P25 Hydrogen sulfide attenuates angiotensin II-induced hypertension, proteinuria and renal damage

Sodium thiosulfate attenuates angiotensin II-induced hypertensionproteinuria and renal damage 1

Pauline M Snijder ab1 Anne-Roos S Frenay a1 Anne M Koning ab Matthias Bachtler cAndreas Pasch c Arjan J Kwakernaak d Else van den Berg d Eelke M Bos abJan-Luuk Hillebrands a Gerjan Navis d Henri GD Leuvenink b Harry van Goor aa Department of Pathology and Medical Biology University Medical Center Groningen University of Groningen Groningen The Netherlandsb Department of Surgery University Medical Center Groningen University of Groningen Groningen The Netherlandsc Department of Nephrology Hypertension and Clinical Pharmacology University Hospital Bern Inselspital Bern Switzerlandd Kidney Center Groningen University Medical Center Groningen University of Groningen Groningen The Netherlands

A R T I C L E I N F O

Article historyReceived 1 June 2014Revised 5 October 2014Available online 16 October 2014

KeywordsHydrogen sulfideThiosulfateAngiotensin IIProteinuriaFibrosis

A B S T R A C T

Hypertension and proteinuria are important mediators of renal damage Despite therapeutic interven-tions the number of patients with end stage renal disease steadily increases Hydrogen sulfide (H2S) isan endogenously produced gasotransmitter with vasodilatory anti-inflammatory and antioxidant prop-erties These beneficial characteristics make H2S an attractive candidate for pharmacological use inhypertensive renal disease We investigated the protective properties of H2S in angiotensin II (Ang II)-induced hypertensive renal disease in rats Treatment with the H2S donor NaHS and major H2S metabolitesodium thiosulfate (STS) during three weeks of Ang II infusion reduced hypertension proteinuria oxi-dative stress and renal functional and structural deterioration In an ex vivo isolated perfused kidney setupNaHS but not STS reduced intrarenal pressure The effect of NaHS could partially be explained by itsactivation of the ATP-sensitive potassium channels In conclusion treatment with H2S attenuates AngII-associated functional and structural renal deterioration suggesting that intervention in H2S produc-tion pathways has potential therapeutic benefit and might be a valuable addition to the already existingantihypertensive and renoprotective therapies

copy 2014 Elsevier Inc All rights reserved

1 Introduction

Chronic kidney disease (CKD) is a highly prevalent disorder as-sociated with extensive morbidity and mortality worldwideHypertension and proteinuria are major contributors to the pro-gression of CKD Both are important actors in enhancing structuraland functional renal deterioration through changes in intrarenal he-modynamics and inflammation thereby promoting the release ofchemokines and reactive oxygen species (ROS) [1ndash4] This resultsin stimulation of extracellular matrix synthesis and enhancementof cellular apoptosis Increased activity of the renin-angiotensin-aldosterone system (RAAS) resulting in augmented angiotensin II(Ang II) signaling is often the underlying cause of hypertension andproteinuria Functional RAAS modulation has afforded great pro-gress in renoprotection by reducing blood pressure proteinuria and

the rate of renal function loss Although RAAS blockade stands outas the most effective renoprotective treatment in many cases renaldisease ultimately progresses to end-stage renal failure with the de-plorable need for dialysis or transplantation [56] This prompts foradditional modes of intervention by either optimization of RAASblockade based therapies or targeting other pathophysiological path-ways involved in the development of CKD

Hydrogen sulfide (H2S) is acknowledged as the third gaso-transmitter in addition to nitric oxide (NO) and carbon monoxide(CO) and modulates many physiological functions [7] It is endog-enously produced from the amino acid L-cysteine by cystathionineγ-lyase (CSE) and cystathionine β-synthase (CBS) [89] and from3-mercaptopyruvate (3MP) by 3-mercaptopyruvate sulfurtransferase(3-MST) [10] In the vasculature H2S functions as an endothelial cell-derived relaxing factor via direct activation of ATP-sensitivepotassium (KATP) channels [11] Accordingly CSE-deficient mice andCBS heterozygous mice develop hypertension [1213] CSE can actas an endogenous modulator of oxidative stress as CSE-deficientmice have increased renal damage after ischemia-reperfusion [14]Exogenous treatment with the soluble sulfide salt NaHS attenu-ates the hypertensive effects of NO synthase (NOS) inhibition [15]and has preventive and therapeutic effects on renovascular hyper-tension by inhibiting plasma renin activity [16] In addition H2S

Corresponding author Department of Pathology and Medical Biology UniversityMedical Center Groningen Hanzeplein 1 PO Box 30001 9713 GZ Groningen TheNetherlands Fax +31 50 3619107

E-mail address hvangoorumcgnl (H van Goor)1 These authors contributed equally to this manuscript

httpdxdoiorg101016jniox2014100021089-8603copy 2014 Elsevier Inc All rights reserved

Nitric Oxide 42 (2014) 87ndash98

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stimulates cellular proliferation [17] and angiogenesis [18]and reduces inflammation [1920] Endogenous H2S functions asa signaling molecule by regulating protein activity throughS-sulfhydration which is a form of posttranslational modification[2122] Furthermore H2S can play a detoxifying role during oxida-tive stress by direct scavenging of ROS or increasing the formationof the antioxidant glutathione [2324] Progression of renal diseasein a CKD model is associated with depletion of H2S and its produc-ing enzymes [25] Recently urinary sulfur metabolites were foundto associate with a favorable cardiovascular risk profile and evenimproved survival in renal transplant recipients [26] Given thecytoprotective features of H2S its deficiency may contribute to pro-gression of CKD and its systemic complications

H2S can be delivered in vivo via gaseous administration or throughthe use of soluble sulfide salts like NaHS and Na2S In addition severalslow-release H2S donors have been developed Another possibilityis the use of thiosulfate (TS) a major metabolite of H2S Increasingevidence grounds the idea that a dynamic conversion exists betweenthe two substances [27ndash29] In humans the short term therapeu-tic use of sodium TS (STS) has been proven safe [30] for the treatmentof calciphylaxis [3132] STS is also proposed to be an antioxidant[32] and useful in case of cyanide poisoning [33] or cisplatin tox-icity [34] Furthermore vasodilating properties of TS itself have beendescribed [35]

The vasodilating and cytoprotective features of H2S make it anattractive therapeutic candidate for reducing the damaging effectsof hypertension and proteinuria In the experimental setting AngII infusion causes hypertension proteinuria and renal damage [36]We used this model to investigate the renoprotective properties ofsulfide containing compounds

2 Materials and methods

21 Animals

Male Sprague Dawley rats (240ndash280 gram Harlan Zeist the Neth-erlands) were housed under standard conditions with a 12 hourlight-dark cycle at the animal research facility with ad libitum accessto food and water Experimental procedures were in agreement withinstitutional and legislator regulations and approved by the localethics committee for animal experiments

22 Ang II infusion and NaHS or STS treatment

Osmotic minipumps (model 2004 Alzet Cupertino CA USA) wereplaced subcutaneously under general anesthesia (2 IsofluraneO2) for continuous administration of Ang II (435 ngkgmin n = 7group Bachem Weil am Rhein Germany) or vehicle (09 NaCln = 6) Post-operatively all rats received a subcutaneous injectionof 50 μgkg buprenorphin (Schering-Plough Houten the Nether-lands) for analgesic purposes and were allowed to recover fromsurgery at 37 degC in a ventilated incubator At placement of the pumpsAng II-infused rats were randomized to either 09 NaCl NaHS(56 mgkgday Sigma Zwijndrecht the Netherlands) or STS (1 gkgday Sigma Zwijndrecht the Netherlands) treatment During thethree weeks of infusion rats received intraperitoneal (ip) injec-tions with one of the compounds twice a day Control rats received09 NaCl infusion via osmotic minipumps as well as they were dailyadministered with 09 NaCl via ip injections At baseline blood wascollected via orbital puncture On a weekly basis body weight wasmeasured and rats were placed in metabolic cages for collection of24-hour urine Chlorhexidin was added to the urine as an antisep-tic agent to prevent bacterial growth After three weeks blood pressurewas measured under general anesthesia (2 IsofluraneO2) via anintra-aortic probe (Cardiocap5 GE Healthcare Little Chalfont Buck-inghamshire UK) Subsequently rats were sacrificed and blood was

collected in heparin and EDTA containing tubes and centrifuged for10 minutes at 1000 rcf Plasma was collected and stored at minus80 degCKidneys were perfused with 09 NaCl Coronal slices were fixed in4 paraformaldehyde and paraffin embedded for immunohisto-chemical analysis or immediately snap frozen in liquid nitrogen andstored at -80 degC for molecular analysis

23 Plasma and urine biochemical analysis

Plasma and urine levels of creatinine urea and electrolytes weredetermined by standard assays from Roche on the Roche Modular(Roche Diagnostics GmbH Mannheim Germany) according to routineprocedures in our clinical chemical laboratory Urinary protein levelswere determined with the pyrogallol red molybdate method [37]Urinary TS was determined by a specific HPLC method as de-scribed previously [3038] In short 25 μL of urine was derivatizedwith 5 μL of 46 mM monobromobimane 25 μL of acetonitrile and25 μL of 160 mM HEPES16 mM EDTA pH 8 buffer (Invitrogen Carls-bad CA USA) for 30 minutes in the dark Derivatization of thiolgroups was stopped by 50 μL of 65 mM methanosulfonic acid (FlukaBuchs Switzerland) and proteins were removed by recentrifugation

24 Qualitative real-time polymerase chain reaction

Rat renal tissue containing cortex and medulla was homog-enized in lysis buffer and total RNA was extracted using the TRIZOLmethod (Invitrogen Carlsbad USA) RNA concentrations were mea-sured by a nanodrop UV-detector (Nanodrop Technologies WilmintonDE) cDNA was synthesized using Superscript II with random hexamerprimers (Invitrogen Carlsbad USA) Gene expression (AppliedBiosystems Foster City CA USA) was determined by qualitativerealtime-PCR (qRT-PCR) based on the Taqman methodology HPRTwas used as a housekeeping gene with the following primers (Inte-grated DNA Technologies) and probe (Eurogentec) Forward 5rsquo-GCC CTT GAC TAT AAT GAG CAC TTC A-3rsquo Reverse 5rsquo-TCT TTT AGGCTT TGT ACT TGG CTT TT-3rsquo and Probe 6-FAM 5rsquo-ATT TGA ATC ATGTTT GTG TCA TCA GCG AAA GTG-3rsquo TAMRA The other primers wereobtained from Applied Biosystems as Assays-on-Demand (AOD) geneexpression products The AOD IDs used were Coll3a1 (Collagen 3)Rn01437683_m1 Acta2 (αSMA) Rn01759928_g1 Havcr1 (KIM-1)Rn00597703_m1 CTH (CSE) Rn00567128_m1 CBS Rn00560948_m1Mpst (3-MST) Rn00593744_m1 Renin Rn00561847_m1 TGF-β1Rn00572010_m1 and Cybb (NOX2) Rn00576710_m1 The qRT-PCRreaction mixture contained 20 ng cDNA template and 5 μl PCR-mastermix Nuclease free water was added to a total volume of 10μl All assays were performed in triplicate The thermal profile was15 minutes at 95 degC followed by 40 cycles of 15 seconds at 95 degC and1 minute at 60 degC The average Ct values for target genes were sub-tracted from the average housekeeping gene Ct values to yield thedelta Ct Results were expressed as 2-ΔCt

25 Immunohistochemistry

For immunostaining deparaffinized sections were subjected toheat-induced antigen retrieval by overnight incubation with 01 MTrisHCl buffer (pH 90) at 80 degC (ED1 αSMA KIM-1 desmin) or byincubation with EDTA buffer (pH 80) heated by a microwave (Col-lagen 3) Endogenous peroxidase was blocked with 0075 H2O2 inphosphate buffered saline (PBS pH 74) for 30 minutes Primary an-tibodies for macrophages (mouse anti-CD68 ED1 MCA341R AbD1750 Serotec Ltd Oxford UK) αSMA (mouse anti-SMA clone 1A4A2547 110000 Sigma Zwijndrecht the Netherlands) Collagen 3(goat anti-type 3 Collagen 1330-01 175 Southern Biotech Bir-mingham Alabama USA) Desmin (mouse anti-desmin NCL-DES-DER11 1500 Novocastra Rijswijk the Netherlands) or KIM-1 (rabbit

88 PM Snijder et alNitric Oxide 42 (2014) 87ndash98

anti-KIM-1 peptide 9 1400 gift V Baily) were incubated for 60minutes at room temperature Binding was detected using sequen-tial incubation with peroxidase-labeled secondary and tertiaryantibodies (Dakopatts Glostrup Denmark) for 30 minutes Allantibodies were diluted with PBS supplemented with 1 BSA Atthe secondary and tertiary antibody dilutions 1 normal rat serumwas added Peroxidase activity was developed using 33rsquo-diaminobenzidine tetrachloride for 10 minutes containing 003H2O2 Counterstaining was performed using Mayerrsquos hematoxylinAppropriate isotype and PBS controls were consistently negative

26 Analysis of histopathological changes

Kidney sections were scanned using an Aperio Scanscope GS(Aperio Technologies Vista CA USA) The extent of fibrotic changes(α-SMA Collagen 3) glomerular damage (desmin) and proximaltubular ischemic damage (KIM-1) were determined using the Aperiopositive pixel analysis v91 algorithm For α-SMA Collagen 3 andKIM-1 the ratio between the relative cortical staining intensity andthe total cortical surface area was used For desmin the ratio betweenglomerular staining intensity and total cortical glomerular area wascalculated Interstitial macrophages were counted manually byrandom selection of thirty renal cortical high powered fields His-topathological analysis was performed in a blinded fashion

27 Urinary malondialdehyde measurements

Malondialdehyde (MDA) a major breakdown product of lipid per-oxides is generated after oxidative stress MDA is a thiobarbituricacid-reactive substance and can be fluorescently measured afterbinding to thiobarbituric acid Twenty μL urine was incubated with90 μL of 3 SDS and 10 μL of 05 M butylated hydroxytoluene fol-lowed by addition of 400 μL 01 N HCl 50 μL 10 phosphotungsticacid and 200 μL 07 2-Thiobarbituric acid The reaction mixture wasincubated for 30 minutes at 95 degC After adding 800 μL of 1-butanolthe samples were centrifuged at 960 g for 10 minutes Two hundredμL of the 1-butanol phase was fluorescently measured using 530 nmexcitation and 590 nm emission wavelengths

28 Isolated perfused kidney setup

To investigate the effect of NaHS and STS on intrarenal pres-sure we used an ex vivo isolated perfused kidney (IPK) setup Afterinduction of anesthesia with 2 IsofluraneO2 both kidneys and renalvessels from five healthy rats were isolated via a midline incisionand subsequently a cannula was placed in the renal artery De-pending on the renal vascular anatomy either the right or left kidneywas used After placement in the IPK setup the kidney was con-

tinuously perfused via the renal artery with warmed (37 degC) andoxygenated (95 O2 and 5 CO2 gas mixture) Krebs-Ringer Bicar-bonate (KRB) solution complemented with albumin and creatinineat a pH of 75 plusmn 005 and a PO2 asymp 60 kPa by using a roller pump(Ismatec mv-ca04 Ismatec Glattbrugg Switzerland) delivering aconstant flow of 8 mLmin throughout the experiment The com-position of the perfusion solution was as follows 1186 mM NaCl47 mM KCl 25 mM CaCl2 12 mM KH2PO4 12 mM MgSO4 25 mMNaHCO3 61 mM glucose 71 mM creatinine and 50 gL albumin Afterconnecting the kidney the flow was gradually increased to 8 mLmin Vascular responses were monitored by an electromechanicalpressure transducer (Cobe Arvada CO) connected to a computerinterface (LabView National Instruments Austin TX) After an equil-ibration period when renal vascular pressure had stabilized 1ndash2 μMof phenylephrine (PE) (Sigma Zwijndrecht the Netherlands) wasadded to the perfusate to obtain a stable pre-contraction pressureof 200ndash250 mmHg When the PE-induced vasoconstriction hadreached a plateau kidneys were subjected to subsequent doses ofNaHS (1 μM 10 μM 100 μM 1 mM and 5 mM (n = 5)) or STS (1 μM(n = 5) 10 μM 100 μM (n = 2) 1 mM 5 mM (n = 5)) for 1 minuteTo investigate the role of KATP channels in NaHS-induced vasodila-tion kidneys (n = 5) were continuously perfused with 1 mM ofGlibenclamide (Sigma Zwijndrecht the Netherlands) and sub-jected to subsequent doses of NaHS After every dose we conducteda washout period of 4 minutes or continued when the intrarenalpressure had returned to baseline

29 Statistical analysis

Data were analyzed and graphed using GraphPad Prism 50 soft-ware (GraphPad San Diego CA USA) Statistical analyses wereperformed using t-tests MannndashWhitney U tests two-way ANOVAone-way ANOVA or KruskalndashWallis tests where appropriateBonferroni Dunnettrsquos or Dunnrsquos postcorrection was applied wheremultiple comparisons where made Normality was tested using theKolmogorovndashSmirnov test Statistical significance was accepted atp lt 005 All data are expressed as the mean plusmn standard error ofthe mean (SEM) unless indicated otherwise

3 Results

31 Rat characteristics ndash body weight and electrolytes

At baseline there were no significant differences in body weightbetween the groups (Table 1) After 3 weeks vehicle treated AngII-infused rats had a significantly lower body weight compared toNaCl-infused controls (p lt 0001) Treatment with NaHS partiallyprevented Ang II-induced weight loss (NaHS p lt 005) (Table 1)

Table 1Weight and biochemical parameters at baseline and the end of the study

Minipump treatment NaCl Ang II

Daily injection NaCl NaCl NaHS STSBody weight (gram)

Baseline 266 plusmn 7 261 plusmn 4 269 plusmn 4 262 plusmn 5Week 3 304 plusmn 20 211 plusmn 5 266 plusmn 16 257 plusmn 16

Plasma sodium (mmolL) 145 plusmn 09 139 plusmn 09 142 plusmn 10 142 plusmn 08Plasma potassium (mmolL) 41 plusmn 02 38 plusmn 02 37 plusmn 04 37 plusmn 03Plasma calcium (mmolL) 21 plusmn 01 22 plusmn 01 22 plusmn 01 23 plusmn 01Urinary sodium (mmol24 h) 22 plusmn 07 26 plusmn 03 17 plusmn 04 49 plusmn 05Urinary potassium (mmol24 h) 18 plusmn 03 19 plusmn 01 17 plusmn 02 20 plusmn 03Urinary calcium (mmol24 h) 001 plusmn 001 008 plusmn 001 005 plusmn 002 010 plusmn 002

Data are expressed as mean plusmn SEM p lt 005 vs control

p lt 0001 vs control p lt 005 vs Ang II + NaCl

p lt 001 vs Ang II + NaCl

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Plasma sodium was significantly lower in vehicle treated ratscompared to controls (p lt 0001) No differences between groupswere observed in plasma potassium and calcium levels (Table 1)In STS treated rats urinary sodium excretion was significantly highercompared to vehicle treated rats (p lt 001) Urinary excretion ofcalcium was increased in Ang II-infused rats treated with vehiclecompared to controls (p lt 005) Urinary excretion of potassium didnot differ between groups (Table 1)

32 NaHS and STS treatment attenuated AngII-induced hypertension

Ang II infusion increased both systolic (211 plusmn 9 mmHg vs143 plusmn 2 mmHg p lt 0001) and diastolic (127 plusmn 10 mmHg vs84 plusmn 2 mmHg p lt 001) blood pressure compared to controls Si-multaneous treatment with either NaHS or STS decreased systolicblood pressure (SBP) by 22 (164 plusmn 3 mmHg p lt 0001) and 18(173 plusmn 7 mmHg p lt 0001) and diastolic blood pressure (DBP) by26 (93 plusmn 8 mmHg p lt 005) and 30 (89 plusmn 7 mmHg p lt 001)respectively (Fig 1A and B) Infusion with Ang II reduced mRNA levelsof renin in all groups compared to control (NaCl and NaHS p lt 005STS p lt 001) (Fig 1C)

33 Improved renal function and reduced proteinuria in NaHS andSTS treated rats

Renal function ndash reflected by creatinine clearance andplasma urea ndash and proteinuria were similar in all groupsat baseline (data not shown) After three weeks of Ang II infusionvehicle treated rats had an impaired renal function as reflected bya 67 reduction in creatinine clearance (14 plusmn 01 mLmin vs42 plusmn 02 p lt 0001) and a 29-fold increase in plasma urea(190 plusmn 10 mmolL vs 65 plusmn 02 mmolL p lt 0001) compared tocontrol rats (Fig 2A and B) Furthermore proteinuria was signifi-cantly increased from week 2 onwards (346 plusmn 35 mg24 h vs28 plusmn 11 mg24 h p lt 0001) (Fig 2C) After three weeks of treat-ment with NaHS or STS renal function loss was partially preventedas evidenced by a higher creatinine clearance (NaHS 25 plusmn 03 mLmin p lt 005 STS 29 plusmn 05 mLmin p lt 0001) and lower plasmaurea levels (NaHS 14 plusmn 2 mmolL p lt 001 STS 14 plusmn 2 mmolLp lt 001) compared to vehicle treated rats (Fig 2A and B) Fromweek 2 onwards the 24-hour urinary protein excretion was sig-nificantly moderated by NaHS and STS treatment (NaHS179 plusmn 75 mg24 h p lt 0001 STS 197 plusmn 58 mg24 h p lt 001)(Fig 2C)

Fig 1 Treatment with NaHS and STS attenuated Ang II-induced hypertension and Ang II infusion caused downregulation of renin mRNA expression Infusion with Ang IIincreased (A) systolic blood pressure by 48 and (B) diastolic blood pressure by 51 Treatment with NaHS and STS significantly attenuated the Ang II-induced hyperten-sion (C) Infusion with Ang II caused downregulation of renin mRNA levels in all groups (p lt 005 p lt 001 p lt 0001 vs control p lt 005 p lt 001 p lt 0001vs Ang II + NaCl)

90 PM Snijder et alNitric Oxide 42 (2014) 87ndash98

34 STS increased the excretion of urinary TS

From week 1 onwards the excretion of urinary TS (p lt 0001)was significantly increased in Ang II-infused rats treated with STSNo differences were observed in the other groups (Fig 3)

35 Effect of NaHS and STS treatment on tubular andglomerular damage

After 3 weeks kidney injury molecule-1 (KIM-1) mRNA andprotein expression were increased in Ang II-infused rats treated withvehicle compared to controls (p lt 0001) Both NaHS and STS treatedrats showed less tubular damage as evidenced by a 2-fold reduc-tion in proximal tubular damage at mRNA and protein levelcompared to vehicle treated animals (p lt 005) (Fig 4A B and C)Also the glomerular damage marker desmin was increased afterthree weeks of Ang II infusion (p lt 0001) Treatment with NaHSbut not STS decreased the glomerular protein levels of desmin by40 (p lt 001) (Fig 5A and B)

Fig 2 Reduction of renal function loss and proteinuria in NaHS and STS treated rats Three weeks of Ang II infusion decreased renal function as evidenced by a 67 de-crease in (A) creatinine clearance and a 29-fold increase in (B) plasma urea Furthermore (C) proteinuria was significantly increased from week 2 onwards Treatment withNaHS and STS reduced renal function loss by preserving the creatinine clearance and plasma urea levels In addition the development of proteinuria was diminished inNaHS and STS treated rats from week 2 onwards (p lt 0001 vs control p lt 005p lt 001 p lt 0001 vs Ang II + NaCl)

Fig 3 Elevated urinary TS excretion in STS treated animals In rats treated with STSthe excretion of urinary TS was increased from week 1 onwards In all other groupsno differences in urinary TS levels were observed (p lt 0001 vs Ang II + NaCl)

91PM Snijder et alNitric Oxide 42 (2014) 87ndash98

36 Influx of macrophages is reduced by treatment with STS

Ang II infusion increased the number of ED1 positive cells in therenal interstitium more than 2-fold compared to NaCl infused con-trols (204 plusmn 46 vs 86 plusmn 12 p lt 005) STS and NaHS decreased thenumber of interstitial macrophages to near control levels (STS73 plusmn 21 p lt 001 NaHS 114 plusmn 18 p = 006) (Fig 6A and B)

37 Treatment with NaHS and STS reduced oxidative stress

Expression of NOX2 mRNA was increased 16-fold after Ang IIinfusion (p lt 005) (Fig 7A) Furthermore the urinary excretion ofmalondialdehyde (MDA) was increased more than 2-fold in Ang IIrats treated with vehicle (p lt 0001) (Fig 7B) Simultaneous treat-ment with either NaHS or STS decreased NOX2 mRNA expressionby 38 (p lt 001) and 39 (p lt 001) and urinary MDA levels by35 (p lt 005) and 30 (p lt 005) respectively (Fig 7A and B)

38 Protective effects of NaHS and STS treatment against renalinterstitial changes

TGF-β a growth factor with proliferative and fibrotic effects onmyofibroblasts is significantly upregulated during Ang II infusion(p lt 005) In rats treated with NaHS and STS levels of TGF-β mRNAreturn to near control values (p lt 001) (Fig 8A) Ang II infusion sig-nificantly increased mRNA (p lt 005) and protein (p lt 0001)expression of the pre-fibrotic marker α-smooth muscle actin (αSMA)indicating ongoing interstitial myofibroblast transformation STS butnot NaHS decreased the mRNA and protein expression of αSMA by50 (p lt 005) (Fig 8B C and D) The pre-fibrotic effects of Ang IIwere accompanied by increased fibrotic damage as evidenced bya significantly higher expression of Collagen 3 mRNA and protein(p lt 005) Treatment with NaHS and STS reduced the develop-ment of fibrosis resulting in a reduction of Collagen 3 mRNA(p lt 005) and protein (NaHS p lt 005 STS p lt 0001) to nearcontrol levels (Fig 8E F and G)

Fig 4 Effect of NaHS and STS treatment on tubular damage (A) KIM-1 mRNA and (B) KIM-1 protein levels were increased by Ang II infusion Rats treated with NaHS andSTS had less tubular damage as evidenced by reduced KIM-1 protein and mRNA levels (C) Representative photomicrographs of KIM-1 stained renal sections (p lt 0001vs control p lt 005 vs Ang II + NaCl)

92 PM Snijder et alNitric Oxide 42 (2014) 87ndash98

39 Restoration of CSE CBS and 3-MST mRNA expression with NaHSand STS treatment

Renal mRNA expression of CSE CBS and 3-MST was signifi-cantly decreased after 3 weeks of Ang II infusion (p lt 001)Expression levels were partially restored in Ang II-infused rats thatreceived NaHS and STS (p lt 005) (Fig 9A B and C)

310 Vasodilatory effects of NaHS in the isolated perfusedkidney setup

Administration of 1 and 5 mM NaHS lowered intrarenal pres-sure by 44 (p lt 0001) and 39 (p lt 005) compared to baselinepressure during phenylephrine (PE) constriction Lower concentra-tions of NaHS had no effect on intrarenal pressure To investigatethe role of KATP channels in NaHS-induced vasodilation kidneys wereperfused with Glibenclamide which diminished the vasodilatorypotency of 1 mM NaHS (p lt 005) STS showed no effect on intrarenalpressure (Fig 10)

4 Discussion

Exogenous treatment with NaHS and STS reduces hyperten-sion proteinuria renal damage and renal function loss associatedwith Ang II infusion Furthermore we are the first to show that STSa clinically applicable compound has promising renoprotectiveproperties These data suggest that intervention in sulfur path-ways has protective potential in hypertension and hypertensive renaldisease

The effects of exogenous H2S on blood pressure reduction arein line with previous literature showing its preventive and thera-peutic properties in experimental hypertension Endogenous H2Sshortage is suggested to be involved in the pathogenesis of hyper-tension [11ndash131539] One of the underlying mechanisms in bloodpressure reduction by H2S is its direct effect on vascular smoothmuscle cells by sulfhydration and subsequent activation of KATP-channels [11] The effects of H2S on proteinuria are describedless clearly and not in a rectilinear fashion [4041] The antiprotei-nuric effects of H2S in our model as structurally evidenced by

Fig 5 Effect of NaHS and STS treatment on glomerular damage (A) Desmin protein levels were increased in glomeruli of Ang II-infused animals treated with vehicle Treat-ment with NaHS reduced glomerular desmin levels by 40 Treatment with STS only showed a trend towards decreased desmin levels (B) Representative photomicrographsof desmin stained renal sections (p lt 0001 vs control p lt 001 vs Ang II + NaCl)

Fig 6 Interstitial inflammation is reduced by treatment with STS (A) The influx of macrophages was increased 2-fold in Ang II-infused rats treated with vehicle Treat-ment with STS but not NaHS reduced the number of interstitial macrophages 3-fold (B) Representative photomicrographs of ED-1 stained renal sections (p lt 005 vscontrol p lt 001 vs Ang II + NaCl)

93PM Snijder et alNitric Oxide 42 (2014) 87ndash98

preservation of podocytes can be partially explained by the reduc-tion of systemic and intrarenal pressures Mechanistic evidence forthis comes from our ex vivo isolated perfused kidney (IPK) experi-ments in which we observed a reduction in intrarenal pressure byNaHS Blockage of the KATP-channels partly decreased the vasodilatorypotency of NaHS suggesting that other mechanisms are involvedas well One of these might be crosstalk between H2S and NO sincethe vasorelaxant effects of H2S are partly NO-dependent Inhibi-tion of NO using NG-nitro-L-arginine methyl ester (L-NAME)decreased the potency of H2S in aortic rings [42] The in vivo effectsof STS were unmistakable but could not be related to direct changesin intrarenal pressure in our IPK setup Since it is known thatTS can be converted to H2S in vivo we suggest that the IPK setuplacks the proper physiological conditions necessary for thisconversion

H2S directly affects the RAAS as shown by its capacity to inhibitrenin activity and ACE activity [16] Since we infused Ang II the con-tribution of this mechanism is probably low or undetectable becauseof the existing negative feedback loop between Ang II and reninIndeed we found low mRNA levels of renin in all the Ang II treatedgroups Furthermore H2S can decrease Ang II-induced activation ofmitogen-activated protein kinases and the binding affinity of theangiotensin-1 (AT-1) receptor in a dose-dependent manner [43]

Although it is plausible that blood pressure regulation is theprimary mechanism of action of H2S in our model it is known thatrenal injury in Ang II-induced hypertension can be independent ofan elevated blood pressure This suggests additional protective modesof action of H2S Since we did not include a group receiving con-ventional antihypertensive drugs during Ang II infusion we cannotdetermine whether the protective effects of H2S are solely medi-ated by blood pressure reduction

Inflammation is one of the major consequences of renal expo-sure to hypertension and proteinuria [4] STS completely preventedAng II-induced influx of interstitial macrophages whereas NaHS onlyshowed a trend H2S is widely known for its anti-inflammatory prop-erties and its role in modulation of leukocyte influx [1944ndash48] Sinceproteinuria causes tubular damage and subsequent production ofchemokines and attraction of macrophages [49] the anti-inflammatory effects of H2S can also be explained by a reductionin proteinuria and a subsequent decline in tubular damage as evi-denced by decreased KIM-1 mRNA and protein levels

Treatment with H2S influences renal fibrotic pathways as evi-denced by a reduction in collagen 3 mRNA and protein and a

downregulation of TGFβ mRNA in both treatment groups The at-tenuation of fibrosis by H2S treatment is in concordance withliterature showing decreased fibrosis in various organs followingH2S exposure [4650ndash52] STS outperformed NaHS with regard todecreased αSMA levels which might be related to the lower levelof interstitial macrophages in the former Other studies showed aninhibiting effect of H2S on αSMA formation [5354]

Another functional property of H2S relates to the inhibition ofROS production Ang II induces oxidative stress by activating NADPHoxidases via the AT-1 receptor [55] We found diminished ROS pro-duction in H2S treated animals as evidenced by lower urinary MDAlevels This might be related to the prevented upregulation of NOX2in both H2S treated groups which is in line with literature showingthe effect of H2S on NADPH oxidases [56] H2S can also directly scav-enge ROS increase the intracellular glutathione levels and reducethe amount of ROS produced through modulation of mitochon-drial ROS production [142324] Loss of endogenous H2S-productioncauses increased susceptibility to renal ischemia [14] Productionof ROS plays an important role in the development of hyperten-sion [57] and treatment with antioxidants reduces blood pressurein experimental models for hypertension [5859]

Interestingly we observed a decline in CSE CBS and 3-MST mRNAlevels in the Ang II-infused animals treated with vehicle Consid-ering the vasorelaxing properties of H2S we expected acompensatory increase in these enzymes However levels of H2Sandor its producing enzymes were also decreased in other modelsof disease in which one might expect compensatory upregulation[142560ndash62] There is a concordance between progression of renaldisease and the decline in H2S producing capacity of renal tissue[25] suggesting that depleted levels of endogenous H2S produc-tion enhance renal damage We have not measured H2S since thereliability of the available techniques is controversial Treatment withNaHS and STS restored the expression of CSE CBS and 3-MST whichis probably related to a reduction in proteinuria and subsequentsalvage of tubular cells which are major producers of these enzymesThese expression data suggest that there is a shortage in renal en-dogenous H2S production in hypertensive renal disease whichimplies that intervention in the H2S producing pathway might bevaluable to increase renal levels of sulfide

One detailed study on the production of H2S from thiosulfateshowed that the amount and rate of H2S production varies signifi-cantly between species and organs Interestingly the magnitude andrate of H2S production was greatly amplified by the reducing agent

Fig 7 Treatment with NaHS and STS reduced oxidative stress After three weeks of Ang II infusion (A) NOX2 mRNA and (B) urinary MDA levels were increased 16-foldand 2-fold respectively Treatment with NaHS and STS reduced NOX2 mRNA expression by 38 and 39 and urinary MDA levels by 35 and 30 respectively (p lt 005p lt 0001 vs control p lt 005 p lt 001 vs Ang II + NaCl)

94 PM Snijder et alNitric Oxide 42 (2014) 87ndash98

Fig 8 Effect of NaHS and STS on renal fibrosis (A) In rats infused with Ang II and treated with vehicle renal TGF-β mRNA levels were upregulated Treatment with NaHSand STS prevented the upregulation of TGF-β Ang II infusion caused upregulation of (B) αSMA mRNA and (C) αSMA protein expression STS but not NaHS decreased thelevels of αSMA protein and mRNA (D) Representative photomicrographs of αSMA stained renal sections (E) Collagen 3 mRNA and (F) Collagen 3 protein levels were in-creased in Ang II-infused rats treated with vehicle In NaHS and STS treated rats the formation of Collagen 3 was decreased (G) Representative photomicrographs of Collagen3 stained renal sections (p lt 005 p lt 0001 vs control p lt 005 p lt 001 p lt 0001 vs Ang II + NaCl)

95PM Snijder et alNitric Oxide 42 (2014) 87ndash98

DTT in the presence of tissue with the most notable effects occur-ring in the liver H2S production was influenced by the amount ofoxygen present with increased H2S production in hypoxic circum-stances [29] These interesting results give us a small clue on the

relative H2S production from thiosulfate However absolute valuesmust be interpreted with caution because of the lack of a reliablemeasurement method for H2S The observation that STS as a majorH2S metabolite [27ndash29] has similar protective effects as NaHS pro-vides us with exciting possibilities for the translation into clinicaluse While short term treatment with STS is well tolerated the longterm side effects should be further explored One of the draw-backs for long term administration to renal patients is the currentlyavailable route of delivery To date STS is only given intravenouslyto patients with calciphylaxis because oral forms of the com-pound with a validated intestinal uptake have not been developedIt is unknown whether the observed effects of STS are solely me-diated by conversion to H2S or by unknown direct effects of STS itselfAnimals treated with STS had significantly increased sulfate levelsindicating that conversion to H2S and sulfite took place beforeforming sulfate [28] Effects of STS on blood pressure in humans havenot been described extensively however there are indications thatSTS exhibits vasodilating effects and antioxidant properties [323563]probably via the conversion to H2S [27ndash29] but also direct effectsof STS have been suggested [32]

Taken together our data reveal novel protective modalities of H2Streatment in experimental renal disease Therefore intervention inH2S related pathways may have therapeutic potential in hyperten-sion and hypertensive renal damage and deserves further explorationfor clinical application

Fig 9 Treatment with NaHS and STS restored the expression of CSE CBS and 3-MST mRNA After three weeks of Ang II infusion renal mRNA levels of (A) CSE (B) CBS and(C) 3-MST are decreased Treatment with NaHS and STS partially prevented this down regulation (p lt 001 p lt 0001 vs control p lt 005 vs Ang II + NaCl)

Fig 10 NaHS lowered intrarenal pressure partly via activation of KATP-channels Inan IPK setup a stable pre-constriction pressure of 200ndash250 mmHg was induced byperfusion with 1ndash2 μM PE Administration of 1 and 5 mM NaHS lowered intrarenalpressure Simultaneous perfusion with 1 mM Glibenclamide reduced the vasodilatoryproperties of 1 mM NaHS significantly Concentrations of NaHS below 1 mM and allconcentrations of STS had no effect on intrarenal pressure (p lt 005 p lt 0001vs baseline pressure p lt 005 vs NaHS)

96 PM Snijder et alNitric Oxide 42 (2014) 87ndash98

Disclosure

AP has support from an unrestricted research grant providedby Koumlhler Chemie HvG has support from two unrestricted re-search grants from the Dutch Kidney Foundation None of the otherauthors report a conflict of interest financially or otherwise re-garding this paper

Acknowledgments

The authors would like to express their gratitude towards SippieHuitema Marian Bulthuis Pieter Klok Petra Ottens Susanne Veldhuisand Jacco Zwaagstra for their excellent technical support Further-more we would like to thank Beatrix Blanchard for her valuable helpin measuring urinary thiosulfate concentrations This work was sup-ported by Grants (C08-2254 P13-114) from the Dutch KidneyFoundation and COST Action BM1005 ENOG European Networkon Gasotransmitters (wwwgasotransmitterseu)

References

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[19] RC Zanardo V Brancaleone E Distrutti S Fiorucci G Cirino JL WallaceHydrogen sulfide is an endogenous modulator of leukocyte-mediatedinflammation FASEB J 20 (2006) 2118ndash2120

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[22] N Krishnan C Fu DJ Pappin NK Tonks H2S-Induced sulfhydration of thephosphatase PTP1B and its role in the endoplasmic reticulum stress responseSci Signal 4 (2011) ra86

[23] Y Kimura Y Goto H Kimura Hydrogen sulfide increases glutathione productionand suppresses oxidative stress in mitochondria Antioxid Redox Signal 12(2010) 1ndash13

[24] Y Kimura H Kimura Hydrogen sulfide protects neurons from oxidative stressFASEB J 18 (2004) 1165ndash1167

[25] MA Aminzadeh ND Vaziri Downregulation of the renal and hepatic hydrogensulfide (H2S)-producing enzymes and capacity in chronic kidney diseaseNephrol Dial Transplant 27 (2012) 498ndash504

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[27] U Sen TP Vacek WM Hughes M Kumar KS Moshal N Tyagiet al Cardioprotective role of sodium thiosulfate on chronic heart failureby modulating endogenous H2S generation Pharmacology 82 (2008) 201ndash213

[28] C Szabo Hydrogen sulphide and its therapeutic potential Nat Rev Drug Discov6 (2007) 917ndash935

[29] KR Olson ER Deleon Y Gao K Hurley V Sadauskas C Batz et alThiosulfate a readily accessible source of hydrogen sulfide in oxygensensing Am J Physiol Regul Integr Comp Physiol 305 (2013) R592ndashR603

[30] S Farese E Stauffer R Kalicki T Hildebrandt BM Frey FJ Frey et al Sodiumthiosulfate pharmacokinetics in hemodialysis patients and healthy volunteersClin J Am Soc Nephrol 6 (2011) 1447ndash1455

[31] RP Singh H Derendorf EA Ross Simulation-based sodium thiosulfate dosingstrategies for the treatment of calciphylaxis Clin J Am Soc Nephrol 6 (2011)1155ndash1159

[32] MR Hayden DJ Goldsmith Sodium thiosulfate new hope for the treatmentof calciphylaxis Semin Dial 23 (2010) 258ndash262

[33] L Coentrao D Moura Acute cyanide poisoning among jewelry and textileindustry workers Am J Emerg Med 29 (2011) 78ndash81

[34] M Sooriyaarachchi A Narendran J Gailer The effect of sodium thiosulfate onthe metabolism of cis-platin in human plasma in vitro Metallomics 4 (2012)960ndash967

[35] JE Thomas G McGinnis Safety of intraventricular sodium nitroprusside andthiosulfate for the treatment of cerebral vasospasm in the intensive care unitsetting Stroke 33 (2002) 486ndash492

[36] Z Wang L Tang Q Zhu F Yi F Zhang PL Li et al Hypoxia-induciblefactor-1alpha contributes to the profibrotic action of angiotensin II in renalmedullary interstitial cells Kidney Int 79 (2011) 300ndash310

[37] N Watanabe S Kamei A Ohkubo M Yamanaka S Ohsawa K Makino et alUrinary protein as measured with a pyrogallol red-molybdate complexmanually and in a Hitachi 726 automated analyzer Clin Chem 32 (1986)1551ndash1554

[38] GL Newton R Dorian RC Fahey Analysis of biological thiols derivatizationwith monobromobimane and separation by reverse-phase high-performanceliquid chromatography Anal Biochem 114 (1981) 383ndash387

[39] A Roy AH Khan MT Islam MC Prieto DS Majid Interdependencyof cystathione gamma-lyase and cystathione beta-synthase in hydrogensulfide-induced blood pressure regulation in rats Am J Hypertens 25 (2012)74ndash81

[40] HD Francescato EC Marin Q Cunha Fde RS Costa CG Silva TM CoimbraRole of endogenous hydrogen sulfide on renal damage induced by adriamycininjection Arch Toxicol 85 (2011) 1597ndash1606

[41] U Sen P Basu OA Abe S Givvimani N Tyagi N Metreveli et al Hydrogensulfide ameliorates hyperhomocysteinemia-associated chronic renal failure AmJ Physiol Renal Physiol 297 (2009) F410ndashF419

[42] W Zhao R Wang H(2)S-induced vasorelaxation and underlying cellular andmolecular mechanisms Am J Physiol Heart Circ Physiol 283 (2002) H474ndashH480

[43] X Zhao LK Zhang CY Zhang XJ Zeng H Yan HF Jin et al Regulatory effectof hydrogen sulfide on vascular collagen content in spontaneously hypertensiverats Hypertens Res 31 (2008) 1619ndash1630

[44] EM Bos HG Leuvenink PM Snijder NJ Kloosterhuis JL HillebrandsJC Leemans et al Hydrogen sulfide-induced hypometabolism preventsrenal ischemiareperfusion injury J Am Soc Nephrol 20 (2009) 1901ndash1905

[45] EM Bos PM Snijder H Jekel M Weij JC Leemans MC van Dijk et alBeneficial effects of gaseous hydrogen sulfide in hepatic ischemiareperfusioninjury Transpl Int 25 (2012) 897ndash908

[46] PM Snijder RA de Boer EM Bos JC van den Born WP Ruifrok IVreeswijk-Baudoin et al Gaseous hydrogen sulfide protects against myocardialischemia-reperfusion injury in mice partially independent from hypome-tabolism PLoS ONE 8 (2013) e63291

[47] S Fiorucci E Antonelli E Distrutti G Rizzo A Mencarelli S Orlandi et alInhibition of hydrogen sulfide generation contributes to gastric injury causedby anti-inflammatory nonsteroidal drugs Gastroenterology 129 (2005) 1210ndash1224

[48] LF Hu PT Wong PK Moore JS Bian Hydrogen sulfide atte-nuates lipopolysaccharide-induced inflammation by inhibition of p38

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mitogen-activated protein kinase in microglia J Neurochem 100 (2007)1121ndash1128

[49] C Zoja A Benigni G Remuzzi Protein overload activates proximal tubular cellsto release vasoactive and inflammatory mediators Exp Nephrol 7 (1999)420ndash428

[50] LP Fang Q Lin CS Tang XM Liu Hydrogen sulfide suppresses migrationproliferation and myofibroblast transdifferentiation of human lung fibroblastsPulm Pharmacol Ther 22 (2009) 554ndash561

[51] HN Fan HJ Wang CR Yang-Dan L Ren C Wang YF Li et al Protective effectsof hydrogen sulfide on oxidative stress and fibrosis in hepatic stellate cells MolMed Rep 7 (2012) 247ndash253

[52] HJ Lee MM Mariappan D Feliers RC Cavaglieri K Sataranatarajan HEAbboud et al Hydrogen sulfide inhibits high glucose-induced matrix proteinsynthesis by activating AMP-activated protein kinase in renal epithelial cellsJ Biol Chem 287 (2012) 4451ndash4461

[53] CI Schwer P Stoll U Goebel H Buerkle A Hoetzel R Schmidt Effects ofhydrogen sulfide on rat pancreatic stellate cells Pancreas 41 (2012) 74ndash83

[54] G Tan S Pan J Li X Dong K Kang M Zhao et al Hydrogen sulfide attenuatescarbon tetrachloride-induced hepatotoxicity liver cirrhosis and portalhypertension in rats PLoS ONE 6 (2011) e25943

[55] R Agarwal RC Campbell DG Warnock Oxidative stress in hypertension andchronic kidney disease role of angiotensin II Semin Nephrol 24 (2004)101ndash114

[56] N Tyagi KS Moshal U Sen TP Vacek M Kumar WM Hughes Jr et al H2Sprotects against methionine-induced oxidative stress in brain endothelial cellsAntioxid Redox Signal 11 (2009) 25ndash33

[57] M Rathaus J Bernheim Oxygen species in the microvascular environmentregulation of vascular tone and the development of hypertension Nephrol DialTransplant 17 (2002) 216ndash221

[58] BA Mullan IS Young H Fee DR McCance Ascorbic acid reduces bloodpressure and arterial stiffness in type 2 diabetes Hypertension 40 (2002)804ndash809

[59] S Racasan B Braam DM van der Giezen R Goldschmeding P Boer HAKoomans et al Perinatal L-arginine and antioxidant supplements reduce adultblood pressure in spontaneously hypertensive rats Hypertension 44 (2004)83ndash88

[60] K Wang S Ahmad M Cai J Rennie T Fujisawa F Crispi et al Dysregulationof hydrogen sulfide producing enzyme cystathionine gamma-lyase contributesto maternal hypertension and placental abnormalities in preeclampsiaCirculation 127 (2013) 2514ndash2522

[61] D Kovacic N Glavnik M Marinsek P Zagozen K Rovan T Goslar et alTotal plasma sulfide in congestive heart failure J Card Fail 18 (2012) 541ndash548

[62] Y Zhang ZH Tang Z Ren SL Qu MH Liu LS Liu et al Hydrogen sulfidethe next potent preventive and therapeutic agent in aging and age-associateddiseases Mol Cell Biol 33 (2013) 1104ndash1113

[63] MR Hayden SC Tyagi L Kolb JR Sowers R Khanna Vascular ossification-calcification in metabolic syndrome type 2 diabetes mellitus chronic kidneydisease and calciphylaxis-calcific uremic arteriolopathy the emerging role ofsodium thiosulfate Cardiovasc Diabetol 4 (2005) 4

98 PM Snijder et alNitric Oxide 42 (2014) 87ndash98