Part2: Protective Effect Of Hydrogen Sulfide On The Kidney (Review)

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5. Role of H2S in renal disease

Renal injury. Our previous study revealed that the expression levels of CBS and CSE, two enzymes that produce H2S, were decreased in renal tissues following urinary tract obstruction (42). In vivo studies also demonstrated that supplementation with an H2S donor, to provide sufficient H2S, improved renal injury (42); the mechanisms and molecular pathways involved are relevant to the disease model studied. Kidney injury can be divided into two categories: Acute kidney injury(AKI) and CKD.AKI may occur as a result of ischemia-reperfusion (hemorrhagic or septic shock)or after exposure to toxic substances (such as iodized contrast agents, aminoglycosides, and cisplatin).CKD occurs in glomerular and tubular interstitial lesions, such as diabetic nephropathy (DN)and hypertensive nephropathy, amongst other causes (43).

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Ischemia-reperfusion injury(IRI).In the process of kidney transplantation, the temporary cessation of renal blood flow leads to acute ischemic injury, and reperfusion further enhances the functional and structural damage to human kidneys, namely renal ischemia-reperfusion injury (IRI). Animal experiments have shown that following renal ischemia-reperfusion, serum and tissues exhibit markedly increased levels of IL and tumor necrosis factor-α(TNF-α), alongside other inflammatory indicators, significantly elevated malondialdehyde(MDA)concentrations, significantly reduced superoxide dismutase (SOD) activity and renal tubular necrosis; conversely, the H2S donor Na, S has been shown to significantly reduce inflammation, oxidative stress, and kidney damage, as shown in Fig.4(44). Increased levels of MDA and reduced activity of SOD have been shown to promote lipid peroxidation and upregulate nuclear factor-KB(NF-KB), IL-2, and Toll-like receptor-4(TLR-4), which can stimulate an inflammatory response, thereby increasing renal cell apoptosis(45). The CSE inhibitor, propargyl glycine, or the CBS inhibitor, hydroxylamine, have been shown to aggravate AKI and apoptosis, presenting with higher levels of pro-inflammatory factors, and significantly increased levels of NF-xB(P65), and phosphorylated(p)-apoptosis signal-regulating kinase 1 and p-TNFR-associated factor 2. These changes were accompanied by the increased expression levels of TLR-2 and TLR-4, indicating that a TLR-mediated inflammatory response and apoptosis are also involved in renal IRI (46).

The mitochondrial-targeted H2S donor, AP39, has been reported to significantly improve the survival and function of donor kidney transplantation, and reduce cell apoptosis and necrosis(47,48). H2S has been shown to attenuate apoptosis and necrosis during cryopreservation of a donor's kidney and may increase the survival rate and function of transplanted kidneys by regulating the mitochondrial membrane potential and reducing reactive oxygen species(ROS)production (47). Oxidative stress-induced by glucose oxidase can lead to mitochondrial dysfunction, which reduces the levels of ATP in renal epithelial cells, increases the formation of cellular ROS at a relatively high concentration, and promotes cell necrosis. A previous study using both in vitro and in vivo experiments found that AP39 pretreatment has a concentration-dependent protective effect on renal IRI, with the most significant effect observed at a concentration of 300 nml-(48). The H2S protection of AP39 was 1,000X higher than that of GYY4137, a nonspecific exogenous H2S donor (47).In addition, H2S may reduce the inflammatory response by inhibiting activation of the Nod2 signaling pathway and suppressing the type A macrophage scavenger receptor signaling pathway to upregulate endoplasmic reticulum stress-induced autophagy to protect the kidney from IRI (49). However, how H2S acts on these targets are unclear.

Studies on renal transplantation storage also demonstrated that long-term static storage of donation after cardiac death (DCD) kidneys at 21℃ in UW solution supplemented with AP39 may increase the activity of renal tubular epithelial cells and reduce tissue necrosis compared with long-term static storage at 4℃C in UW solution. However, the experimental results also revealed that the UW solution supplemented with AP39 exhibited improved cell-protective effects at 4°C compared with that at 21°C(50). This is consistent with static cryogenic storage(SCS) and continuous cryogenic machine perfusion commonly used in our clinic. However, it is worth noting that organ preservation at a physiological temperature (37C), such as normal temperature machine perfusion, may be worthy of study to better prevent the damage to transplanted organs caused by low temperatures(51). Renal function was revealed to be improved in transplanted kidneys stored at a normal physiological temperature compared with those stored in the cold state (52).In a previous study, kidneys from expanded criteria donors(ECD) were normally perfused in vitro for 63±16 min with a plasma-free red cell-based solution at an average temperature of 34.6°C and compared with 47 ECD kidneys with CSCin a control group; the results showed that all donor's kidneys were successfully transplanted with good renal function (53).In addition, sub-normothermic machine perfusion of DCD porcine grafts at 20℃ has been shown to improve graft prognosis compared with hypothermic machine perfusion and SCS (54). Therefore, the effects of H2S and the storage temperature of transplanted kidneys should be studied further to determine the ideal storage conditions.

Drug nephrotoxicity. Cisplatin is a common chemotherapeutic drug that is widely used in the clinic. Cisplatin, by downregulating the expression levels of CSE, is known to disrupt H_S generation and lead to the death of proximal tubular cells, thereby causing renal toxicity. The HS donors, NaHS and GYY4137, have been reported to reduce cisplatin-induced cell death and renal toxicity (55). A previous study revealed that H2S can increase S-sulfhydrylation of the Cys256, Cys259, Cys280, and Cys283 residues of NAD-dependent deacetylase sirtuin-3(SIRT3), which induces the deacetylation of its target proteins,dynamin-like 120 kDa protein (OPAl), ATP synthase and SOD2, thus reducing mitochondrial division and increasing ATP production, and thereby reducing oxidative damage (56).In addition, H2S may inhibit the generation of intracellular ROS and MAPKs by inhibiting the activity of NADPH oxidase, which is related to the vulcanization effect of HS on the NADPH oxidase subunit P47PHOx(55). Whilst reducing NADPH oxidase activity, H2S may also induce nuclear translocation of the nuclear factor erythroid 2-related factor 2(Nrf2) to inhibit the production of ROS in cells. Further experiments have revealed that exogenous H2S donors lead to the phosphorylation of Akt and dimerization of Kelch-like ECH-related protein 1(Keapl); the inhibition of Akt activation has been reported to not only weaken the nuclear translocation of Nrf2 but also reduce the protective effects of exogenous H2S donors (57).H2S can activate Nrf2 translocation to the nucleus by dimerizing Keapl, thus promoting the expression of antioxidant genes(58). Therefore, H2S is hypothesized to inhibit ROS production in cells via Akt/Keapl and the activation of MAPKs, thereby mediating the nuclear translocation of Nrf2. It may also inhibit the production of ROS in cells by reducing the activity of NADPH oxidase. Recent studies have shown that exogenous H2S serves a renoprotective role in cyclophosphamide-induced nephrotoxicity, which is associated with increased expression of Nrf2 and downstream antioxidant proteins, such as heme oxygenase-1(HO-1), and reduced glutathione and SOD in renal tissues (57,59), as shown in Fig.4.

The histopathological results of a previous study revealed that the renal tissues of a cisplatin group were positive for desmin protein expression, with notable podocyte injury, increased quantities of the mesangial matrix, and increased proliferation of mesangial cells. Notably, NaHS therapy could improve podocyte injury and increase nephrin protein levels(60). These findings suggested that H2S may improve cisplatin-induced renal injury by protecting renal podocyte cells. In gentamicin-induced kidney injury in rats, NaHS significantly reduced renal NO and TNF-α levels, whilst increasing total antioxidant capacity(T-AOC), HO-1, and IL-10 levels, and reducing the increase in renal inducible NOS (iNOS), whilst upregulating eNOS levels. Zinc protoporphyrin (a selective HO-1 inhibitor)could reverse these changes, and block the anti-inflammatory and antioxidant effects of H2S(61). Therefore, H2S may serve an anti-inflammatory and antioxidant role in protecting AKI, partly by relying on the CO/NO pathway, and this mechanism may function to primarily downregulate NO levels, or to downregulate the effects of NO by increasing CO levels (61).

DN. Streptomycin-induced DN rats have been shown to exhibit notable inflammation and oxidative stress, with obvious renal decline and insufficiency, decreased activities of SIRT1 and SOD, and increased relative expression of caspase-3,p53, and MDA; however, NaHS may improve renal function, manifested as significantly reduced serum urea and creatinine levels, and markers of renal injury, and reversal of the aforementioned indicators of DN(62,63). ATP-sensitive potassium(Karp)channels and L-type calcium channels have been shown to be related to the increase in ROS levels and oxidative stress in DN renal cells. NaHS may increase T-AOC and reduce the total NO levels in a rat model of DN, and the use of Kap inhibitors may further increase T-AOCand reduce NO levels(62). Therefore, the renoprotective mechanism of H2S on DN may be partly dependent on KATp channel activation-mediated effects on renal tissue antioxidants and NO.

In a previous study, the renal injury was simulated in C57BL/6J and Akita(C57BL/6JIns2Akita)mice under a high-glucose environment, and the experiment showed increased cytoplasmic Ca²+influx， activation of the mitochondrial matrix protein cyclophilin D(CypD), increased mitochondrial permeability transition opening, loss of mitochondrial membrane potential and oxidative burst. The H2S donor GYY4137 could reduce the aforementioned effects following treatment. Similar results were also observed with the N-methyl-D-aspartate receptor-R1 (NMDA-RI)blocker MK-801, which further confirmed that H2S function may involve NMDA-R1(64).H2S has been reported to reduce intracellular Ca2 by inhibiting NMDA-R1-mediated inflow of Ca2*ions, and thus reducing Ca2+-dependent CypD activation to result in mitochondrial permeability transition pore opening and loss of mitochondrial membrane potential. These effects may avoid damage to the mitochondrial morphology and function, could cause outbreaks of active oxygen substances, and protect diabetic kidney cells from oxidative stress injury, as shown in Fig.4.In dose-response experiments assessing the protective effects of H2S on DN kidney, it was found that at a dose of 100 mol/kg/day, the activity/expression of SIRT1 returned to normal, and the kidney function of DN rats was improved (63). However, this previous study did not elaborate on the relationship between SIRT1 and oxidative stress and inflammation, and the molecular mechanism of action between them still remains to be further explored.

Renal fibrosis. Long-term damage to the kidney by various factors can lead to the occurrence of renal fibrosis. In the kidney of diabetic rats, NaHS therapy downregulated the expression of transforming growth factor-β1 (TGF-β1), extracellular signal-regulated kinase 1/2(ERK1/2), tissue inhibitors of metalloproteinases(TIMPs), and matrix metalloproteinases (MMPs), leading to the improvement of renal fibrosis (65,66). Renal fibrosis is associated with TGF-β/Smad signaling, AMP-activated protein kinase (AMPK)activation, ERK1/2 expression, and MMP/TIMP dysregulation(65,66), as shown in Fig.5.

The novel H2S-releasing compound, S-propylcysteine, was revealed to inhibit the mRNA expression levels of hyperglycemic fibulin and type IV collagen, as well as the over-proliferation and hypertrophy of mesangial cells. Further experiments confirmed that this was related to the inhibition of TGF-β1-and Smad3-related signaling pathways (67).

Following unilateral ureteral obstruction(UUO)in male Lewis rats, H2S treatment was shown to reduce serum creatinine and urine protein/creatinine excretion rate, and tissues exhibited reduced expression of EMT-related proteins, including fibronectin, vimentin, Smad2, TGF-β1 and TGF-β1 receptor (TβR)I. The pathological analysis also showed that H2S alleviated cortical loss, inflammatory damage, and renal tubulointerstitial fibrosis(68). Previous studies have shown that H2S-mediated Smad7 expression may reduce TβRII expression, and improve UUO renal fibrosis in a rat model via the upregulation of cadherin expression and downregulation of vimentin expression in endothelial cells(69-71).In this mechanism, TβRII binds to and activates TβRI, which can increase the activation of downstream Smad expression, leading to the upregulation of vimentin expression and downregulation of cadherin expression in endothelial cells; Smad7 can interact with TβRI/TβRII to prevent this process(70,71).In vitro experiments using human recombinant active TGF-β1 to induce EMT also found that H2S cleaved the disulfide bonds in the active dimer of TGF-β1, and promoted the formation of inactive TGF-β1 monomers(72).In addition, NaHS reduced the increase in expression of β-catenin induced by TGF-β1, increased the phosphorylation of ERK, and inhibited the nuclear translocation of β-catenin induced by TGF-β1. Using the ERK inhibitor U0126 or β-catenin small interfering RNA (siRNA)agent XAV939 abrogated the effects of NaHS on fibronectin, E-cadherin, and TGF-βRI. These findings indicated that H2S may block TGF-β-induced EMT by inhibiting ERK activation andβ-catenin translocation, thus preventing renal fibrosis (73).

In diabetic Akita mice, the levels of plasma H2S, ROS and its regulator ROS modulator l, and the expression of collagen cross-linking proteins(prolyl 4-hydroxylase subunit a 1 and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2) were increased, and the activity and the expression levels of poly ADP-ribose-polymerase-1(PARP-1), hypoxia-inducible factor-1(HIF-1), and MMP-9,-13 and-14wereincreased. These findings may be related to the downregulation of microRNA (miR)-194. Notably, GYY4137 was shown to restore the expression of miR-194. In addition, in vivo, and in vitro experiments revealed that cells transfected with miR-194 mimic exhibited alleviation of high glucose-induced ROS production(74). A high-glucose environment may increase ROS levels and lead to PARP activation, whereas PARP-1 deficiency may alleviate DN(75). Furthermore, blocking HIF-1 may reduce glomerular hypertrophy, ECM deposition, and urinary albumin excretion in diabetic kidneys(76). These results suggested that H2S may alleviate diabetic renal ECM deposition and thereby reduce renal fibrosis by regulating MMPs/PARP-1/HIF-1 expression to reduce ROSlevels and increase collagen cross-linking.

The increase in matrix protein content involved in renal fibrosis has been reported to be associated with AMPK activity and activation of the insulin receptor(IR)/IR substrate (IRS)-2/Akt/mammalian target of rapamycin complex 1 (mTORC1)/mRNA transcriptional signaling axis(77).In proximal renal tubular epithelial cells, high glucose levels inhibited AMPK phosphorylation and activity, increased NADPH oxidase 4(NOX4)expression and activity, and the production of ROS and matrix protein synthesis, which was reversed by NaHS.In further experiments, an AMPK inhibitor prevented NaHS from reducing the expression of NOX4 induced by high glucose (78).In addition, it was revealed that N(o)-nitro-L-arginine methyl ester (a NOS inhibitor) could abolish NaHS inhibition of NOX4 expression induced by high glucose. NaHS enhanced the expression of iNOS instead of eNOS. Further experiments showed that iNOS siRNA and 1400W(a selective iNOS inhibitor) eliminated the favorable effects of NaHS on the expression of high glucose-induced NOX4, ROS, and matrix laminin expression(78). Therefore, NaHS may regulate oxidative stress and the expression of renal interstitial matrix protein by inducing NO production and mediating the AMPK pathway to inhibit hyperglycemic renal fibrosis and protect diabetic renal function. Two gas transmitters, H2S and NO, and their interactions can be used as therapeutic targets for DN (78).

Hypoxia and inflammation can lead to renal fibrosis, and renal hypoxia is associated with methylation and silencing of the Klotho promoter. Notably, NaHS treatment has been reported to significantly reduce hypoxia, reverse Klotho promoter methylation to increase Klotho expression, and thereby improve renal tubular interstitial fibrosis in mice(79). Inhibition of M1/M2 macrophage infiltration and NLRP3 inflammasome activation, and subsequent inactivation of the NF-kB and IL-4/STAT6 signaling pathways can also exert anti-inflammatory and anti-fibrotic roles in the protection against renal fibrosis and renal injury following obstruction (80). In renal tubular epithelial cells, H2S has been shown to sulfurize the two conserved domains of SIRT1(Cys371/374 and Cys395/398), and induce the dephosphorylation and deacetylation of its target proteins NF-kB(p65)and STAT3, thereby reducing oxidative stress, inflammation and EMT caused by high glucose (81). Renal fibrosis is also associated with aging and obesity. Notably, NaHS restored AMPK activity, inhibited activation of the IR/IRS-2/Akt/mTORCl/mRNA translation axis, and improved renal function in aged mice(77).In addition, miR-21 has been shown to be associated with renal injury in the elderly. After inhibition of miR-21 expression, the expression levels of H2S-generating enzymes, CBS and CSE, in mouse endothelial cells were upregulated, and the expression levels of MMP-9 and type IV collagen were downregulated(82).In a high-fat diet(HFD)-induced model of kidney injury, H2S reduced the phosphorylation levels of IR and Akt in the renal cortex of male mice, which may suggest that obesity-related kidney injury is related to the IR/Akt pathway; however, this association was not observed in female mice, and whether it is related to sex-related factors remains to be studied(83). Furthermore, H2S significantly reduced lipid accumulation in the kidneys of HFD-induced obese mice, and studies have shown that H2S may downregulate NF-kB(P65)expression to reduce renal inflammation and alleviate HFD-induced renal injury in obese mice(84). These findings suggested that obesity may aggravate inflammatory-mediated renal fibrosis and renal injury.

6. Conclusions

H2S deficiency is a potential risk factor for the development and progression of renal diseases. A variety of renal injuries, including IRI, drug nephrotoxicity, and DN, exhibit metabolic imbalances of H2S during their pathological development. Supplementing exogenous H2S can alleviate renal injury caused by these diseases, delay the progression of renal fibrosis and improve renal function. The signaling pathways and molecules in which H2S serves an antioxidant, anti-inflammatory, anti-apoptotic and anti-fibrotic role in renal protection are being increasingly better understood. In addition, organelles serve a notable role in the progression of AKI and CKD. At present, studies on the damage to organelles, such as mitochondrial homeostasis, mitochondrial autophagy, and endoplasmic reticulum oxidative stress, are relatively limited with regard to the pathological mechanism of AKI and CKD, and more in-depth studies are required. In terms of exogenous HS donors, research into drugs targeting the mitochondria and inducing the controlled release of agents may form an indispensable means of treatment for AKI and CKD.