. NaHS administration decreases lung injury in haemorrhagic shock and lead to increased survival (64). This is achieved through inhibiting apoptosis pathways and reducing radical oxygen species formation (100). In addition to this, H2S also protects against lung injury in ischaemic re-perfusion and in traumatic limb injury, through similar mechanisms (101, 102).

CENTRAL NERVOUS SYSTEM

In the brain, H2S is thought to be produced mainly in the astrocytes using the enzyme CBS, and reaches relatively high concentrations of up to 160 µM (103).

As previously reviewed by Hu et al. and Zhou et al., endogenous H2S is a neuromodulator. It potentiates N-methyl-D-aspartate receptor (NMDA) mediated currents and modulates long term potentiation; stimulate uptake of synaptic glutamate by astrocytes; potentiates the effect of GABA and regulates intracellular calcium levels (104, 105). H2S has also been shown have T type calcium channel mediated effects in peripheral nerves, which includes altering protein expression in the spinal cord. (29).

In CNS pathology H2S is thought to have anti-apoptotic, anti-inflammatory and anti-oxidative benefits. Whiteman et al. found that H2S could directly scavenge reactive oxygen species as effectively as reduced glutathione, that H2S significantly increased human neuroblastoma cell survival and protein oxidation after exposure to superoxide (35, 106). H2S administration is also associated with significantly increased superoxide dismutase activity and tissue GSH level (107, 108). Lu et al. showed that this may be achieved by potentiation of the mitochondrial uncoupling protein 2 (UCP2), which reduces the generation of ROS in the mitochondria (109). Administration of H2S prevents apoptosis by inhibiting mediators such as caspase, BAX and up regulating Bcl, it also down regulates cytokines such as TNF-α and interleukin-6 (IL-6) (110). This neuroprotective effect has been shown in acute neuronal injuries secondary to other modalities of injury including LPS, glutamate and trauma where H2S donor administration significantly increases cell viability (37, 108, 111, 112).

Hydrogen sulfide is thought to play a role in the protection against Alzheimer’s disease. Eto et al. showed that post-mortem H2S level in the brain tissue of Alzheimer’s patients on average half that of the control population (113). Similar results were also found in studies involving animal models of Alzheimer’s disease, where H2S donors significantly reduced amyloid plaque formation, memory impairment and learning impairment (110). This is achieved through preventing apoptosis and alleviating oxidative stress (114, 115). A study by Nagpure suggests that H2S also directly reduces β-amyloid production by down regulating the production of amyloid precursor protein and the activity of β and γ secretase (116).

Similar findings have also been seen in Parkinson’s disease where H2S exposure in a Parkinson’s disease mice model prevented the development of movement disorders and substantia nigra degeneration (117). The therapeutic benefits have also been demonstrated in human cell lines (107). In addition to direct anti-oxidative and anti-apoptotic effects, H2S has also been demonstrated to reduce the expression of TNF-α and IL-6 from glia cells, and reduces neurotoxicity of glia cell secretion (37).

Similar to its effect in myocardium ischaemia, H2S also protects neuronal cells against hypoxia. Gheibi et al. showed that treatment with NaHS significantly reduced infarct size, also significantly reduced brain edema and apoptosis associated with reperfusion injury (118). This can also be attributed to the anti-oxidative and anti-inflammatory effect of H2S, which reduces cytokine release by microglia, inhibits inducible nitric oxide synthase (iNOS) expression, and inhibits phosphorylation of MAPK and ERK (119, 120). H2S has also been demonstrated to promote angiogenesis after hypoxia via up regulation of angiopoietin-1 and 2, VEGF and p-AKT, which may further improve recovery (121).

LIVER

Hydrogen sulfide producing enzymes are expressed in hepatocytes and hepatic stellate cells, but not in the sinusoidal endothelial cells (122). The ability of H2S in regulating blood flow has been implicated in treating portal hypertension, which is characterised by increased hepatic vascular resistance and increased splanchnic blood flow (123). Due to the dynamic and reversible nature of intrahepatic vasculature in cirrhotic liver, the interplay between vasoconstrictors and vasodilators plays an important role in the progression of the condition. Fiorucci et al. demonstrated that infusion of NaHS prevented the increase of hepatic resistance induced by noradrenaline infusion (124), preserving hepatic blood flow, suggesting that H2S acts as a vasodilator and an important endogenous modulator of the hepatic microcirculation.

The relationship between the concentrations of H2S and the severity of portal hypertension was also studied. Wang et al. reported a significantly lower endogenous H2S levels in patients with portal hypertension than those in the healthy controls (125). In the same study, the authors reported lower concentrations of H2S in rabbit liver tissue as well as lower expressions of CSE.

Hydrogen sulfide has also been shown to reduce hepatic fibrosis. It has been shown that administration of NaHS reduces the hepatic stellate cell proliferation as well as collagen I expression (126).

In a study examining the role of CSE in acute liver failure induced by D-GalN and LPS, the authors reported an attenuation of liver injury in mice with congenital deficiency of CSE and inhibition of CSE using PAG. In mice with congenital deficiency of CSE, there are markedly elevated homocysteine and thiosulfate levels, up regulation of Nrf2 and antioxidant proteins, activation of Akt-dependent anti-apoptotic signaling, and inhibition of GalN/LPS-induced c-JNK phosphorylation in the liver (127).

Hydrogen sulfide has also been reported to demonstrate potent anti-hepatocellular carcinoma activity (128). The authors examined the effect of H2S with GYY4137, a H2S donor. The exact mechanisms involved remain to be investigated. They demonstrated the effects of GYY4137 in suppressing cell proliferation in human hepatocellular carcinoma cell lines.

Hydrogen sulfide has also been shown to be beneficial in the events of ischaemic/reperfusion injury. In a hepatic ischaemic/reperfusion model, it was reported that animals treated with H2S showed reduction in serum ALT and AST levels and necrotic lesions at 24 hours after ischaemia/reperfusion. H2S was also reported to reduce the TNF-α level and IL-6 mRNA level increases induced by ischaemic/reperfusion injury and in turn, H2S may suppress cell necrosis, apoptosis and inflammation (129). Its preconditioning is shown to protect rat liver against ischemia/reperfusion injury (130). The proposed mechanism is via the activation of Akt-GSK-3β. The levels of serum ALT and AST were lower compared to the ischaemic/reperfusion group without NaHS treatment, probably through the mechanism of suppressing cytochrome c release and caspase activation.

Hydrogen sulfide has also been shown to reduce liver damage in the case of acitaminophen-induced hepatotoxicity. In a rodent acitaminophen-induced hepatotoxicity model, H2S was reported to cause significant decrease in serum alanine aminotransferase and hepatic malondialdehyde and nitric oxide levels, with a concurrent increase in hepatic glutathione content compared to acetaminophen only group (131). The authors proposed that the therapeutic benefits of H2S are comparable to N-acetylcysteine in alleviating hepatotoxicity caused by acitaminophen.

Interestingly, H2S has been reported to cause vasoconstriction in intrahepatic vasculature during sepsis. Portal infusion of Na2S causes a small but significant decrease in sinusoidal diameter (132). Norris et al. also proposed the unique role of H2S in pathophysiological states by demonstrating that the metabolism of H2S is prioritised over the availability of oxygen during sepsis in rats (15). Understandably, sepsis causes multi-organ dysfunction and the roles of H2S in sepsis have been explored in many reviews (36, 39-41, 133-137).

KIDNEY

Both CSE and CBS are abundantly present in renal cells. Within the kidneys, H2S is produced from L-cysteine via the activity of aforementioned CSE and CBS. In diabetic nephropathy, one of the complications from diabetes mellitus, the production of endogenous H2S is greatly reduced. The reduction of H2S production is greater as the severity of type 2 diabetes mellitus progresses. Treatment with H2S donor has shown to improve the outcome suggesting the protective roles of H2S in kidney conditions. H2S was shown to rescue the mesangial cells by high glucose-induced damage, partly due to the reduced production of ROS (138). One other proposed mechanism was via the increased expression of heme oxygenase-1 (HO-1) in both mesangial and podocyte cells. HO-1 is an antioxidant enzyme and is the rate-limiting enzyme in the conversion of heme to carbon monoxide (CO) and bilirubin (139), which is well known to have direct anti-oxidant properties (140).

Chronic kidney disease is associated with significant reduction in plasma H2S concentration, diminished remnant kidney and liver tissue H2S-producing capacity and down regulation of the H2S-producing enzymes (141). The exact relationship between H2S and CKD is poorly defined. It has been recently reported that the decrease in H2S in CKD is related to the reduced gene expression of CBS and CSE, and decreased protein levels of both CBS and CSE in the liver, kidney, brain of a CKD rat model (142).

In hyperhomocysteinaemia found in end-stage renal failure, the level of endogenous H2S production is reduced. Sen et al. demonstrated the use of H2S donors to prevent hyperhomocysteinemia related renal injury (143). They reported that in mice with hyperhomocysteinaemia, there is an increase in superoxide production, but this is suppressed by H2S donors. Supplementation with H2S donors also causes an increase in intracellular SOD and CAT levels, which are believed to be inhibiting the oxidative stress related injury.

Compared with normal rats, obstructive injury decreased the plasma H2S level. CBS was dramatically reduced in the ureteral obstructed kidney, but another enzyme CSE was increased. The authors reported that NaHS inhibited renal fibrosis by reducing the production of collagen, extracellular matrix, and the expression of α-smooth muscle actin (144). This study suggests a therapeutic role of H2S in preventing chronic kidney failure.

In a pneumonia associated kidney injury, it was reported that NaHS reversed the fall in glomerular filtration rate and renal function. The proposed mechanism was that H2S led to decreased protein leakage, thereby preserving the endothelial barriers (36). Similarly, H2S has shown to be protective in renal ischaemic/reperfusion injury. Hunter et al. reported that H2S can reduce creatinine levels in a swine model of renal ischaemic/reperfusion model as well as achieve a shorter time in reaching levels of creatinine of less than 250 µM compared to the control (145). Another study reported that H2S decreases blood pressure and oxidative stress and improves renal haemodynamic and secretory function in spontaneously hypertensive rats, which are used as a model for human essential hypertension (53).

Interestingly, H2S plays an opposite role in nephrotoxicity. In animal models of gentamicin associated tubular necrosis, PAG is found to reduce creatinine levels. The authors proposed that the reduction of renal injury is partly due to the decreased H2S formation (146). In another report on a rat model of cisplatin and adriamycin nephrotoxicity, the PAG reduces the renal injury (147, 148). The exact roles of H2S in kidney diseases are still being discovered today and the mechanisms involved warrant further study.

SUMMARY

In summary, in healthy tissue, H2S generally has pro-apoptotic and anti-proliferative effects. However, it is also involved in a number of organ specific functions such as thermoregulation, modulating myocardial activity and broncho-dilation. This is further complicated by the concentration-dependent nature of the H2S action, and the fact that H2S synthesis itself is affected by the H2S level and the interaction between different gaseous mediators. Much like NO before, H2S has been successfully added to existing drug molecules without compromising their efficacy, and it is likely that H2S releasing modification of current drugs will be the main mode of H2S therapy in the future.

In ischaemia induced injurious settings, studies into central nervous system, heart, lungs, liver and kidney all show that administration of H2S donor reduces the extent of the ischaemic damage and improves organ function through a combination of reducing oxygen consumption and promoting cell survival. This could be valuable as a therapeutic tool as there are presently limited treatments available in established tissue ischaemia. Additionally, H2S also promotes angiogenesis, which should limit the extent of the secondary tissue injury and further promote healing from ischaemia. Theoretically, H2S donor could be a viable adjuvant treatment in myocardium ischaemia, cerebral vascular accidents and other organ ischaemia.

Conversely, as H2S promotes angiogenesis, drugs which reduce H2S level may be of use in conditions associated with undesired angiogenesis, examples of which include cancers and diabetic retinal neovascularisation.

Hydrogen sulphide s is also a vasodilator and negative inotrope which has been demonstrated to alleviate hypertension as well as its associated organ damage. While it might not add much to the myriad of anti-hypertensive drugs already available, its benefit in pulmonary and portal hypertension are likely to be of clinical interest as there are currently very limited treatment available for those.

Hydrogen sulphide generally has a protective effect in acute inflammation and oxidative stress from causes such as allergy and toxins. H2S administration has been shown to be beneficial in lungs, brains and livers against a large range of different offending agents. In the kidneys, inhibiting H2S seems to protect from nephrotoxicity. Clinically, patients with chemical poisoning may benefit from H2S administration of H2S in addition to other supportive treatment.

At higher levels, H2S does have anti-proliferative and apoptotic effects. H2S has been shown to cause cell death of both lung and liver cancer. However, as it is not known if that level of H2S exposure is safe for healthy cells, more studies need to be conducted before H2S can be considered as a possible anti-cancer chemical.

In chronic organ pathology, low H2S level has been observed in a number of different diseases of the brain, heart, lungs, liver and kidney. In the cardiovascular system, lungs, liver and kidney, H2S inhibits fibrosis and pathological remodeling. In neuro-degenerative conditions, H2S promotes cell survival by reducing oxidative stress and prevents apoptosis. However, a common problem between all such studies is the lack of consideration of the difference in the timeline; it is possible that H2S acutely alleviates the cell injury in those disease models, as it does in other acute insults, but will not have much benefit in the long term. This is arguably the most difficult aspect of H2S therapeutic potential to investigate, however, if successful, may result in disease modifying or curative treatment for a large number of diseases.

In conclusion, H2S donors have consistently shown to be beneficial in acute ischaemia, and may have an important role in acute organ injury from toxins and in chronic organ pathology. Considering the success in animal models andin vitro work, trials on patients are warranted to further explore the potential of H2S modulators in clinical medicine as well as to further elucidate the role of H2S in human pathophysiology.

Abbreviations: 3-MST: 3-mercaptopyruvate sulphurtransferase; ANG1: angiopoietin-1; AOAA: aminooxyacetic acid; BAX: Bcl-2 associated X protein; BKCa: calcium activated BK potassium channel; CAT: catalase; CBS: cystathionine β-synthase; CKD: chronic kidney disease; CLP: cecal ligation and puncture; CO: carbon monoxide; COPD: chronic obstructive pulmonary disease; COX-2: cyclooxygenase 2; CSE: cystathionine γ-lyase; D-GalN: D-galactosamine; ERK: extracellular-signal-regulated kinase; FEV1: forced expiratory volume in first second; GSH: glutathione; GSK3β: glycogen synthase kinase 3 beta; H2S: hydrogen sulphide; HO-1: heme oxygenase-1; HPV: hypoxic pulmonary vasoconstriction; ICAM: intercellular adhesion molecule; IL: interleukin; InsP3: inositol-1,4,5-trisphosphate; IP: ischaemic pre-conditioning; IPO: ischaemic post-conditioning; JNK: C-Jun N-terminal kinase; KATP: ATP-sensitive potassium channels; LDH: lactate dehydrogenase; LDL: low density lipoprotein; LPS: lipopolysaccharide; MAPK: mitogen-activated protein kinase; MCP: monocyte chemoattractant protein; MIP2: macrophage inhibitory protein 2; MIPa: macrophage inhibitory protein-a; MMP: matrix metalloproteinases; NF-κB: nuclear factor kappa beta; NMDA: N-methyl-D-aspartate receptor; NO: nitric oxide; Nrf2: NF-E2 p45-related factor 2; PAG: DL-propargyl glycine; PI3K: phosphoinositide 3-kinase; PKC: protein kinase C; ROS: reactive oxygen species; SOD: superoxide dismutase; SP: substance P; TGF-β: transforming growth factor-β; TNF-α: tumour necrosis factor-α; TRPV-1: transient receptor potential cation channel subfamily V member 1; UCP2: mitochondrial uncoupling protein 2; VEGF: vascular endothelial growth factor.

Conflict of interests: None declared.

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