
 cis - diamminedichloroplatinum ( cisplatin ) is one of the most potent anticancer drugs and is widely used as the front - line therapy for the treatment of tumors of head , neck , lungs , and genitourinary tract .
however , the clinical application of cisplatin is limited by its serious adverse effects , particularly irreversible nephrotoxicity [ 2 , 3 ] , which occurred in about one - third of patients with cisplatin treatment .
nephrotoxicity induced by cisplatin has been ascribed to several mechanisms , including inflammation , oxidative stress , and apoptosis [ 5 , 6 ] .
hydrogen sulfide ( h2s ) is the third gasotransmitter besides nitric oxide ( no ) and carbon monoxide ( co ) and attracted more and more attention because of its pleiotropic physiological effects .
endogenous h2s is generated mainly from l - cysteine by two key enzymes named cystathionine -synthase ( cbs ) and cystathionine -lyase ( cse ) .
recently , emphasis has been placed on investigating whether h2s pathway is involved in different physiological or pathological processes by endogenous or exogenous perturbation .
exogenous h2s donor is reported to have renoprotective effects in prolonged warm renal ischemia - reperfusion injury , obstructive nephropathy , and gentamicin - induced renal injury .
the dysregulation of endogenous cbs or cse also contributes to kidney ischemia - reperfusion injury   and diabetic nephropathy . by reviewing the literatures , there
one study showed that the inhibition of endogenous h2s production by dl - propargylglycine ( pag ) could reduce renal damage induced by cisplatin through the restriction of inflammation in wistar rats .
oppositely , ahangarpour et al . demonstrated that exogenous h2s donor sodium hydrosulfide ( nahs ) was protective via antioxidant property in cisplatin - treated rats . to address these controversies
, we employed morpholin-4-ium 4-methoxyphenyl ( morpholino ) phosphinodithioate ( gyy4137 ) , a novel slow releasing h2s donor , to investigate the role of exogenous h2s in cisplatin - induced nephrotoxicity and the underlying mechanisms .
gyy4137 was purchased from cayman chemical company ( ann arbor , mi , usa ) , which was dissolved in 1  :  1 mixture of dimethyl sulfoxide ( dmso ) and polyethylene glycol ( peg ) .
male c57bl/6 mice ( jackson laboratory ) aged 810 weeks old were maintained on a standard rodent chow with free access to food and water and were kept on a 12  h ( light )  :  12  h ( dark ) cycle .
the animals were divided into three groups : control ( ctr ; n = 5 ) , cisplatin alone ( cp ; n = 10 ) , and cisplatin plus gyy4137 ( cp + gyy4137 , n = 10 ) .
cisplatin was freshly prepared in ultrapure water at a concentration of 2  mg / ml and used to treat the mice by a single intraperitoneal ( i.p . )
injection ( 20  mg / kg ) , in both cp and cp + gyy4137 groups .
the mice were pretreated for 72  h with a mixture of dmso and peg ( ctr and cp groups ) or gyy4137 ( cp + gyy4137 group ) at a dose of 21  mg / kg / d via a microosmotic pump ( durect corporation , cupertino , ca , usa ) . to figure out the effect of gyy4137 alone on kidney morphology and function , another experiment was designed by the application of gyy4137 or vehicle to the mice for 6 days ( n = 5 in each group ) .
all protocols employing mice were conducted in accordance with the principles and guidance of sun yat - sen university institutional animal care and use committee .
blood urinary nitrogen ( bun ) was determined to assess renal function . for histology ,
kidneys were fixed in 4% paraformaldehyde and stained with periodic acid - schiff ( pas ) .
the tissue damage was indicated by tubular lysis , dilation , disruption , necrosis , and cast formation .
the degree of tissue damage was scored according to the percentage of damaged tubules as previously described : 0 , no damage ; 1 , < 25% ; 2 , 2550% ; 3 , 5075% ; and 4 , > 75% .
the plasma tnf- level was determined by an enzyme immunoassay kit ( catalog number 559732 , bd opteia , bd biosciences , san jose , ca , usa ) according to the manufacturer 's instructions .
the measurement of plasma thiobarbituric acid - reactive substances ( tbars ) was based on the formation of malondialdehyde by using a commercially available tbars assay kit ( catalog number 10009055 , cayman chemical ) according to the manufacturer 's instructions .
protein lysates were denatured at 100c for 10  min , separated by sds - polyacrylamide gel electrophoresis , and transferred onto pvdf membranes .
the bolts were blocked with 5% nonfat dry milk for 1  h and then probed with primary antibodies directed to cbs ( santa cruz ) , cse ( abcam ) , or -actin ( sigma - aldrich ) overnight at 4c , washed three times with tris - buffered saline ( tbs ) containing 0.1% v / v tween-20 , and incubated for 1  h at room temperature ( rt ) with secondary antibodies ( goat anti - rabbit igg and goat anti - mouse igg , santa cruz ) .
the immunoreactive bands were visualized using chemiluminescent reagent ( thermo scientific ) and exposed to x - ray film .
total rna was isolated using trizol ( invitrogen ) and first - strand cdnas were synthesized from 4  g of total rnas in a 20  l reaction using superscript ( invitrogen ) .
the first - strand cdnas served as the template for quantitative pcr ( qpcr ) performed in the applied biosystems 7900 real time pcr system using sybr green pcr reagent .
oligonucleotides were designed using primer3 software ( available at http://frodo.wi.mit.edu/primer3/ ) and the sequences are shown in table 1 .
cycling conditions were 95c for 10  min , followed by 40 repeats of 95c for 15  s , and 60c for 1  min .
the statistical analysis was performed using anova followed by bonferroni 's test or unpaired student 's t - test with spss 13 statistical software .
to evaluate the effects of gyy4137 on renal function in cisplatin - treated mice , we measured bun level and found that bun was robustly elevated in cisplatin - treated mice ( cp : 87.4  3.3 versus ctr : 14.8  0.4  mg / dl , p < 0.01 ) .
however , gyy4137 administration resulted in a greater elevation of bun ( 104.6  6.6  mg / dl , p < 0.01 , versus cp group ) ( figure 1(c ) ) .
microscopically , the mice treated with cisplatin displayed severe pathological changes , characterized by the distortion of the overall renal morphology , dilation of renal tubules , and appearance of protein casts .
remarkably , these histological changes were more severe in gyy4137-pretreated animals ( figures 1(a ) and 1(b ) ) .
these data suggested that this h2s donor played a detrimental role in cisplatin - induced nephrotoxicity .
it is well known that the activation of inflammation was involved in the pathogenesis of cisplatin nephrotoxicity .
particularly , tumor necrosis factor- ( tnf- ) has shown a central role in mediating cisplatin - induced inflammation .
as shown by the data , renal tnf- mrna was increased by 9.2-fold in cisplatin group as compared to the control mice , while gyy4137 pretreatment resulted in a much higher renal tnf- mrna expression than cisplatin alone group ( figure 2(b ) ) .
consistent with the regulation of tnf- in kidney , gyy4137 also promoted circulating tnf- level after cisplatin treatment ( figure 2(a ) ) .
meanwhile , the expressions of renal interleukin-6 ( il-6 ) and il-1 mrna showed similar patterns as renal tnf- regulation ( figures 2(c ) and 2(d ) ) .
oxidative stress is another important factor responsible for cisplatin nephrotoxicity besides inflammation [ 18 , 19 ] .
the level of plasma tbars , a marker of oxidative stress , was increased from 4.1  1.0 ( control group ) to 7.2  1.3 ( cisplatin alone group ) , while the tbars level was further increased to 13.3  2.1 in gyy4137 plus cisplatin group ( figure 3(a ) ) .
moreover , we found that the cisplatin - induced oxidative stress was associated with the downregulation of antioxidative enzymes superoxide dismutase 1 ( sod1 ) , sod2 , and sod3 , among which sod3 was further decreased by gyy4137 .
these findings indicated that gyy4137 pretreatment aggravated cisplatin - induced oxidative stress possibly via a suppression of sod3 following a challenge of cisplatin .
apoptotic pathway is also reported to be a molecular mechanism of cisplatin - induced nephrotoxicity , and the bak and caspase activation served as potential key elements [ 20 , 21 ] .
here we measured renal mrna expression of caspase-3 , bak , and bax in mice from different groups .
we found the enhanced mrna expressions of caspase-3 and bak after cisplatin treatment were further increased in gyy4137-pretreated animals ( figures 4(a ) and 4(b ) ) . in contrast , the induction of bax showed no difference between cisplatin alone group and gyy4137 plus cisplatin group ( figure 4(c ) ) .
overall , these data suggested that gyy4137 could aggravate apoptotic response in cisplatin nephrotoxicity . to demonstrate
whether gyy4137 affected the expression of endogenous h2s - producing enzymes , we detected the mrna and/or protein levels of cbs and cse in the kidney .
both cbs ( figures 5(a)5(c ) ) and cse ( figure 5(d ) ) were significantly decreased after cisplatin treatment , which was unaffected by gyy4137 .
thus the reduction of cbs and cse might act as a protective mechanism against cisplatin - induced renal injury . to exclude the effect of the drug alone on renal structure and function , we set up the gyy4137 alone group and measured plasma creatinine and observed the morphological changes .
the plasma creatinine level in gyy4137 group was not significantly different from that in ctr group ( gyy4137 : 0.392  0.023 versus ctr : 0.357  0.030  mg / dl ,
microscopically , the mice treated with gyy4137 did not display any pathological changes ( figures 6(a ) and 6(b ) ) .
hydrogen sulfide ( h2s ) has been known as a toxic gas with  rotten egg  smell for a long period of time .
it shows its toxicity possibly via inhibiting mitochondrial cytochrome c oxidase ( cco ) and oxidative phosphorylation and thus decreasing the production of adenosine triphosphate ( atp ) .
its physiological relevance was firstly reported by abe and kimura where endogenous h2s serves as a neuromodulator in the brain and facilitates the hippocampal long - term potentiation by enhancing nmda receptor - mediated responses .
in addition , h2s also displays important functions in cardiovascular system , kidney , liver , gastrointestinal , and endocrine systems [ 2327 ] . in this
regard , h2s has been widely acknowledged as the third gasotransmitter besides no and co. in kidney , h2s promotes urinary sodium excretion via both tubular and vascular mechanisms .
h2s can also inhibit renin - angiotensin system ( ras ) by decreasing reactive oxygen species ( ros ) generation   or cyclic adenosine monophosphate ( camp ) generation .
meanwhile , h2s modulates renal oxidative stress response through upregulating antioxidant haem oxygenase-1 ( ho-1 ) in human mesangial cells   and acts as an oxygen sensor in renal medulla .
therefore , aberrant regulation of renal h2s might contribute to the pathogenesis of kidney diseases and thus modulation of endogenous or exogenous h2s could be potentially effective for treating renal diseases .
watanabe et al . reported that homozygous cbs mutants have about 40 times plasma homocysteine levels as normal and a majority of them died within 5 weeks after birth .
mutant mice lacking cse displayed pronounced hypertension in line with diminished endothelium - dependent vasorelaxation .
the aortic h2s production was decreased by half in spontaneously hypertensive rats ( shrs ) compared to normotensive rats and exogenous nahs supplement attenuated hypertensive vascular collagen remodeling .
endogenous h2s production was decreased during obstructive nephropathy and nahs treatment attenuated unilateral ureteral obstruction- ( uuo- ) induced renal fibrosis [ 9 , 35 ] .
moreover , recent report demonstrated that suppressed cse / h2s pathway contributed to the pathogenesis of streptozotocin- ( stz- ) induced dn .
's results proved that h2s alleviated stz - induced dn via attenuating oxidative stress and inflammation and inhibiting renin - angiotensin system activity .
considering the anti - inflammatory and antioxidant effects of h2s , we tend to expect protective effects of h2s in kidney diseases . however , the facts are not that simple as expected .
inhibition of endogenous h2s formation by pag ( an irreversible inhibitor of cse ) could attenuate both cisplatin- and gentamicin - induced nephrotoxicities in rat models [ 13 , 3840 ] , which suggested that endogenous h2s may aggravate kidney injury .
in contrast , exogenous h2s donors , including nahs and sodium thiosulfate ( sts ) , were reported to protect against both cisplatin- and gentamicin - induced nephrotoxicities [ 10 , 14 , 41 ] . in consideration of these conflicting results ,
the role of h2s in kidney injury is hard to conclude and thus deserves further evaluation . in the present study , we employed a novel h2s donor , gyy4137 , to study its role in cisplatin - induced nephrotoxicity .
our results showed that gyy4137 further exacerbated cisplatin - induced renal injury by aggravating inflammation , oxidative stress , and apoptosis in the kidney .
renal , circulatory , and urinary tumor necrosis factor- ( tnf- ) and other proinflammatory cytokines including interleukin 1 ( il-1 ) were known to be upregulated by cisplatin injection [ 17 , 42 , 43 ] .
tnf- seemed to play a central role in the activation of inflammatory cascade since that inhibition of tnf- via genetic or pharmacological approach strikingly attenuated cisplatin nephrotoxicity .
our results showed a significant increase of circulating tnf- and renal tnf- mrna expression after cisplatin administration .
these increments of inflammatory cytokines were further increased in cisplatin plus gyy4137 group , which indicated that gyy4137 , a h2s donor , exacerbated the inflammatory response induced by cisplatin .
antioxidants are shown to be protective against cisplatin treatment both in cultured renal tubular cells [ 19 , 44 ] and in animal models [ 45 , 46 ] .
our results also showed an elevation of plasma tbars level accompanied by significantly decreased renal expressions of sod1 , sod2 , and sod3 .
similar to gyy4137 effects on inflammation , this h2s donor aggravated oxidative stress in cisplatin - treated animals in line with a further reduction of sod3 . in agreement with promoted inflammation and oxidative stress , gyy4137
further enhanced the mrna expressions of caspase-3 and bak , suggesting that apoptotic response was also deteriorated .
considering the aggravated effect of this h2s donor on cisplatin - induced renal injury , we detected the expression of endogenous h2s - producing enzymes in the kidney .
the mrna expressions of cbs and cse were significantly decreased after cisplatin treatment , which was not affected by gyy4137 .
the previous study demonstrated the consistent results that gyy4137 increased the levels of h2s but had little effect on h2s - synthesizing activity .
the apparent downregulation of cbs and cse probably would result in reduced h2s production , which might be a protective mechanism against cisplatin injury . in agreement with our findings
, previous study showed that inhibition of endogenous h2s formation ameliorated the injury after cisplatin administration .
gyy4137 is a novel slow - releasing h2s donor while the conventional donors such as nahs release h2s instantaneously in aqueous solution .
reported that nahs ameliorated the kidney dysfunction and damage in cisplatin - induced nephrotoxicity in sprague - dawley rats , which disagreed with our results .
we think the difference of the species ( mouse and rat ) and/or h2s donors might result in the discrepancies .
bolus intravenous or intraperitoneal administration of gyy4137 to anesthetized rats increased plasma h2s concentration at 30 minutes and remained elevated during the 180-minute time course , while nahs delivery to rats did not elevate plasma h2s levels .
thus it is reasonable to expect that plasma h2s was increased and remained at a steady level in the current experiment with the use of microosmotic pump for gyy4137 delivery . in summary , this study firstly examined the role of a novel exogenous h2s donor gyy4137 in a mouse model of cisplatin nephrotoxicity .
following cisplatin administration , the mice showed severe renal injury accompanied with increased oxidative stress , inflammation , and apoptotic response , which was further aggravated by gyy4137 , suggesting a detrimental role of exogenous h2s in cisplatin - induced kidney injury in mouse .
 accumulating evidence demonstrated that hydrogen sulfide ( h2s ) is highly involved in inflammation , oxidative stress , and apoptosis and contributes to the pathogenesis of kidney diseases . 
 however , the role of h2s in cisplatin nephrotoxicity is still debatable . 
 here we investigated the effect of gyy4137 , a novel slow - releasing h2s donor , on cisplatin nephrotoxicity in mice . 
 male c57bl/6 mice were pretreated with gyy4137 for 72  h prior to cisplatin injection . 
 after cisplatin treatment for 72  h , mice developed obvious renal dysfunction and kidney injury as evidenced by elevated blood urea nitrogen ( bun ) and histological damage . consistently , these mice also showed increased proinflammatory cytokines such as tnf- , il-6 , and il-1 in circulation and/or kidney tissues . 
 meanwhile , circulating thiobarbituric aid - reactive substances ( tbars ) and renal apoptotic indices including caspase-3 , bak , and bax were all elevated . 
 however , application of gyy4137 further aggravated renal dysfunction and kidney structural injury in line with promoted inflammation , oxidative stress , and apoptotic response following cisplatin treatment . 
 taken together , our results suggested that gyy4137 exacerbated cisplatin - induced nephrotoxicity in mice possibly through promoting inflammation , oxidative stress , and apoptotic response . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion