Exploring Redox Biology in physiology and disease
Koning, Anne
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Chapter 7
H
2
S treatment
in renal ischemia-reperfusion injury
and renal metabolism in rats
Anne M. Koning Harry van Goor Dane H. Hoeksma Andreas Pasch Henri G.D. Leuvenink
Abstract
Ischemia-reperfusion injury (IRI) is a major cause of renal transplant dysfunction. Hydrogen sulfide (H2S) can attenuate IRI in mice by metabolic suppression. In larger animals applicability
of hypometabolism is unlikely. However, in these species H2S may be protective independent
of hypometabolism, e.g. by its vasodilatory, anti-inflammatory and anti-oxidant properties. This study investigates the effects of sodium hydrosulfide (NaHS) and sodium thiosulfate (STS) in a model of unilateral renal IRI in rats and the potential of NaHS to induce hypometabolism in isolated rat kidneys.
In vivo, rats underwent 35 min of unilateral renal warm ischemia (WI) and received vehicle, NaHS (5.6 mg/kg/day) or STS (1g/kg/day) via intra-peritoneal injections twice daily from 48 h before WI until sacrifice at 24 or 96 h after reperfusion (n=6-7/group). Kidneys were studied for overall structural (PAS staining) and tubular epithelial damage (KIM-1), oxidative stress (MDA), inflammation (macrophages; ED1) and early fibrotic changes (αSMA). Ex vivo, kidneys were exposed to 100 μM or 1 mM NaHS for 30 min and oxygen consumption and renal function parameters, including sodium reabsorption were compared to controls (n=2-5/group).
In kidneys from the in vivo renal IRI experiment, histology (PAS staining) and immunohistochemistry (KIM-1, ED1, αSMA) showed no benefit from NaHS or STS treatment. Also, at the mRNA level no differences were found (KIM-1, αSMA). MDA levels were the same for all treatment groups. Ex vivo, H2S reduced oxygen consumption and sodium reabsorption
both by over 50% (p<0.05).
In conclusion, in vivo, NaHS and STS treatment by twice daily intra-peritoneal injections did not protect against renal IRI in rats. Ex vivo, NaHS-induced metabolic suppression in isolated rat kidneys, indicating a possible role for H2S as a treatment modality for kidney transplant
1 Introduction
In end-stage renal disease, patients rely on replacement therapy and transplantation is the treatment of choice. Unfortunately, the transplant process is inherently associated with ischemia-reperfusion injury (IRI), which is a major cause of kidney transplant dysfunction.(1)
Gaseous signalling molecule hydrogen sulfide (H2S) can attenuate renal IRI in mice by
metabolic suppression, through reversible inhibition of complex IV of the mitochondrial electron transport chain.(2,3) Although the applicability of H2S-induced hypometabolism in
larger animals is improbable, H2S is also considered to be protective independent of
metabolic suppression, e.g. by exerting vasodilatory, anti-inflammatory and anti-oxidant effects.(4–10) Accordingly, benefit of H2S treatment in the context of renal IRI has not only
been described in mice, but also in rats and pigs.(11–24)
In this study, apart from sulfide salt sodium hydrosulfide (NaHS), sodium thiosulfate is used as a treatment modality. Although thiosulfate (TS) is generally dismissed as a break down product of H2S, there is evidence to support its reconversion into H2S in vivo.(25–28) Also,
previous studies have shown TS to offer protection in various models of disease, including Angiotensin II-induced hypertensive renal damage.(29–33) This form of H2S treatment is of
particular interest as TS can safely be administered to humans. In fact, it is clinically applied, e.g. for the treatment of calciphylaxis in dialysis patients.(34)
The aim of this study is to investigate the effects of H2S treatment, in the form of NaHS and
STS, in a model of unilateral renal IRI in rats and to determine the potential of NaHS to induce a hypometabolic state in isolated rat kidneys. Hereby, we wish to contribute to the expansion of knowledge on the protective properties of H2S and their applicability in renal
transplantation.
2 Methods
2.1 Animals
Male Sprague Dawley (200-220 gram, in vivo experiment) and Fisher 344 (240-270 gram, ex vivo experiment) rats (Harlan, Zeist, the Netherlands) were housed at the animal research facility under standard conditions with a 12 h light-dark cycle and access to food and water ad libitum. Experimental procedures were in agreement with institutional and legislator regulations and approved by the local ethics committee for animal experiments.
2.2 Ischemia-reperfusion protocol and NaHS and STS treatment
Under general anesthesia (2% isoflurane in O2), a midline abdominal incision was made and
rats underwent 35 min of unilateral renal warm ischemia (WI) by clamping of the left renal artery and vein using non-traumatic clamps.
From 48 h before WI until sacrifice at 24 of 96 h after reperfusion rats received vehicle (0.9% NaCl, n=6/group), NaHS (5.6 mg/kg/day, Sigma, Zwijndrecht, the Netherlands, n=7/group) or STS (1 g/kg/day, Sigma, Zwijndrecht, the Netherlands, n=7/group) twice daily via intra-peritoneal injections.
When rats were sacrificed, the left kidney was perfused with 0.9% NaCl and harvested. Transverse slices were fixed in 4% paraformaldehyde and paraffin embedded for histological and immunohistochemical analysis or immediately snap frozen in liquid nitrogen and stored at -80 °C for molecular analysis.
2.3 Histology
Periodic acid-Schiff (PAS) staining was performed according to the standard protocol. Stained sections were subjected to blinded evaluation by light microscopy, resulting in a tubular damage score ranging from 1 to 6 (<5% to 75-100% damage).
2.4 Immunohistochemistry
Deparaffinised kidney sections were incubated overnight with 0.1 M Tris/HCl buffer (pH 9.0) at 80 °C for antigen retrieval. Endogenous peroxidase was blocked with 0.075% H2O2 in
phosphate buffered saline (PBS, pH 7.4) for 30 min. Section were incubated with primary antibodies for KIM-1 (rabbit anti-KIM-1 peptide 9, 1:400, gift V. Baily), macrophages (mouse anti-CD68 ED1, MCA341R AbD, 1:750, Serotec Ltd, Oxford, UK) or αSMA (mouse anti-SMA, clone 1A4 A2547, 1:10.000, Sigma, Zwijndrecht, the Netherlands) for 60 min at room temperature. Binding was detected by sequential incubation with peroxidase-labelled secondary and tertiary antibodies (Dakopatts, Glostrup, Denmark) for 30 min at room temperature. All antibodies were diluted with PBS supplemented with 1% bovine serum albumin. At the secondary and tertiary antibody dilutions 1% normal rat serum was added. Peroxidase activity was developed for 10 min using 3,3’-diaminobenzidine tetrachloride containing 0.03% H2O2. Counterstaining was performed using Mayer’s hematoxylin.
Kidney sections were scanned using an Aperio Scanscope slide scanner (Aperio Technologies, Vista, CA, USA). Tubular epithelial damage (KIM-1), macrophages (ED1) and early fibrotic changes (αSMA) were determined using the Aperio positive pixel analysis v9.1 algorithm and expressed as the number of positive pixels per μm of the cortical and corticomedullary border surface area. Analysis was performed in a blinded fashion.
2.5 Qualitative real-time polymerase chain reaction
Kidney tissue, consisting of cortex and medulla, was homogenized in lysis buffer and total RNA was extracted using the TRIZOL method (Invitrogen, Carlsbad, USA). RNA concentrations were measured by means of a nanodrop UV-detector (Nanodrop Technologies, Wilminton, DE). cDNA was synthesized using Superscript II with random hexamer primers (Invitrogen, Carlsbad, USA). Gene expression (Applied Biosystems, Foster City, CA, USA) was determined by qualitative real-time PCR (qRT-PCR) based on the Taqman methodology. HPRT was used as a housekeeping gene with the following primers (Integrated DNA Technologies) and probe (Eurogentec): Forward: 5’- GCC CTT GAC TAT AAT GAG CAC TTC A-3’, Reverse: 5’-TCT TTT AGG CTT TGT ACT TGG CTT TT-3’ and Probe: 6-FAM 5’-ATT TGA ATC ATG TTT GTG TCA TCA GCG AAA GTG-3’ TAMRA. Other primers were obtained from Applied Biosystems as Assays-on-Demand (AOD) gene expression products. The AOD IDs used were: Acta2 (αSMA) Rn01759928_g1 and Havcr1 (KIM-1) Rn00597703_m1. The
qRT-PCR reaction mixture contained 20 ng cDNA template and 5 μl qRT-PCR-master mix. Nuclease free water was added to a total volume of 10 μl. All assays were performed in triplicate. The thermal profile was 15 min at 95 °C, followed by 40 cycles of 15 sec at 95 °C and 1 min at 60 °C. The average Ct values for target genes were subtracted from the average housekeeping gene Ct values to yield the delta Ct. Results were expressed as 2-ΔCt.
2.6 Tissue malondialdehyde measurement
Malondialdehyde (MDA) was measured in kidney tissue after binding to thiobarbituric acid (TBA). Twenty μl of tissue homogenate was mixed with 2% sodium dodecyl sulfate and 5 mM butylated hydroxytoluene, followed by 400 μl 0.1 N HCL, 50 μl 10% phosphotungstic acid and 200 μl 0.7% TBA. The mixture was incubated for 1 h at 97 °C. Subsequently, 800 μl 1-butanol was added and the sample was centrifuged at 960 g. Fluorescence of 200 μl of the 1-butanol supernatant was measured at 480 nm excitation and 590 nm emission wavelengths. Results were expressed as μmol/g protein.
2.7 Isolated perfused kidney setup
Under general anesthesia (2% isoflurane in O2), through a midline abdominal incision, kidneys
of rats were isolated. Depending on the vascular anatomy, either the left or right kidney was chosen to be used for the experiment. Of this kidney, the artery, vein and ureter were cannulated. Subsequently, the kidney was placed in the isolated perfused kidney (IPK) setup and continuously perfused at a constant pressure of 100 mmHg with a warmed (37 °C) and oxygenated (95% O2 and 5% CO2 gas mixture) Krebs-Ringer Bicarbonate solution
complemented with albumin and creatinine, at a pH of 7.5 ± 0.05 and a PO2 ≈ 60 kPa. The
perfusion solution was composed of 118.6 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM
KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 6.1 mM glucose, 50 g/l albumin and 7.1 mM
creatinine. The pressure was regulated by means of a roller pump (Ismatec mv-ca/04, Ismatec, Glattbrugg, Switzerland) in combination with an electromechanical pressure transducer (Cobe; Arvada, CO), connected to a computer interface (LabView, National Instruments, Austin, TX).
Control kidneys (n=4) did not receive NaHS and were perfused for 60 min. Other kidneys were exposed to 100 μM (n=4) or 1 mM (n=5) NaHS for 30 min - from 30 min after the start of perfusion until the end of the experiment at 60 min. To assess reversibility of the effect of NaHS, a final set of kidneys (n=2) was also exposed to 1 mM NaHS for 30 min, yet from 15 min after the start of perfusion, followed by a wash out period of 30 min.
Oxygen consumption by the kidney was determined every 15 min, on the basis of the flow and the decline of the oxygen concentration of the perfusate passing through the kidney. The perfusate’s oxygen concentration was measured using flow-through oxygen sensors, incorporated in the afferent and efferent line of the IPK setup, in combination with optical oxygen meter Fibox 4 and PreSens Datamanager software (PreSens - Precision Sensing GmbH, Regensburg, Germany). At the same time points, afferent and efferent perfusate samples and a timed urine sample were taken. Urine samples were weighed and all samples were snap frozen and stored at -20 °C until analysis. Perfusate and urine levels of creatinine,
sodium and potassium were determined by standard assays from Roche on the Roche Modular (Roche Diagnostics GmbH, Mannheim, Germany) according to routine procedures in our clinical chemical laboratory.
2.6 Statistical analysis
Statistical analysis was performed with and graphs were drawn in GraphPad Prism (version 5.0, GraphPad Software, La Jolla, California, USA).
All data are presented as median (interquartile range (IQR)). Treatment groups were compared by means of the Kruskal-Wallis test for unrelated samples or the Friedman test for repeated measures. In both cases the test was complemented with Dunns post test to compare all pairs of columns. Statistical analysis was performed for groups with n ≥ 4. Values of P<0.05 were considered statistically significant.
3 Results
3.1 NaHS and STS treatment had no effect on renal ischemia-reperfusion injury in vivo Assessment of structural damage (PAS staining), tubular epithelial damage (KIM-1 mRNA and protein) and oxidative stress (lipid peroxidation, measured as tissue MDA) 24 hours after reperfusion, as well as influx of macrophages (ED1 protein) and early fibrotic changes (αSMA mRNA and protein) 96 hours after reperfusion revealed no differences between controls and NaHS and STS treated rats (Fig. 1-4).
Figure 1: IRI-induced structural damage
24 hours after reperfusion of the kidney Periodic acid-Schiff staining (A), KIM-1 protein (B), and KIM-1 mRNA (C) levels showed no differences in tubular damage between controls (n=6) and NaHS (n=7) or STS (n=7) treated rats.
Data are presented as median (IQR)
Figure 4: IRI-induced early fibrotic changes
7
96 hours after reperfusion αSMA mRNA (A) and protein (B) levels showed no differences in early fibrotic changes between controls (n=6) and NaHS (n=7) or STS (n=7) treated rats.
Data are presented as median (IQR).
NaHS; sodium hydrosulfide, STS; sodium thiosulfate, IRI; ischemia-reperfusion injury
24 hours after reperfusion of the kidney there were no differences in lipid peroxidation - as determined by tissue MDA levels - between controls (n=6) and rats treated with NaHS (n=7) or STS (n=7).
Data are presented as median (IQR).
NaHS; sodium hydrosulfide, STS; sodium thiosulfate, IRI; ischemia-reperfusion injury, MDA; malondialdehyde
96 hours after reperfusion of the kidney ED1 staining revealed no differences in influx of macrophages between controls (n=6) and NaHS (n=7) or STS (n=7) treated rats. Data are presented as median (IQR).
IRI; ischemia-reperfusion injury, NaHS; sodium hydrosulfide, STS; sodium thiosulfate
3.2 NaHS-induced hypometabolism ex vivo
In the IPK setup NaHS was found to decrease renal oxygen consumption in a dose dependent manner (Fig. 5, A-C). Data from two kidneys suggest reversibility of this effect (Fig. 5, D). Furthermore, 1 mM NaHS was found to significantly decrease the fractional reabsorption of sodium by the kidney (P<0.01), whereas other renal function parameters (urine production, creatinine clearance and excretion of potassium) were unaffected (Fig. 6).
Figure 5: Oxygen consumption by the isolated perfused kidney
NaHS decreased renal oxygen consumption in a dose dependent manner (A-C, n=4 for controls, n=4 for kidneys exposed to 100 μM NaHS and n=5 for kidneys exposed to 1 mM NaHS). Data from two kidneys suggest this effect to be reversible (D). Data are presented as median (IQR), **P<0.01. NaHS; sodium hydrosulfide, STS; sodium thiosulfate
Figure 6: Function of the isolated perfused kidney
After 30 min NaHS did not significantly change urine production (A) or creatinine clearance (B). 1 mM NaHS significantly decreased sodium reabsorption (A). Potassium excretion was not changed by NaHS (B). n=4 for controls, n=4 for kidneys exposed to 100 μM NaHS, n=5 for kidneys exposed to 1 mM NaHS, data are presented as median (IQR), **P<0.01.
NaHS; sodium hydrosulfide, STS; sodium thiosulfate
4 Discussion
In the context of renal IRI in rats, this study shows no benefit of NaHS and STS treatment through twice daily intra-peritoneal injections. Ex vivo, however, NaHS did significantly supress metabolism in the isolated rat kidney, as evidenced by the decrease of renal oxygen consumption and tubular sodium reabsorption.
The treatment regimens applied in our in vivo IRI experiment have previously been shown by our group to protect against Angiotensin II-induced hypertensive renal damage.(30) The absence of any sign of protection in our model was unexpected and, at first glance, does not comply with existing evidence on H2S treatment in renal IRI and transplantation. Indeed, NaHS
has repeatedly been shown to protect the kidney from IRI, even in rats and when administered through intra-peritoneal injections.(11,13,17,18,21,24) However, notably, in all of these studies H2S treatment was applied close to or during reperfusion, whereas in our case the
time between restoration of blood flow and administration of treatment was much longer.(11,13,17,18,21,24) Thus, although in the context of cardiac IRI positive results of H2S
pre-treatment have been described, application of therapy close to or even during the reperfusion phase may be the key to protection of rat kidneys in vivo.(35,36) This may be
related to the unique way in which renal blood flow is regulated. Indeed, while in most organs vascular tone is adapted in response to tissue oxygenation, in the kidney blood flow is relatively unresponsive to hypoxia or hyperoxia and controlled by mechanisms that regulate renal function, i.e. glomerular filtration and tubular reabsorption.(37) Under normal conditions, arterial-to-venous (AV) oxygen shunting helps to retain renal tissue oxygenation within a physiological range. However, the same mechanism causes the kidney to be vulnerable in case of hypoxia. Conversely, during reperfusion AV oxygen shunting is likely to be inadequate. As a result, the kidney is susceptible to hyperoxia and consequent oxidation.(37)
Aside from the warm ischemia and reperfusion that occur in vivo, ex vivo preservation of the kidney is another phase of the transplant process in which renal damage arises. Traditionally, during this phase the evident mismatch between oxygen supply and demand is confined by suppression of the kidney’s metabolism through the induction of hypothermia. However, hypothermia itself is a cause of renal damage and, accordingly, prolonged cold storage significantly contributes to kidney transplant dysfunction.(38) H2S therapy possibly
represents an opportunity to accomplish metabolic suppression in the kidney without the detrimental effects associated with hypothermia. While in mice H2S can safely induce a
reversible hypometabolic state, studies in larger animals, including pigs and sheep, have questioned the applicability of this phenomenon in large mammals, including humans. As previously described by Haouzi et al., differences in mechanisms controlling resting metabolic rate in relation to body mass may explain discrepancies in the response to H2S, dosed
according to body mass. Possibly, much higher concentrations of H2S would induce metabolic
suppression in larger animals, however, considering H2S toxicity at higher levels, this would
not be without side-effects.(39) Even though a therapeutic dose of H2S may not affect whole
body metabolism in larger animals as it does in mice, our data show that NaHS is certainly able to decrease metabolism in isolated rat kidneys. STS was not applied in the IPK setup because previous research suggests that the reconversion of TS into H2S predominantly
occurs in the liver and under hypoxic circumstances.(28) The unlikeliness of TS conversion in the IPKis supported by an earlier IPK experiment by our group in which STS, in contrast to NaHS, was found to have no effect on intra-renal pressure.(30) One might argue that 1 mM NaHS - the dose that resulted in the most significant suppression of renal metabolism - is high and potentially toxic. However, cell damage can not have caused the observed decrease of renal oxygen consumption as this was found to be reversible. Our finding indicates a possible role for H2S therapy in the kidney transplant preservation phase and warrants confirmation in
porcine and eventually human kidneys. While our ex vivo experiment was not followed by transplantation of the kidneys and, therefore, outcome could not be assessed, previous studies by Lobb et al. have shown beneficial effects of adding 150 μM NaHS to University of Wisconsin preservation solution.(18,23) NaHS treatment was shown to increase graft function, attenuate necrosis, apoptosis and inflammation and increase recipient survival. However, it is unclear from these studies whether the improved outcome is the result of H2S-induced
hypometabolism, other protective properties of H2S or both. Notably, all kidneys were stored
at 4 °C.(18,23) Thus, whether and at which dose H2S therapy actually permits normothermic
Apart from the caveats mentioned elsewhere in this discussion, the limitations of our study include the use of different strains of rats for the in vivo and the ex vivo experiment. We can not be sure NaHS would similarly have induced a hypometabolic state in the kidneys from Sprague Dawley rats. At the same time, NaHS and STS treatment, as applied in this study, may have shown protection against renal IRI in Fisher 344 rats. Furthermore, the unilateral IRI model, chosen to be used for animal welfare reasons, did not permit assessment of renal function following IR. Therefore, any functional benefit of treatment that may have been present could not be detected.
In conclusion, NaHS and STS therapy by twice daily intra-peritoneal injections did not protect against renal IRI in rats. Possibly, application of treatment close to or even during the reperfusion phase is essential to in vivo protection of the kidney. Ex vivo, NaHS induced metabolic suppression in isolated rat kidneys. This points towards a possible role for H2S as a
treatment modality for kidney transplant protection during the preservation stage.
Funding
This work was supported by grants from the Dutch Kidney Foundation (IP13-114, to HvG) and the Groningen University Institute for Drug Exploration (to AMK).
Acknowledgements
The authors would like to express their gratitude to Pieter Klok, Annemiek Schaafsma, Sander Twickler, Petra Ottens, Susanne Veldhuis, Sippie Huitema and Marian Bulthuis for their excellent technical assistance.
Conflicts of interest None declared.
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