Exploring Redox Biology in physiology and disease
Koning, Anne
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Chapter 5
Review:
H
2
S in renal physiology, disease
and transplantation
The smell of renal protection
Anne M. Koning*
Anne-Roos S. Frenay*
Henri G.D. Leuvenink Harry van Goor
Nitric Oxide 2015 Apr 30; 46: 37-49.
Abstract
Hydrogen sulfide (H2S), the third gasotransmitter, next to nitric oxide and carbon monoxide,
is a key mediator in physiology and disease. It is involved in homeostatic functions, such as blood pressure control, electrolyte balance and apoptosis, and regulates pathological mechanisms, including oxidative stress and inflammation. Besides, it is believed to serve as an oxygen sensor under ischemic conditions. The kidney plays a decisive role in many of these processes, indicating an interplay between H2S and renal (patho)physiology.
In this review we focus on the (protective) functions of H2S in the kidney. We first discuss
endogenous renal H2S production and signaling and elaborate on its regulatory functions in
renal physiology. Next, we present data on the role of aberrant H2S levels in the onset and
progression of renal disease and suggest the use of H2S metabolites as biomarkers. Finally,
we describe that exogenous H2S can protect the kidney against various forms of injury and
conclude that modulation of renal H2S levels holds promise for renal patients in the future.
Highlights
• H2S is a key regulator in renal physiology.
• H2S is involved in the onset and progression of renal disease.
• H2S can prevent or breach the vicious circle of chronic hypoxia in renal disease.
1 Introduction
Gasotransmitters are considered to be important physiological molecules. Next to nitric oxide
(NO) and carbon monoxide (CO), hydrogen sulfide (H2S) has now been recognized as a key
regulator in physiology and disease.
As a matter of fact, in the beginning of life on earth cells depended on H2S as their primary
source of energy. Even though today cells primarily use oxygen (O2), H2S is believed to remain
important by functioning as an O2 sensor.(1) Moreover, H2S producing enzymes, which
normally reside in the cytosol, were found to translocate to mitochondria during hypoxia, allowing the body to fall back on the ancient process of sulfide metabolism when necessary.(2,3)
Furthermore, H2S is involved in many homeostatic functions, such as blood pressure
control, electrolyte balance and apoptosis, and regulates various pathological mechanisms, including oxidative stress and inflammation. As the kidney plays a decisive role in many of
these processes, an interplay between H2S and renal (patho)physiology is easy to
comprehend.
H2S exerts its actions through various mechanisms, many of which are incompletely
understood. Recently, sulfhydration of proteins has emerged as an important mechanism of H2S signalling.(4,5) Also, H2S is believed to act in interaction with other gasotransmitters,
especially NO.(6–9)
In this review we first describe the role of H2S in physiological processes in the kidney.
Next, we consider H2S as a factor in the onset and progression of renal disease and suggest
the use of H2S metabolites as biomarkers. Furthermore, we attempt to elucidate some of the
mechanisms behind the actions of H2S in the kidney. Finally, we evaluate H2S based
therapeutic interventions to attenuate compromising processes in renal disease and transplantation.
2 H
2S production and biological functions
In the past centuries H2S was merely known for its toxicity and rotten egg odor. It wasn’t until
the 1990s that it was recognized as a physiological molecule, produced in many cells of the
mammalian body. Since then the perception of H2S has shifted rapidly, as by now it is
generally acknowledged as the third gasotransmitter, next to NO and CO, with which it shares several targets and functions.(10,11)
The endogenous production of H2S is catalyzed by three enzymes:
cystathionine-beta-synthase (CBS), cystathionine-gamma-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST), all of which are expressed abundantly in the kidney. (Fig. 1 and 2) In the trans-sulfuration pathway, CBS uses homocysteine (Hcy), derived from methionine, to generate cystathionine. Converting cystathionine to L-cysteine is the primary task of CSE. L-cysteine serves as a substrate for both CBS and CSE to form H2S. Also, CSE but not CBS can generate
H2S from Hcy alone.(12) In the recently discovered secondpathway, cysteine aminotransferase
(CAT) and D-amino acid oxidase (DAO) provide 3-mercaptopyruvate (3MP) for 3MST to produce H2S. In the presence of α-ketoglutarate, CAT metabolizes L-cysteine to form 3MP,
whereas DAO produces 3MP from D-cysteine (Fig. 1, next page). In the kidney, H2S production
from D-cysteine has been reported to be much more pronounced than from L-cysteine.(13)
Enzymes that produce H2S from L-cysteine (CBS, CSE and CAT) depend on pyridoxal
5’-phosphate (PLP) and are suppressed by hydroxylamine (HA). In contrast, DAO is PLP-independent and HA-resistant.(14,15)3MST, in turn, requires the presence of a dithiol, such as thioredoxin or dihydrolipoic acid, to release H2S.(16)H2S is oxidized in mitochondria and
secreted as sulfite, thiosulfate (TS), and sulfate.(12) Sulfite and TS, but not sulfate, can also be converted back into H2S (Fig. 1).(16–19)
Figure 1: Endogenous H2S production, metabolism and sulfhydration of proteins
1. H2S production from L-cysteine, derived from essential amino acid L-methionine, via the trans-sulfuration pathway by CBS, CSE and CAT/3MST. Under resting conditions CBS and CSE are localized in cytoplasm. Under hypoxic conditions CBS and CSE can translocate to mitochondria. 2. H2S production from D-cysteine by DAO/3MST. Peroxisomal DAO metabolizes D-cysteine to form 3MP, which in turn is metabolized by mitochondrial 3MST to produce H2S. A. Sulfhydration of a protein by formation of a persulfide (-SSH) bond to the reactive cysteine
residue of the target protein. B. H2S metabolism by mitochondrial oxidation to form sulfite (SO32-), thiosulfate
(S2O32-) and sulfate (SO42-). Sulfite and thiosulfate but not sulfate, may also be converted back into H2S. H2S;
hydrogen sulfide, CBS; cystathionine-beta-synthase, CSE; cystathionine-gamma-lyase, CAT; cysteine aminotransferase 3MST; 3-mercaptopyruvate sulfurtransferase, DAO; D-amino acid oxidase
Figure 2 shows messenger ribonucleic acid (mRNA) levels of the three H2S producing enzymes
under physiological conditions in total kidney tissue homogenates of Sprague Dawley rats. These kidneys express CSE to a greater extent compared to 3MST (± 1.8 : 1) and CBS (± 2.5 : 1) (Fig. 2). Data on the expression of these enzymes by different renal cell types have predominantly been derived from murine kidneys. These data have shown expression of CBS, CSE and 3MST in proximal tubular epithelial cells (PTECs) and, in contrast, of none of these enzymes in distal tubular epithelial cells and glomeruli.(20,21) Besides, 3MST has been found in vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs).(22) CSE has
similarly been shown in VSMCs and, inconsistently, in VECs, whereas CBS has not been shown in either of these two cell types.(21–24) In one study of human renal tissue, CSE protein was shown in glomeruli (in endothelial and mesangial cells) and distal tubular epithelium, as well as in proximal tubular epithelium, and vascular endothelium of arterioles and peritubular
capillaries.(25)To our knowledge, data on the expression of CBS and 3MST by cells of the
human kidney are not available as yet. The scarcity and inconsistency of data warrant further investigation. Laser-capture microdissection may be used to overcome some of the shortcomings of immunohistochemistry and provide more reliable information on the expression of H2S producing enzymes by different cell types.
Figure 2: Global renal mRNA expression of H2S producing enzymes
Under physiological conditions, in total kidney tissue homogenates of Sprague Dawley rats (n=10) all three H2S
producing enzymes are present. CSE mRNA is expressed more abundantly than 3MST and CBS mRNA. (unpublished data) Data are shown as mRNA abundance relative to housekeeping gene HPRT. mRNA; messenger ribonucleic acid, H2S; hydrogen sulfide, HPRT; Hypoxanthine guanine phosphoribosyltransferase, CSE;
cystathionine-gamma-lyase, CBS; cystathionine-beta-synthase, 3MST; 3-mercaptopyruvate sulfurtransferase
Under resting conditions, CBS and CSE are localized in cytoplasm, whereas 3MST is mainly found in mitochondria.(15) However, both CBS and CSE can translocate to mitochondria
under stress conditions, to stimulate mitochondrial H2S and adenosine triphosphate (ATP)
production (Fig. 1 and Fig. 4).(2,3)This reminds of the beginning of time when there was no O2 and organisms depended on H2S as an electron donor for photosynthesis. Possibly, under
ischemic conditions cells continue to revert to this early evolutionary mechanism.
Since its recognition as a gasotransmitter many biological functions have been attributed
to H2S. These include metabolic modulation, vasodilation, and angiogenic, anti-apoptotic,
anti-inflammatory, and anti-oxidant responses.(26–28) These physiological, cytoprotective
effects are generally driven by low concentrations of H2S, in contrast to its cytotoxic effects,
which typically occur at high concentrations. Note that many of these properties directly counter the mechanisms involved in the vicious circle of chronic renal hypoxia described in section 4.1 (Fig. 5).
Unfortunately, reliable methods to measure endogenous H2S are lacking and accordingly,
there is no consensus on the physiological levels of free H2S in blood and tissues. However,
most likely these are in the low nM range.(29–31) When interpreting data on H2S levels,
shortcomings of H2S measurements should be taken into account.
How H2S exerts its biological functions is incompletely understood. One way of H2S
signaling is by formation of a persulfide (-SSH) bond to reactive cysteine residues of target proteins (Fig. 1). This reversible, post-translational process, called sulfhydration, bears resemblance to nitrosylation by NO in the sense that both occur on reactive cysteine residues and both are reversible by the thioredoxin system. Sulfhydration, however, seems to be more prevalent than nitrosylation. Also, nitrosylation typically inhibits protein function, whereas sulfhydration often activates enzymatic activity.(4,5,32) Sulfhydration is dependent on the acid dissociation constant (pKa) of Cys residues, which determines their reactivity.(5) Interestingly,
as administration of H2S precursor L-cysteine to wild-type mice results in a significant increase
of sulfhydrated liver proteins, sulfhydration seems to be a modifiable process.(4) It has been found to play a role in several (patho)physiological processes. The downstream effects of sulfhydration include anti-inflammatory and anti-apoptotic actions of nuclear factor kappa B (NF-κB), regulation of endoplasmic reticulum stress responses through protein tyrosine
phosphatase 1B, vasodilation by the opening of ATP-sensitive K+ (KATP) channels,
suppression of cellular senescence through 8-nitro-guanosine-3′,5′-cyclic monophosphate and Kelch-like ECH-associated protein 1, and anti-oxidant actions of mitochondrial redox
signaling activator p66Shc.(27,32–37) H2S treatment to modulate sulfhydration seems to be
eligible for various pathological conditions. Whether H2S donors are able to influence the
process of sulfhydration has to be elucidated. However, in this respect the before mentioned
increase of sulfhydrated proteins following administration of L-cysteine holds great
promise.(4)
Recently, the idea surfaced that the rate of H2S production, because of insufficient
substrate concentrations, is inadequate to induce physiological responses. Instead, H2S is
suggested to be stored as sulfane sulfur (the uncharged form of sulfur, which is bound to proteins through a covalent bond with other sulfur atoms) and in acid-labile pools (that consist of proteins with iron-sulfur clusters) and released from proteins when prompted. Also, H2S is
oxidized to polysulfides (H2Sn), that are more stable than H2S and in itself can act as signaling
molecules. In fact, because of their oxidation state polysulfides are thought to be more potent to signal through sulfhydration than H2S.(5,15,38)
3 H
2S in renal physiology
H2S plays an important role in renal homeostasis (Fig. 3). Its substrate L-cysteine, infused into
the renal arteries of rats, has been shown to cause an increase of the glomerular filtration rate (GFR) and the urinary excretion of sodium (Na+) and potassium (K+). Simultaneous infusion of
CBS and CSE inhibitors, aminooxyacetic acid (AOAA) and DL-propargylglycine (PAG), was found to block these effects, indicating that they are caused by endogenously produced H2S.
Conversely, infusion of AOAA and PAG in the absence of L-cysteine decreased the same
Figure 3: H2S in renal physiology
In renal physiology, H2S causes vasodilation and increases RBF and the GFR. These changes cause an indirect
increase of the urinary excretion of Na+ and K+. In addition, H
2S exerts direct inhibitory effects on specific Na+ and
K+ transporters in the kidney, thereby further increasing the excretion of these electrolytes in the urine. H 2S;
hydrogen sulfide, RBF; renal blood flow, GFR; glomerular filtration rate, Na+; sodium, K+; potassium
significant effect on renal function, suggesting compensatory H2S production by the unaltered
enzyme.(39)This compensatory mechanism has also been posited in other studies.(40) Arterial infusion of sodium hydrosulfide (NaHS) was shown to lead to an increase of the renal blood flow (RBF), the GFR and the urinary excretion of Na+ and K+. A discrepancy was found between
the induced urinary excretion of Na+ and K+ and the rise in RBF and GFR; the first is more
evident than one would expect from the latter. This is explained by in vivo data demonstrating direct inhibitory effects of H2S on specific transporters in the kidney, including the Na+/K+/2Cl
-cotransporter and the Na+/K+-ATPase. In contrast, H
2S shows no involvement in Na+/H+
exchange or Na+/Cl- co-transport.(39) To elucidate the effects of H
2S on specific transporters
in the kidney an isolated perfused kidney setup can be very useful. As a consequence of its direct effects on GFR and renal Na+ handling, H
2S reduces blood
pressure. H2S is also presumed to control blood pressure by direct regulation of vascular tone
and interference with the renin angiotensin aldosterone system (RAAS) (e.g. through regulation of renin production). H2S is acknowledged as an endothelium-derived relaxing and
hyperpolarizing factor.(24,34,41) One of its vasodilatory mechanisms is sulfhydration of KATP channels on VSMCs.(23) Recently, our group has demonstrated that this property is only
responsible for a small portion of the total vasodilatory effect of H2S.(42) Accordingly, other
mechanisms have been described. These include activation of small and intermediate
conductance calcium activated K+ channels and upregulation of cyclic guanosine
monophosphate (cGMP) by inhibition of cGMP phosphodiesterases.(41,43)
Besides, H2S is believed to regulate vascular tone in synergy with NO. Cyclic GMP-mediated
upregulation of H2S production has been found after treatment with a NO donor.(6)
Conversely, blocking of NO synthase has been reported to reduce plasma H2S levels.(7)
Moreover, administration of100 μM NaHS alone causes only 25% relaxation of VSMCs in the
thoracic aorta, whereas 30 μM NaHS – which by itself does not cause relaxation – enhances
NO-induced VSMCs relaxation by up to 13 fold.(8) H2S-induced rapid upregulation of NO
production through Akt-dependent phosphorylation of endothelial NO synthase (eNOS) has
been described as the molecular mechanism behind H2S and NO synergy.(9) However,
administration of low doses of NaHS (10-100 μM) has also been reported to downregulate NO and consequently cause vasoconstriction in aortic rings of rats. In this study, unphysiologically high concentrations (200-1600 μM) caused vasorelaxation.(44)
The observation that CSE knockout mice develop hypertension indicates that
endogenously produced H2S modulates blood pressure.(24)However, there is no consensus
on the regulatory role of H2S regarding normotension. Some studies have shown no significant
effect of NaHS treatment or CSE and CBS inhibition on normal blood pressure.(45,46) One group has shown CSE inhibition with PAG to induce systolic hypertension, whereas another group has reported an increase of the mean arterial pressure (MAP) only when a combination of PAG and AOAA is used. The increased MAP was preceded by decreased urinary sulfate excretion, an indicator of protein intake as well as endogenous H2S production.(40) Also, a
study of two-kidney-one-clip and normal rats has demonstrated that H2S decreases systolic
blood pressure (SBP) and plasma renin levels under hypertensive, but not under normotensive
conditions. This suggests that H2S regulates renin activity only when the RAAS is
overactivated.(45)
H2S has also been posited to act as an O2 sensor in the kidney, especially under hypoxic
circumstances. Oxidation of H2S in mitochondria is O2-dependent. Since the availability of O2
in the kidney is lower in the medulla compared to the cortical region, medullary H2S activity
is expected to be higher. During renal hypoxia, H2S can accumulate and help to restore O2
balance by increasing medullary blood flow, decreasing the energy needed for tubular transport and blocking of mitochondrial respiration.(47) Also, under hypoxic circumstances CBS and CSE can translocate to mitochondria to stimulate mitochondrial H2S production.(2,3)
H2S can serve as an electron donor in the mitochondrial electron transport chain and thereby
increase ATP production.(3) Interactions between H2S and O2 have been determinative for
eukaryotic evolution. O2 controls H2S inactivation and activation of H2S largely takes place
under hypoxic circumstances (Fig. 4).(1) Furthermore, H2S-mediated O2 sensing has been
demonstrated in various O2 sensing tissues in cardiovascular systems, including VSMCs,
adrenal medulla and several chemoreceptors.(1) However, the consequences of this process still have to be elucidated.
Figure 4: Interactions of H2S and O2 in the kidney
A. Under normoxic circumstances, O2 causes H2S to be oxidized in mitochondria to form sulfite (SO32-), thiosulfate
(S2O32-) and sulfate (SO42-). In the kidney, the availability of O2 is lower in the medulla compared to the cortical
region. Thus, medullary H2S activity is expected to be higher. B. Under hypoxic circumstances, both CBS and CSE
can translocate to mitochondria to stimulate mitochondrial H2S production. Also, because of decreased O2
-dependent mitochondrial oxidation, H2S accumulates, resulting in increased H2S levels in both the medulla and
the cortex. O2; oxygen, H2S; hydrogen sulfide, CBS; cystathionine-beta-synthase, CSE; cystathionine-gamma-lyase
The above mentioned physiological processes in the kidney have been shown to be regulated by H2S in one way or another. Despite the challenges of sorting out the exact role of H2S in
renal physiology, the body of knowledge is expanding. Likely, other regulatory functions are to be revealed.
4 Renal disease mechanisms
Chronic kidney disease (CKD) is a worldwide public health problem. Both early stages of CKD and end-stage renal disease (ESRD) are associated with high morbidity and increased healthcare utilization. Especially patients with ESRD are at increased risk of mortality, particularly from cardiovascular disease (CVD).(48)CKD is defined as abnormalities of kidney structure or function, present for 3 or more months and is classified based on cause, and
estimated GFR and albuminuria category. (49)Even though CKD is typically asymptomatic in
the early stages, as the disease progresses more and more signs and symptoms arise. Eventually, patients enter a uremic state in which, to continue life, they are dependent on replacement therapy; hemodialysis and transplantation.(50)
For several years, tubulointerstitial hypoxia has been recognized as the final common pathway
by which CKD progresses to ESRD.(51)The ‘chronic hypoxia hypothesis’ was first posited by
Fine et al.(52)Up to then, glomerulosclerosis was believed to account for disease progression, as explained by Brenner’s hyperfiltration theory.(53)However, the discovery that functional impairment of the kidney has a stronger correlation with tubulointerstitial damage than with glomerular pathology urged for
an alternative explanation.(52) The
chronic hypoxia hypothesis argues that all events involved in the progression of renal disease contribute to a vicious circle of chronic hypoxia and tubulointerstitial injury that leads to ESRD. In chronic renal disease or after an acute renal insult, loss of peritubular capillaries, as well as an imbalance of vasoactive substances, reduce tubulointerstitial blood flow. Local hypoxia stimulates inflammation, the production of reactive oxygen species (ROS) and fibrogenesis, which in turn further limit the supply and diffusion of oxygen within the kidney. ROS also contribute by increasing the oxygen demand, thereby promoting cellular susceptibility to hypoxia.(51,54–56) Indeed, even though to date treatment of renal disease typically focuses on preserving renal function (i.e. GFR), it has been posited that instead it should focus on preserving renal tissue oxygenation.(54)
Figure 5: Breaching of the vicious circle of chronic hypoxia in renal disease by H2S
The chronic hypoxia hypothesis argues that all events involved in the progression of renal disease contribute to a vicious circle of hypoxia and tubulointerstitial damage leading to ESRD. H2S may breach this circle as many of
its biological functions directly counteract the mechanisms involved in disease progression. ESRD; end stage renal disease, H2S; hydrogen sulfide
Despite the increased understanding of renal disease mechanisms and substantial improvements in the quality of existing therapies, including dialysis and transplantation, patients continue to experience significant mortality and morbidity and a reduced quality of
life. In the next part of this review we discuss why H2S is a promising source of novel
therapeutic modalities to prevent or breach the vicious circle of chronic hypoxia in renal disease (Fig. 5).
4.1 H2S in the onset and progression of renal disease
Abnormalities of the CBS and CSE gene have been described in humans and in experimental mouse models. Human CBS and CSE deficiency are both recessive genetic disorders.(57,58) Human CBS deficiency is characterized by hypermethioninemia and hyperhomocysteinemia
(HHcy) and entails a high risk of developing vascular diseases.(57,59–61) Human CSE
deficiency is marked by hypercystathioninemia and cystathioninuria. This rare disorder seems to be relatively benign, although an increased risk to develop atherosclerotic disease has been implied.(58)
In mice, Yang et al. showed homo- as well as heterozygous knockout of the CSE gene to
lead to pronounced, age-dependent, hypertension.(24) In contrast, in homozygous CSE
knockouts (CSE-/-) bred by Ishii et al. hypertension was not observed. Apart from an increased
susceptibility to oxidative stress, these mice seemed to develop normally.(62)Heterozygous
CBS knockouts (CBS+/-) develop endothelial dysfunction similarly to the CSE knockouts bred
by Yang et al., whereas the majority of homozygous CBS knockouts (CBS-/-) suffer from severe
growth retardation and die within 4 weeks.(63,64)Although the hypertensive CSE knockout
mice show no structural damage to the kidneys, at least not within 10 weeks, CBS+/-mice
do.(24,65)In these animals, an increase of activity of matrix metalloproteinase-2 and -9 (MMP-9), apoptosis, and superoxide production and a decrease of the glutathione (GSH) to oxidized glutathione (GSSG) ratio have been found.(65) All of the CBS and CSE knockouts experience
HHcy.(24,62–64) Interestingly, expression of mutant human CBS in CBS-/- mice prevents
mortality but not HHcy, indicating that elevated Hcy per se is not responsible for the lethality
observed in CBS-/- animals.(66) To our knowledge, neither human 3MST deficiency nor
susceptibility to vascular and renal disease in 3MST knockout mice has been described.
Elevated levels of H2S precursors, Hcy and cysteine in the plasma of CKD patients were
already reported in the 1970s. These increased levels strongly correlate with the extent of
renal failure (serum creatinine).(67–70) A causative role for HHcy in the development of
cardiovascular and renal disease is the subject of ongoing debate.(71,72) However, several
papers have reported that impaired conversion of Hcy into H2S and consequent H2S
deficiency may, at least in part, be responsible for HHcy associated pathology.(73–75)
In accordance with this statement, the availability of H2S itself has been reported to be
decreased is renal disease. In rats, mass reduction-induced CKD is associated with a
decreased plasma concentration of H2S and decreased H2S producing capacity of both the
kidney and the liver, but not the brain. The H2S depletion results from downregulation of H2S
producing enzymes CBS, CSE and 3MST in the kidney and CBS and CSE in the liver.(76)In
CKD patients on hemodialysis, decreased plasma concentrations of H2S have also been
found, as well as lower sulfhemoglobin levels. Circulating mononuclear cells were used to
determine the expression of H2S producing enzymes. CBS was found not be expressed by
these cells, while CSE was reduced and 3MST was upregulated in CKD patients compared
to healthy controls.(77)The increased expression of 3MST may be related to its role in the
conversion of uremic toxin cyanide into the less noxious molecule thiocyanate.(78)
Furthermore, decreased CSE expression and plasma concentrations of H2S have also been
shown in spontaneously hypertensive rats (SHR). NaHS treatment enhanced CSE and H2S
levels, reduced blood pressure and attenuated structural remodeling of the aorta.(79)A small study of 25 children suffering from essential hypertension and 30 healthy controls has shown
an imbalance of plasma Hcy and H2S concentrations. In hypertensive children Hcy was
increased while H2S was decreased compared to controls. Moreover, a negative correlation
was found between the H2S/Hcy ratio and SBP.(80)
In Dahl salt sensitive hypertensive rats, HHcy has been reported to contribute to the development of arterial and glomerulosclerosis. However, the HHcy is associated with decreased expression of CBS by renal proximal epithelial cells and both the relative CBS deficiency and the susceptibility of these rats to renal damage are attenuated by substitution of their chromosome 13 by that of a Brown Norway rat. Thus, the damaging properties
ascribed to HHcy may in fact be explained by H2S deficiency.(81)On the other hand, there
are data that indicate that Hcy causes rather than results from decreased H2S production. In
mouse glomerular mesangial cells, Hcy decreased H2S generation and up-regulated
inflammatory molecules monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (through phosphorylation of extracellular signal-regulated kinase 1
and 2 and c-Jun N-terminal kinase 1 and 2) and oxidative molecule NAD(P)H p47phox. Both
NaHS treatment and CBS and CSE overexpression attenuated these effects.(82)
Low levels of plasma H2S are thought to accelerate the process of vascular calcification in
CKD patients. This is supported by the fact that calciphylaxis has been treated successfully with intravenous sodium thiosulfate (STS) even before this hypothesis was ever posed.(83,84)
In human aortic smooth muscle cells (HAoSMCs), inhibition of endogenous H2S production
by PAG was found to increase extracellular calcium deposition and upregulate osteoblast-specific proteins (alkaline phosphatase (ALP) and osteocalcin (OC)) in response to high extracellular phosphate. Silencing of the CSE gene similarly induced calcification. NaHS treatment was shown to prevent phosphate uptake in HAoSMCs by inhibition of the sodium-dependent phosphate cotransporter, decrease calcium deposition, and downregulate APL, OC and osteoblast-specific transcription factor core-binding factor alpha-1.(85)
Recently, progression of renal fibrosis has been attributed to H2S deficiency. In rats,
unilateral ureteral obstruction (UUO) was shown to lead to marked downregulation of CBS in
the obstructed kidney and a decreased plasma concentration of H2S. In contrast, CSE
expression by the affected kidney was increased. Inflammation (interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), MCP-1) and the development of fibrosis (collagen, alpha-smooth muscle actin (αSMA), fibronectin, transforming growth factor beta 1 (TGF-β1))
accompanied these changes and were attenuated by low doses of NaHS.(86)Another study
expression of both CBS and CSE and the concentration of H2S in renal tissue. NaHS treatment
reversed these effects and attenuated the development of fibrosis by inhibition of oxidative stress and inflammation, e.g. by averting TGF-β and NF-κB activation.(46)
Low levels of H2S have also been shown to contribute to the development of diabetic
nephropathy. In streptozotocin induced diabetes in rats, 3 weeks after streptozotocin
injection, plasma H2S and CSE mRNA and protein in the renal cortex were reduced. The
simultaneous increase of pro-fibrotic factors, TGF-β1 and collagen IV was attenuated by NaHS treatment. Also, in rat mesangial cells, exposure to hyperglycemia decreased CSE expression and induced ROS formation, cell proliferation and up-regulation of TGF-β1 and collagen IV, as did PAG. NaHS treatment reversed these effects. Together, these data
indicate that hyperglycemia-induced down-regulation of CSE-catalyzed H2S production by
renal mesangial cells contributes to the pathogenesis of diabetic nephropathy.(87) The
decreased production of H2S associated with hyperglycemia has been found to be mediated
by excessive MMP-9. This study showed upregulation of MMP-9 and downregulation of both CBS and CSE in renal tissue of diabetic (Akita, Ins2Akita) mice, whereas none of these changes
occurred in double knockouts (Akita/MMP-9-/-). The relative H
2S deficiency resulting from
downregulation of its producing enzymes in turn is thought to upregulate N-methyl-D
-aspartate receptor 1 and connexins 40 and 43 and thereby contribute to the adverse renal remodeling seen in diabetes.(88) In accordance, NaHS treatment has been reported to oppose the process of adverse renal remodeling in mice by adenosine monophosphate-activated protein kinase-dependent stimulation of autophagy and matrix metabolism.(89) Furthermore, H2S treatment has been found to mitigate over-activity of the RAAS in diabetic
kidney. In the before mentioned model of streptozotocin induced diabetes, renal Angiotensin II, angiotensin II type I receptor, and angiotensin converting enzyme levels were significantly increased in diabetic rats compared to controls. These increases were tempered by NaHS treatment.(90) Also, in a model of diabetes using transgenic mice featuring pancreatic β-cell-specific calmodulin-overexpression, CSE but not CBS expression was found to be decreased in PTECs. Moreover, these mice experienced decreased peritubular capillary (PTC) blood flow velocity, diameter and blood flow compared to non-transgenic littermates. In both groups, NaHS treatment within 5 min increased PTC diameter and blood flow. These data indicate that reduced CSE expression in the proximal tubules is not only involved in
renal scarring, but also directly contributes to ischemic injury in diabetic nephropathy by
dysregulation of the tubulointerstitial microcirculation.(21) Interestingly, metformin, one of
the most widely prescribed anti-diabetic drugs, has been found to increase H2S tissue
concentrations in various mouse organs, including the kidney.(91)
On top of the reduction of H2S levels associated with CKD, the protective potential of
residual H2S has been found to be negatively affected by uremic toxins. For example, cyanate
and thiocyanate accumulate in CKD patients and have been reported toimpede free radical
scavenging by H2S by a process called S-carbamoylation; the reaction of cyanate with -SH
groups.(69,92)
In contrast to the adverse impact of relative H2S deficiency in models of renal disease
described so far, in rat models of cisplatin, adriamycin and gentamicin-induced
nephrotoxicity endogenous H2S has been reported to aggravate renal damage.(93–96)
However, this assumption was based on the finding that PAG reduced renal damage caused by injection of these nephrotoxic substances. In this context, it is important to note that PAG does not selectively inhibit CSE and thereby H2S production, but may also affect other
PLP-dependent enzymes. Therefore, PAG-induced protection against nephrotoxicity may as well be independent of H2S. Moreover, the hypothesis that H2S is involved in the pathogenesis of
renal injury caused by nephrotoxic drugs is challenged by the recent finding that NaHS treatment protects against gentamicin-induced kidney damage. In rats, NaHS administered via i.p. injections for 14 days attenuated oxidative stress (increased GSH and decreased malondialdehyde (MDA) and NO levels), leukocyte infiltration and structural damage in the
kidneys.(97)On the other hand, in a similar experiment conducted by Van Dam et al. NaHS
treatment was not found to be protective.(96)
The data discussed in this section clearly show involvement of H2S in many aspects of the
pathogenesis of kidney disease and advocate application of H2S based therapeutic
modalities in this context.
4.2 H2S metabolites as biomarkers
H2S is involved in the onset and progression of renal disease. Accordingly, in renal
pathology, the expression of H2S producing enzymes and levels of its precursors and
metabolites are aberrant. With this in mind, a role for H2S metabolites as biomarkers of renal
disease severity is easy to imagine. Nevertheless, only a few studies have addressed this possibility.
Recently, a study of 707 renal transplant recipients (RTRs) and 110 controls showed H2S
metabolites, urinary sulfate and TS, to be associated with a favorable cardiovascular profile and to improve survival in RTRs. These positive associations were evident despite the observed direct association of urinary sulfate with the, allegedly adverse, metabolic acid load. Also, urinary TS, but not sulfate, was higher in RTRs compared to controls. (98)
Even though the results from this study are promising, it is clear that the use of H2S
metabolites as measures of its synthesis and metabolism is not flawless. A small study conducted in humans showed that in 33 pre-hemodialysis CKD patients, serum sulfite was positively correlated with serum creatinine.(99) Another study of 40 CKD patients divided into three groups based on creatinine clearance (CrCl) and 11 healthy controls found that plasma sulfate was higher and urinary sulfate was lower in patients suffering from mild (80 > CrCl > 25 ml/min/1.73 m2) and severe (CrCl < 25 ml/min/1.72 m2) renal failure compared to healthy
controls. Interestingly, patients without renal failure (CrCl ≥ 80 ml/min/1.73 m2) tended to
have higher urinary sulfate levels.(100) Taken together, these data reveal that in renal disease the levels of H2S metabolites in serum and urine may reflect decreased renal clearance as well
as (compensatory) synthesis. Furthermore, as Whiteman et al. have nicely summarized, levels of sulfite, TS and sulfate as well as other (by-)products of H2S production and metabolism are
influenced by dietary and environmental factors.(101)Needless to say, when employing these
this larger study populations are required. Large studies investigating the value of H2S
metabolites as predictors of disease in the general population are underway.
4.3 H2S treatment modalities
Several research groups and companies are engaged in the development of new H2S
treatment modalities. The major challenges are the rapid volatility, short half-life, and
bell-shaped concentration-response curve of H2S, and the accompanying toxicity of
concentrations in the high μM and upward ranges. Despite these difficulties, the options for H2S administration in the experimental setting are increasing, with slow release H2S donors,
hybrids of H2S and other drugs and H2S metabolite TS as the new kids on the block. Box 1
lists currently available H2S treatment modalities.
Box 1: H2S treatment modalities
cysteine analogous (L-cysteine, D-cysteine) gaseous H2S
H2S metabolite (STS)
hybrids of H2S donors and other drugs (aspirin, diclofenac, naproxen, sildenafil) modulation of H2S producing enzymes (gene therapy, pharmaceutical inhibition) garlic derivatives (DADS, DATS)
slow release H2S donors (GYY4137, AP39) sulfide-sodium salts (NaHS, Na2S, IK-1001)
H2S; hydrogen sulfide, STS; sodium thiosulfate, DADS; diallyl disulfide, DATS; diallyl trisulfide, NaHS; sodium
hydrosulfide, Na2S; sodium sulfide
Gaseous H2S is often unsuitable for experimental setups and therefore is not commonly used.
In most experiments soluble sources of H2S are applied: the sulfide-sodium salts NaHS and
sodium sulfide (Na2S). These compounds release H2S rapidly, which is useful when a bolus
dose is desired. However, this property hinders reaching stable concentrations in vitro and in
vivo and thus compromises the reliability of experimental data and safe treatment
administration.(102–104) Also, contamination of these compounds with polysulfides and metal ions has been described.(30)
To increase the reliability of H2S treatment for the experimental and clinical setting,
slow-release H2S agents have been developed over the years. GYY4137 was the first to be
successfully used in the experimental setting. It offers sustained H2S release for hours after
both i.v. administration and i.p. injection in rats.(105) Recently, a second slow release H2S
donor has been released. AP39 is a mitochondrially-targeted H2S donor. It shows anti-oxidant
and cytoprotective effects in oxidatively stressed endothelial cells.(106)
Next to the slow-release H2S compounds, hybrids of H2S and other drugs are gaining
ground. These hybrids consist of one or more sulfide molecules either incorporated in an
existing drug, such as diclofenac or aspirin, or build into a newly developed one.(103,107–
110)H2S can be released from these compounds in response to certain (patho)physiological
stimuli or in the presence of co-factors or substrates.
Garlic derivatives can be useful organic sources of H2S.(111,112) The garlic-derived
polysulfides, diallyl disulfide (DADS) and diallyl trisulfide (DATS) can react with thiol groups, thereby releasing H2S.(112)
Another promising treatment option is the H2S metabolite TS in the form of STS.(113,114)
Because of the dynamic conversion between TS and H2S, TS can function as an H2S
donor.(115) The fact that STS is already applied in the clinic, e.g. for the treatment of calciphylaxis, makes this compound an interesting source of H2S therapy. Unfortunately, it is
rapidly degraded in the stomach and therefore can only be administered intravenously. Further investigation is warranted to overcome this practical issue, as well as to explore the (side)effects of long-term STS administration (in humans).
H2S levels can be influenced by modulation of its producing enzymes. CSE and CBS can
be affected pharmacologically or by gene therapy. Pharmacologically, PAG or β-cyano-L-alanine can be used to block CSE activity, and AOAA or HA to block the activity of both CSE and CBS. To our knowledge, neither pharmacological nor gene therapy options targeting the activity of 3MST are available as yet.
With regard to clinical application of H2S therapy, a phase 1 clinical trial has been
completed, testing the pharmacokinetics of IK-1001. This injectable and possibly more stable
form of Na2S was studied in healthy volunteers and patients with impaired renal function
(ClinicalTrials.gov ID: NCT00879645). Participants were infused with IK-1001 for the duration of 3 hours and subsequently followed for 7 days. Publication of the first results is awaited. Aside from this, a derivate of cysteine, N-acetylcysteine (NAC), has been tested in healthy
volunteers, CKD patients and patients undergoing dialysis (ClinicalTrials.gov ID:
NCT01232257). The objective was to investigate the effect of NAC on plasma H2S levels and
markers of oxidative stress, inflammation and endothelial dysfunction. The results of this intervention study are also pending.
There is a strong need for safe, controllable, specific and cost-effective H2S treatment
modalities. Further experimental and translational investigation is required to enable H2S
therapy to evolve and become suitable for clinical application. 4.4 Protective properties of H2S in renal disease
Since more and more studies demonstrate a role for H2S in the onset and progression of
kidney disease, H2S therapy seems to be a viable possibility. Here, we describe the latest
findings regarding the effects of exogenous H2S in experimental models of renal disease.
4.4.1 Hypertension-related kidney disease
SHR develop hypertension, oxidative stress and renal dysfunction , mimicking essential
hypertension in humans. Decreased levels of plasma and urinary H2S have been reported in
these rats. Treatment with NaHS or the anti-oxidant tempol alone improved renal hemodynamics and excretory functions. In combination, NaHS and tempol caused even larger
decreases in blood pressure and oxidative stress. Also, renal function, as measured by creatinine clearance and urinary excretion of sodium, improved even more compared to single treatment. The improved anti-oxidant status is evidenced by increased total superoxide dismutase, total anti-oxidant capacity and NO, and decreased MDA levels in plasma.(116)
A blood pressure lowering effect of H2S has also been shown in a rat model of soluble
fms-like tyrosine kinase 1 (sFlt1) overexpression. This effect was associated with reduced proteinuria and renal damage. NaHS-induced upregulation of free vascular endothelial
growth factor (VEGF) was proposed as the responsible mechanism. This was supported by in
vitro data from the same study, showing that stimulation of podocytes with NaHS results in short-term VEGF release and upregulation of VEGF-A mRNA levels. Pretreatment of mesenteric vessels with a VEGF receptor-neutralizing antibody attenuated NaHS-induced vasodilatation.(117)
Recently, we have found that administration of a key H2S metabolite TS, in the form of STS
attenuates Angiotensin II-induced renal damage, as does NaHS. Both treatments were shown to decrease hypertension, proteinuria and renal dysfunction, as well as tubular damage, oxidative stress, influx of inflammatory cells and fibrosis. Data from our isolated perfused kidney setup demonstrate that the protective effects of NaHS are partly mediated by vasorelaxation through activation of KATP-channels. Also, STS and NaHS treatment were found to restore the levels of H2S producing enzymes.(42)
In an in vitro setup mimicking the condition of salt-sensitive hypertension, NaHS treatment was shown to abolish H2O2-induced activation of epithelial sodium channels (ENac), thereby
preventing Na+ reabsorption. Furthermore, NaHS was found to prevent the H
2O2-induced
increase of intracellular ROS levels and the inactivation of tumor suppressor phosphatase and tensin homolog.(118)
4.4.2 Sepsis-associated kidney disease
Since H2S has been described to exert both anti- and pro-inflammatory effects, the setting in
which H2S treatment is applied and the dosage are probably important determinants of its
net effect.
In a rabbit model of urinary-derived sepsis, a single dose of NaHS attenuated kidney injury, as evidenced by decreased glomerular deformation, tubular epithelial swelling and necrosis
and tubulointerstitial inflammatory cell infiltration. Decreased H2S plasma levels were
recovered by NaHS treatment. On the other hand, the decreased levels of CSE mRNA in the kidney were unaltered by NaHS. The reduction in kidney injury was accompanied by downregulation of pro-inflammatory markers TNF-α and NF-κB and upregulation of anti-inflammatory cytokine interleukin-10. Also, plasma creatinine and blood urea nitrogen levels were decreased.(119) Besides, short-term infusion with NaHS has been found to be associated with decreased kidney injury in a lipopolysaccharide (LPS)-induced kidney injury model and in
a rat model of pneumococcal pneumosepsis.(120,121)
In contrast, in a mouse model of LPS-induced inflammation, H2S plasma levels were
reported to increase in a time- and dose-dependent manner. LPS injection also increased renal expression of the CSE gene. CSE inactivation using PAG before LPS-injection decreased
plasma H2S levels and leukocyte infiltration in the kidney, whereas NaHS administration
caused a marked increase of plasma TNF-α levels.(122) The same study describes elevated plasma levels of H2S in humans in septic shock (n=5).(122) Whether increased H2S levels
contribute to the severity of sepsis or represent a compensatory mechanism is unknown. To answer this question and to establish whether there is role for H2S therapy in sepsis, larger
studies are warranted.
4.4.3 Obstructive kidney disease
The role of H2S in the progression of obstructive kidney disease has been discussed in section
4.2.1. UUO-induced inflammation and fibrosis have been shown to be attenuated by NaHS.(46,86,123) Two studies have found the protective effects of NaHS to be dose dependent.(46,86) These have described protection to start at 5.6 μg/kg/d and to be optimal at 56 μg/kg/day. In contrast, a ten times higher dose of NaHS (560 μg/kg/d), as well as PAG was found to fail to attenuate and even aggravate injury.(86) A third study however has
reported protection by an even higher dose: 5 mg/kg/d NaHS.(123) The capacity of H2S to
regulate pressure is likely to contribute to its protective effects in UUO. However, this factor was not discussed in the above mentioned studies.
4.5 Protective properties of H2S in renal IRI and transplantation
In 2005 Blackstone et al. were the first to show that, in mice, gaseous H2S can induce a
suspended animation-like state by reversible inhibition of complex IV of the mitochondrial
electron transport chain.(124)They hypothesized that regulation of metabolic rate could be
beneficial in several conditions, including ischemia-reperfusion (IR); a major cause of renal transplant dysfunction. Our group showed that in a mouse model of bilateral renal IR particularly pre-treatment with gaseous H2S indeed protects against structuraland functional
renal damage. Moreover, in the control group excessive renal damage led to an impaired 3 day survival of only 30%, whereas in groups pre-treated with H2S 100% survived the IRI.(125)
The applicability of H2S-induced hypometabolism in larger animals is dubious.(105,120,126–
129) However, hypometabolism can be induced in isolated organs of larger species.
Moreover, in the context of renal IRI and transplantation, endogenous as well as exogenous H2S is also considered to be protective via other mechanisms, e.g. by exerting vasodilatory,
anti-inflammatory, anti-apoptotic and anti-oxidant effects.(25,33,130–139)
Topical administration of NaHS onto the kidneys of rats prior to ischemia and before reperfusion was found to prevent apoptosis and inflammation. It attenuated the phosphorylation of several mitogen-activated protein kinases and the (consequent) decline of B-cell lymphoma 2 (Bcl-2), and activation of Caspase-3, BH3 interacting domain death agonist (Bid) and NF-κB (and its dependent proteins inducible nitric oxide synthase (iNOS), cyclooxygenase 2 and intracellular adhesion molecule 1 (ICAM-1)). The clinical relevance of these findings is questionable, since, in the same paper, the authors report complete recovery of renal function without NaHS treatment after 72 h of reperfusion.(33)In a later report, using a mouse model of bilateral IRI, they show that NaHS via intraperitoneal injection prior to ischemia and into reperfusion significantly decreased serum creatinine and urea after 24 h,
without mention of any hypometabolic features.(130) Porcine kidneys subjected to warm ischemia and cold storage, followed by ex vivo reperfusion. NaHS, infused before and after
reperfusion, improved RBF and function, and reduced oxidative damage.(131) In a porcine
model of aortic occlusion-induced renal IR, treatment with Na2S by continuous intravenous
infusion starting before occlusion, attenuated renal dysfunction and glomerular tubularization. These protective effects were associated with reduced inflammation 1β, interleukin-6 (IL-6)), oxidative DNA base damage (tail moment) and nitrosative (nitrite and nitrate, iNOS) stress.(132)In pigs subjected to left renal ischemia followed by right nephrectomy, infusion of
Na2S from before ischemia until after reperfusion, similarly improved renal function and
glomerular integrity 1-5 d after the ischemic event.(133) Following these porcine models,
recently, the effects of H2S on prolonged warm renal IRI were assessed in a model in which
uninephrectomised rats are subjected to 1 h of warm ischemia followed by either 2 h or 7 d of reperfusion. During clamping the abdomen was filled with phosphate buffered saline with or without NaHS. H2S treatment decreased serum creatinine both in the short and in the long
term. Even though necrosis and apoptosis scores were improved in the short term, in the long term they were not. During reperfusion, intravital microscopy showed increased capillary perfusion in the kidneys of H2S treated animals. Furthermore, H2S treatment decreased
pro-inflammatory markers (e.g. toll like receptor 4 (TLR4)), increased anti-apoptotic molecule
Bcl-2 and decreased pro-apoptotic marker Bid at postoperative day 1. Finally, H2S-treated
kidneys showed decreased infiltration of macrophages at day 7.(134,135)
The discovery that H2S regulates TLR4 is of importance, since TLRs are notorious
contributors to the pathogenesis of IRI. These signalling molecules initiate inflammation via adaptor proteins such as myeloid differentiation primary response gene 88 (MyD88). Using a mouse model of renal IRI, an increased expression of TLR4 by tubular epithelial cells and infiltrating leukocytes was shown within the kidney following ischemia. Moreover, both TLR4 and MyD88 knockout mice were shown to be protected against IR induced injury.(140)
Mitochondrial damage is another key element of IRI.(141) In both IR and cisplatin induced acute renal failure in mice, Dynamin-related protein 1 mediated mitochondrial fragmentation was shown to contribute to mitochondrial membrane permeabilization, release of pro-apoptotic factor cytochrome c and tubular cell apoptosis. In vitro (rat proximal tubular cells), mitochondrial fragmentation was shown to be attenuated by overexpression of Bcl-2, an anti-apoptotic factor that, inconsistently, has been reported to be upregulated by H2S.(33,125,132,135,142)Furthermore, in the context of myocardial IRI, H2S has been found
to preserve mitochondrial membrane integrity and function.(143)For renal IRI the same has
been implied.(125,143)
Numerous articles discuss the role of H2S producing enzymes, CSE and CBS in renal IRI. In
rats subjected to bilateral ischemia and reperfusion, pre-treatment with CSE inhibitor PAG significantly increased serum creatinine and urea, indicating that endogenous H2S synthesis
by CSE is essential for the recovery of renal function following IRI.(33)The same authors later reported that, in mice, CSE protein, H2S production by kidney homogenates and H2S plasma
concentration are significantly increased 24 h after IR, pointing to an endogenous mechanism
to confine IR induced damage.(130)Our group described significantly higher mortality after
bilateral renal IRI in CSE knockout mice compared to controls. Both death and renal failure could be prevented by pre-treatment with NaHS via intraperitoneal injection before clamping.
Interestingly, while endogenous H2S has been shown to regulate leucocyte adherence, here
the absence of CSE expression did not lead to increased influx of granulocytes, suggesting that, in this model, the concentrations needed to produce anti-inflammatory effects might be too high to be reached by endogenous H2S production.(25,144)In Wistar rats, following renal
IRI, CSE mRNA is increased in the acute stage, yet after 24 hours both CSE and CBS mRNA as well as protein are significantly reduced. In Sprague-Dawley rats only CBS mRNA and protein have been found to be decreased.(25,136,137,145) These differences between strains may well explain the contradiction between the findings of Tripatara et al. and those of Xu et al. Whereas data from Tripatara et al. suggest CSE to be essential for the recovery of renal function following IRI, data from Xu et al. show the same for CBS.(33,137) In Sprague-Dawley rats subjected to unilateral IRI, CBS activity was markedly reduced, while CSE activity was not. Also, intraperitoneal HA (that inhibits both CBS and CSE), but not PAG (that inhibits CSE but not CBS) significantly increased serum creatinine and renal oxidative stress. Moreover, partial restoration of CBS activity by NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) attenuated the IR induced damage, as did intraperitoneal NaHS
treatment.(137) Our group found an increase of CSE mRNA shortly after reperfusion in
humans, comparable to the acute increase found in Wistar rats. The decrease of CSE and CBS mRNA and protein or the increase of CSE protein found at later time points in rodent models could not be confirmed in the human study due to absence of biopsies late after reperfusion. Moreover, the level of renal CSE mRNA at the time of organ procurement positively associated with GFR 14 d after transplantation, suggesting that CSE could have protective effects in human IRI similar to those that have been found in murine models.(25) Furthermore, methionine sulfoxide reductase A deficient mice, that feature decreased H2S production from
methionine by CBS and CSE via the trans-sulfuration pathway, were also found to be more susceptible to renal IRI compared to wild-type mice.(146)All of these findings are consistent
in the sense that inhibition of enzyme activity aggravates and exogenous H2S treatment
decreases IRI.(136)Since the H2S producing abilities of 3-MST have been discovered only
recently, understandably, its role in IRI has not been elaborated on. However, an important role for this enzyme has been suggested by the finding that D-cysteine, the substrate for H2S
production by DAO and 3-MST attenuates renal IRI more effectively than does L-cysteine, the substrate for H2S production via the trans-sulfuration pathway. Mice were orally administered D-cysteine or L-cysteine before unilateral IRI. Mice administered D-cysteine showed less disintegration of the renal cortex, in particular of the glomeruli. This advantage was ascribed
to the more substantial increase of the levels of bound sulfane sulfur in the kidney by D
-cysteine.(13)
Recently, H2S protection against IRI in mice was reported to be dependent on eNOS
phosphorylation and NO synthesis. The authors of the study first found that CSE-/- mice carried
elevated levels of oxidative stress and were overly susceptible to cardiac and hepatic IRI. Meanwhile, these mice were shown to express dysfunctional eNOS and decreased NO and cGMP levels. Second, H2S therapy was found to attenuate the IRI to wild-type levels and DATS
was shown to restore eNOS function and NO availability in CSE-/- mice. Third, Na
2S and DATS
were found to be unable to attenuate myocardial IRI in eNOS knockout and eNOS phosphor-dead mice, respectively. Combined, these data indicate that the protective effects of H2S in
IRI are eNOS phosphorylation dependent.(138) In this study cardiac and hepatic IRI were
investigated. Conceivably, the same may be true for H2S protection against renal IR induced
damage.
In 2012, a single renal transplantation experiment applying H2S has been published. To
achieve protection against peri-transplant IRI, H2S was administered during organ
preservation. Following bilateral nephrectomy, Lewis rats were transplanted with kidneys that were flushed with and then stored for 24 h in 4 ˚C standard University of Wisconsin
preservation solution with or without NaHS. H2S addition was associated with decreased
serum creatinine, increased urine output and, most strikingly, increased survival (80 compared to 0% after 14 d). Also, H2S treated grafts showed reduced glomerular and tubular necrosis
and apoptosis, attenuated neutrophil and macrophage infiltration and decreased inflammatory cytokines (IFN-γ and ICAM-1).(139)
Whereas the therapeutic potential of H2S in renal IRI has been brought forward by many
studies, only one study actually applying H2S in renal transplantation has been published so
far.(139) The existing body of evidence for the protective role of H2S in renal IRI clearly calls
for expansion of this number. Furthermore, considering its mechanisms of protection, a role for H2S in the prevention or treatment of transplant rejection is feasible as well. Nevertheless,
studies exploring this role are still awaited.
5 Future perspectives
For centuries H2S has had a bad reputation based on its toxicity and rotten egg odor. Today,
it is finally acknowledged as an important regulator in various (patho)physiological conditions. The data discussed in this review clearly show a role for H2S in renal physiology and disease.
Also, they point towards H2S as a promising source of new treatment options for renal disease,
e.g. in the form of slow H2S releasing compounds, H2S metabolite TS or modulation of H2S
producing enzymes. Despite the challenges of H2S research, in particular regarding reliable
measurement in blood and tissue, recent scientific results have substantially contributed to our understanding of its role in biology and disease. As more and more exciting discoveries regarding H2S functions and mechanisms rise to the surface, we expect many innovative H2S
applications to evolve in the near future.
Funding
This work was supported by COST action BM 1005 and the Dutch Kidney Foundation (NSN C08-2254 and IP13-114).
Acknowledgements
Special thanks goes to Else Koning for the artwork she has created for this article. Conflicts of interest
References
1. Olson KR. Hydrogen Sulfide as an Oxygen Sensor. Antioxid Redox Signal. 2014 Jul 30;(Epub ahead of print)
2. Teng H, Wu B, Zhao K, Yang G, Wu L, Wang R. Oxygen-sensitive mitochondrial accumulation of cystathionine -synthase mediated by Lon protease. Proc Natl Acad Sci. 2013 Jul 30;110(31):12679–84. 3. Fu M, Zhang W, Wu L, Yang G, Li H, Wang R. Hydrogen sulfide (H2S) metabolism in mitochondria and its
regulatory role in energy production. Proc Natl Acad Sci U S A. 2012 Feb 21;109(8):2943–8.
4. Mustafa AK, Gadalla MM, Sen N, Kim S, Mu W, Gazi SK, et al. H2S signals through protein S-sulfhydration. Sci Signal. 2009;2(96):ra72.
5. Paul BD, Snyder SH. H₂S signalling through protein sulfhydration and beyond. Nat Rev Mol Cell Biol. 2012 Aug;13(8):499–507.
6. Zhao W, Ndisang JF, Wang R. Modulation of endogenous production of H2S in rat tissues. Can J Physiol Pharmacol. 2003 Sep;81(9):848–53.
7. Zhong G, Chen F, Cheng Y, Tang C, Du J. The role of hydrogen sulfide generation in the pathogenesis of hypertension in rats induced by inhibition of nitric oxide synthase. J Hypertens. 2003 Oct;21(10):1879–85. 8. Hosoki R, Matsuki N, Kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle
relaxant in synergy with nitric oxide. Biochem Biophys Res Commun. 1997 Aug 28;237(3):527–31. 9. Predmore BL, Julian D, Cardounel AJ. Hydrogen sulfide increases nitric oxide production from endothelial
cells by an akt-dependent mechanism. Front Physiol. 2011;2:104.
10. Wang R. Two’s company, three’s a crowd: can H2S be the third endogenous gaseous transmitter? FASEB J Off Publ Fed Am Soc Exp Biol. 2002 Nov;16(13):1792–8.
11. Kajimura M, Fukuda R, Bateman RM, Yamamoto T, Suematsu M. Interactions of multiple gas-transducing systems: hallmarks and uncertainties of CO, NO, and H2S gas biology. Antioxid Redox Signal. 2010 Jul 15;13(2):157–92.
12. Kabil O, Banerjee R. Enzymology of H 2 S Biogenesis, Decay and Signaling. Antioxid Redox Signal. 2014
Feb 10;20(5):770–82.
13. Shibuya N, Koike S, Tanaka M, Ishigami-Yuasa M, Kimura Y, Ogasawara Y, et al. A novel pathway for the production of hydrogen sulfide from D-cysteine in mammalian cells. Nat Commun. 2013;4:1366. 14. Shibuya N, Kimura H. Production of hydrogen sulfide from d-cysteine and its therapeutic potential. Front
Endocrinol. 2013;4:87.
15. Kimura H. The physiological role of hydrogen sulfide and beyond. Nitric Oxide Biol Chem Off J Nitric Oxide Soc. 2014 Feb 1;(Epub ahead of print).
16. Mikami Y, Shibuya N, Kimura Y, Nagahara N, Ogasawara Y, Kimura H. Thioredoxin and dihydrolipoic acid are required for 3-mercaptopyruvate sulfurtransferase to produce hydrogen sulfide. Biochem J. 2011 Nov 1;439(3):479–85.
17. Villarejo M, Westley J. MECHANISM OF RHODANESE CATALYSIS OF THIOSULFATE-LIPOATE OXIDATION-REDUCTION. J Biol Chem. 1963 Dec;238:4016–20.
18. Koj A, Frendo J, Janik Z. (35S)thiosulphate oxidation by rat liver mitochondria in the presence of glutathione. Biochem J. 1967 Jun;103(3):791–5.
19. Olson KR, Deleon ER, Gao Y, Hurley K, Sadauskas V, Batz C, et al. Thiosulfate: a readily accessible source of hydrogen sulfide in oxygen sensing. Am J Physiol Regul Integr Comp Physiol. 2013 Sep 15;305(6):R592– 603.
20. Nagahara N, Ito T, Kitamura H, Nishino T. Tissue and subcellular distribution of mercaptopyruvate sulfurtransferase in the rat: confocal laser fluorescence and immunoelectron microscopic studies combined with biochemical analysis. Histochem Cell Biol. 1998 Sep;110(3):243–50.
21. Yamamoto J, Sato W, Kosugi T, Yamamoto T, Kimura T, Taniguchi S, et al. Distribution of hydrogen sulfide (H₂S)-producing enzymes and the roles of the H₂S donor sodium hydrosulfide in diabetic nephropathy. Clin Exp Nephrol. 2013 Feb;17(1):32–40.
22. Shibuya N, Mikami Y, Kimura Y, Nagahara N, Kimura H. Vascular endothelium expresses 3-mercaptopyruvate sulfurtransferase and produces hydrogen sulfide. J Biochem (Tokyo). 2009 Nov;146(5):623–6.
23. Zhao W, Zhang J, Lu Y, Wang R. The vasorelaxant effect of H(2)S as a novel endogenous gaseous K(ATP) channel opener. EMBO J. 2001 Nov 1;20(21):6008–16.
24. Yang G, Wu L, Jiang B, Yang W, Qi J, Cao K, et al. H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine gamma-lyase. Science. 2008 Oct 24;322(5901):587–90.
25. Bos EM, Wang R, Snijder PM, Boersema M, Damman J, Fu M, et al. Cystathionine γ-lyase protects against renal ischemia/reperfusion by modulating oxidative stress. J Am Soc Nephrol JASN. 2013 Apr;24(5):759– 70.
26. Szabo C. Medicinal Chemistry and Therapeutic Applications of the Gasotransmitters NO, CO, and H2S and their Prodrugs. Burger’s Medicinal Chemistry and Drug Discovery. Seventh Edition. Hoboken, NJ, USA: John Wiley & Sons, Inc.; 2010. p. 265–368.
27. Sen N, Paul BD, Gadalla MM, Mustafa AK, Sen T, Xu R, et al. Hydrogen sulfide-linked sulfhydration of NF-κB mediates its antiapoptotic actions. Mol Cell. 2012 Jan 13;45(1):13–24.
28. Papapetropoulos A, Pyriochou A, Altaany Z, Yang G, Marazioti A, Zhou Z, et al. Hydrogen sulfide is an endogenous stimulator of angiogenesis. Proc Natl Acad Sci U S A. 2009 Dec 22;106(51):21972–7. 29. Furne J, Saeed A, Levitt MD. Whole tissue hydrogen sulfide concentrations are orders of magnitude lower
than presently accepted values. Am J Physiol Regul Integr Comp Physiol. 2008 Nov;295(5):R1479–85. 30. Wedmann R, Bertlein S, Macinkovic I, Böltz S, Miljkovic JL, Muñoz LE, et al. Working with “H2S”: Facts and
apparent artifacts. Nitric Oxide Biol Chem Off J Nitric Oxide Soc. 2014 Sep 15;41:85–96.
31. Olson KR, DeLeon ER, Liu F. Controversies and conundrums in hydrogen sulfide biology. Nitric Oxide Biol Chem Off J Nitric Oxide Soc. 2014 Sep 15;41C:11–26.
32. Krishnan N, Fu C, Pappin DJ, Tonks NK. H2S-Induced sulfhydration of the phosphatase PTP1B and its role in the endoplasmic reticulum stress response. Sci Signal. 2011;4(203):ra86.
33. Tripatara P, Patel NSA, Collino M, Gallicchio M, Kieswich J, Castiglia S, et al. Generation of endogenous hydrogen sulfide by cystathionine gamma-lyase limits renal ischemia/reperfusion injury and dysfunction. Lab Investig J Tech Methods Pathol. 2008 Oct;88(10):1038–48.
34. Mustafa AK, Sikka G, Gazi SK, Steppan J, Jung SM, Bhunia AK, et al. Hydrogen sulfide as endothelium-derived hyperpolarizing factor sulfhydrates potassium channels. Circ Res. 2011 Nov 11;109(11):1259–68. 35. Nishida M, Sawa T, Kitajima N, Ono K, Inoue H, Ihara H, et al. Hydrogen sulfide anion regulates redox
signaling via electrophile sulfhydration. Nat Chem Biol. 2012 Aug;8(8):714–24.
36. Yang G, Zhao K, Ju Y, Mani S, Cao Q, Puukila S, et al. Hydrogen sulfide protects against cellular senescence via S-sulfhydration of Keap1 and activation of Nrf2. Antioxid Redox Signal. 2013 May 20;18(15):1906–19.
37. Xie Z-Z, Shi M-M, Xie L, Wu Z-Y, Li G, Hua F, et al. Sulfhydration of p66Shc at Cysteine59 Mediates the Antioxidant Effect of Hydrogen Sulfide. Antioxid Redox Signal. 2014 Dec 20;21(18):2531–42.
38. Paul BD, Snyder S. Modes of physiologic H2S signaling in the brain and peripheral tissues. Antioxid Redox
Signal. 2014 May 9;(Epub ahead of print).
39. Xia M, Chen L, Muh RW, Li P-L, Li N. Production and actions of hydrogen sulfide, a novel gaseous bioactive substance, in the kidneys. J Pharmacol Exp Ther. 2009 Jun;329(3):1056–62.
40. Roy A, Khan AH, Islam MT, Prieto MC, Majid DSA. Interdependency of cystathione γ-lyase and cystathione β-synthase in hydrogen sulfide-induced blood pressure regulation in rats. Am J Hypertens. 2012
Jan;25(1):74–81.
41. Wang R. Hydrogen sulfide: a new EDRF. Kidney Int. 2009 Oct;76(7):700–4.
42. Snijder PM, Frenay A-RS, Koning AM, Bachtler M, Pasch A, Kwakernaak AJ, et al. Sodium thiosulfate attenuates angiotensin II-induced hypertension, proteinuria and renal damage. Nitric Oxide Biol Chem Off J Nitric Oxide Soc. 2014 Nov 15;42:87–98.
43. Bucci M, Papapetropoulos A, Vellecco V, Zhou Z, Pyriochou A, Roussos C, et al. Hydrogen sulfide is an endogenous inhibitor of phosphodiesterase activity. Arterioscler Thromb Vasc Biol. 2010 Oct;30(10):1998– 2004.
44. Ali MY, Ping CY, Mok Y-Y, Ling L, Whiteman M, Bhatia M, et al. Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide? Br J Pharmacol. 2006 Nov;149(6):625–34. 45. Lu M, Liu YH, Goh HS, Wang JJX, Yong QC, Wang R, et al. Hydrogen Sulfide Inhibits Plasma Renin Activity.
