Protective effect of hydrogen sulfide on gentamicin-induced renal injury

Abstract

Objective: We evaluated the potential protective effect of hydrogen sulfide (H₂S) against GEN-induced nephrotoxicity in rats. Materials and methods: Twenty-four rats were randomly divided into four groups, each consisting of six animals as follows: (1) the rats were control, (2) intraperitoneally injected with GEN 14 consecutive days (100 mg/kg/day), (3) treated with GEN plus %0.9 saline intraperitoneally for 14 days and (4) treated with GEN plus sodium hydrogen sulfide (NaHS)-exogenous H₂S donor (56 µmol/kg/day) for 14 days. After 15 days, rats were sacrificed and their kidneys were taken and blood analysis was performed. Twenty-four hours urine collections were obtained in standard metabolic cages a day before the rats were sacrificed. Tubular necrosis and interstitial fibrosis scoring were determined histopathologically in a part of kidneys; nitric oxide (NO), malondialdehyde (MDA) and reduced glutathione (GSH) levels were determined in the other part of kidneys. Statistical analyses were made by the chi-squared test and one-way analysis of variance. Results: Serum urea and creatinine levels were significantly higher in rats treated with GEN alone, than the rats in control and GEN + NaHS groups. The GSH levels in renal tissue of only GEN-treated rats were significantly lower than those in control group, and administration of NaHS to GEN-treated rats significantly increased the level of GSH. The group that was given GEN and NaHS had significantly lower MDA and NO levels in kidney cortex tissue than those that was given GEN alone. In rats treated with GEN + NaHS, despite the presence of mild tubular degeneration and tubular necrosis are less severe, and glomeruli maintained a better morphology when compared with GEN group. Discussion: We can say that H₂S prevent kidney damage with antioxidant and anti-inflammatory effect.

• Gentamicin
• hydrogen sulfide
• kidney
• nephrotoxicity
• oxidative stress

Introduction

The kidney is a vital organ, which plays an essential role in health, disease, and overall development and growth. The main function of kidney is to maintain total body fluid volume, its composition and acid–base balance. A number of environmental variables including certain drugs influence these functions.1–3 Gentamicin (GEN) derived from Gram-positive bacteria called Micromonospora purpurea present in soil and water having potential in treating aerobic Gram-negative bacteria. Accumulation of gentamicin in proximal renal tubules may cause nephrotoxicity which leads to brush border network damage.4 The nephrotoxicity involves renal free radical production and accumulation, consumption of antioxidant defense mechanisms, glomerular congestion and acute tubular necrosis,5–7 leading to diminished creatinine clearance and renal dysfunction. The pathological mechanisms also involve elevation of endothelin-1, up-regulation of transforming growth factor-beta (TGF-β), significant increase in monocyte/macrophage infiltration into the renal cortex and medulla, augmentation of oxidative stress, and apoptosis and also necrosis.8,9 Moreover, GEN has also been shown to enhance the generation of reactive oxygen species (ROS). Lipid peroxidation (LPO) mediated by ROS has been suggested as a causative agent of cell death in different pathological states including various models of renal diseases.10 Besides their direct damaging effects on tissues, ROS seem to trigger the accumulation of leukocytes in the tissue involved, and thus cause tissue injury indirectly through activated neutrophils. It has been shown that activated neutrophils secrete enzymes such as myeloperoxidase, elastase, and proteases and liberate oxygen radicals. On the other hand, in vivo and in vitro studies have shown that the scavengers of reactive oxygen metabolites are protective in GEN-induced renal failure.11

As previously, an antifibrotic, antiflammatory and antioxidative agent as hydrogen sulfide (H₂S) was described. For decades, hydrogen sulfide (H₂S) has been known as a toxic gas, and, together with nitric oxide (NO) and carbon monoxide (CO), it is currently recognized as an endogenous gaseous physiological molecule.12 H₂S is synthesized from cysteine by two pyridoxal-5′-phosphate dependent enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), and a pyridoxal-5′-phosphate-independent enzyme, 3-mercaptpyruvate sulfurtransferase (3-MST), in most mammalian tissues, including the kidney.13,14 Progression of fibrosis is associated with oxidative stress, inflammatory responses, vascular tone and intracellular signaling pathways. Recent studies in human and animal have demonstrated involvement of H₂S in those factors in various diseases, including atherosclerosis, ischemia and reperfusion (I/R) injury, hypertension and end-stage renal disease (ESRD).13–15 In a previous study, H₂S supplementation was associated with the suppressions of oxidative stress, inflammation and nitrosative stress.16

Because of these effects of H₂S, in this study we investigated the role of H₂S in renal damage with GEN. We evaluated the antifibrotic anti-inflammatory and antioxidative effects of H₂S in rat kidneys. The nephroprotective effect of i H₂S is previously established in many studies, however, to our knowledge this is the first study in literature concerning the protective role of hydrogen sulfide against GEN nephrotoxicity.

Materials and methods

Drugs and animals

Male Wistar albino rats (200–250 g) were housed in clean plastic cages in a temperature and humidity-controlled facility with a constant 12 h light/dark cycle with free access to food and water. The use of animals and the experimental protocol were approved by the Institutional Animal Care and Use Committee and animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals of Research Council. Gentamicin (GEN) was purchased from Bilim Pharmaceuticals. GEN was dissolved in saline and injected intraperitoneally. Like previous study, sodium hydrogen sulfide (NaHS)-exogenous donor of H₂S (Merck, Schuchardt, OHG, Hohenbrunn, Germany), was administered intraperitoneally 56 µmol/kg/day for 14 days.17 NaHS was freshly dissolved with 0.9% saline.

Treatment and experimental design

After a quarantine period of 7 days, 32 rats were randomly divided into four equal groups, each consisting of six animals as follows: (1) controls, (2) intraperitoneally injected with GEN for 14 consecutive days (100 mg/kg/day), (3) treated with GEN plus %0.9 saline intraperitoneally for 14 days and (4) treated with GEN plus NaHS (intraperitoneally 56 µmol/kg/day) for 14 days. Rats were treated for 14 days. NaHS was administered immediately after injection of GEN. After 15 days, rats were killed and their kidneys were taken, and blood analysis was performed. Tubular necrosis and interstitial fibrosis scores were determined histopathologically in a part of kidneys; NO, malondialdehyde (MDA), and reduced glutathione (GSH) levels were determined in the other part of kidneys. Urea, creatinine, sodium (Na) and potassium (K) levels were investigated in a blood analysis.

Biochemical assays

Twenty-four hours after the administration of last doses of NaHS, on 15th day, rats were anesthetized by intraperitoneal injection of ketamine and sacrificed. Renal cortical tissues were separated into two parts for biochemical analysis and light microscopic examination. Blood samples were also taken by cardiac puncture to assess the serum levels of urea and creatinine concentrations. In frozen tissues biochemically malondialdehyde (MDA), end product of lipid peroxidation, reduced glutathione (GSH), non-enzymatic antioxidant, and total nitrite, a stable product of nitric oxide (NO), were evaluated as a means of oxidative stress. Renal impairment was assessed by serum urea and creatinine levels, as well as by the kidney histology. Serum urea, creatinine, Na and K levels were determined with an autoanalyzer (Syncron LX20, Dublin, Ireland) by using commercial Becman Coulter diagnostic kits. Kidney tissue (300 mg) was homogenized in ice-cold tamponade containing 150 mM KCL for determination of MDA. MDA levels were assayed for products of lipid peroxidation. MDA referred to as thiobarbituric acid reactive substance, was measured with thiobarbituric acid at 532 nm using a spectrofluorometer, as described previously.18 GSH was determined by the spectrophotometric method, which was based on the use of Ellman’s reagent.19 Total nitrite (NOx) was quantified by the Griess reaction20 after incubating the supernatant with Escherichia coli nitrate reductase to convert NO₃ to NO₂. Griess reagent (1 ml 1% sulfanilamide, 0.1% naphthyl-ethylenediamine hydrochloride and 2.5% phosphoric acid; Sigma Chemical Co., St. Louis, MO) was then added to 1 ml of supernatant. The absorbance was read at 545 nm after a 30-min incubation. The absorbance was compared with the standard graph of NaNO₂, obtained from the reduction of NaNO₃ (1–100 mmol/l). The accuracy of the assay was checked in two ways; the inter- and intraassay coefficients of variation were 7.52% and 4.61%, respectively. To check conversion of nitrate to nitrite (recovery rate), known amounts of nitrate were added to control plasma samples; these samples were deproteinized and reduced as above.

Histopathological examinations

Histopathological evaluation of the kidney tissues was done. Paraffin embedded specimens were cut into 6 -μm thickness and stained with Hematoxylin–Eosin stain for light microscopic examination using a conventional protocol21 (Olympus, BH-2, Tokyo, Japan). A semi-quantitative evaluation of renal tissues was accomplished by scoring the degree of severity according to previously published criteria.22 All sections of kidney samples were examined for tubular necrosis. Briefly, minimum of 50 proximal tubules associated with 50 glomeruli were examined for each slide and an average score was obtained. Severity of lesion was graded from 0 to 3 according to the percentage of tubular involvement. Slides were examined and assigned for severity of changes using scores on scale in which (0) denotes no change; grade (1) changes affecting <25% tubular damage (mild); grade (2) changes affecting 25–50% of tubules (moderate); grade (3) changes affecting >50% of tubules (severe). Histopathological evaluation was performed on left kidney tissues. Paraffin-embedded specimens were cut into 5-mm thick sections and stained with hematoxylin and eosin, and Masson’s trichrome for examination under the light microscope (BH-2; Olympus, Tokyo, Japan). To evaluate leukocyte infiltration, the widening of interstitial spaces with focal leukocyte infiltration was assessed in five randomly chosen sections prepared from each kidney sample. For each section, the average number of leukocytes per 0.28 mm² was calculated from these leukocyte-infiltrated foci using a high-power microscopic field (*400). To estimate the grade of interstitial fibrosis, the interstitial area that was stained green with Masson’s trichrome was evaluated as a percentage of the total examined area in five randomly chosen sections prepared from each kidney sample using an image analyzer (Leica; Leica Micros Imaging Solutions, Cambridge, UK). For each section, interstitial space widening with focal leukocyte infiltration and interstitial fibrosis was assessed in high-power fields (*400) to quantify the results. The Banff classification of kidney pathology was used for scoring the degree of mononuclear cell infiltration and interstitial fibrosis. The score was graded from 0 to 3, depending on the severity of histological characteristics.23

Statistical analysis

Results of all groups were shown as mean values ± SD. Statistical analyses of the histopathologic evaluation of the groups were carried out by the chi-squared test and biochemical data were analyzed by the one-way analysis of variance (ANOVA). The significance between two groups was determined by the Dunnett’s multiple comparison test, and p < 0.05 was accepted as statistically significant value.

Results

No deaths or remarkable signs of external toxicity were observed in the groups of rats given GEN either alone or combination with PE. The biochemical and histopathological results were similar for the control and PE groups, and we decided to consider them without distinction and report only the control group.

Urinary volume

The 24-h urine volume in group GEN was significantly higher than in control group (p < 0.01), indicating the presence of GEN-induced polyuria, whereas in group GEN-NaHS, it was not different from that in control group, pointing out the protective role of NaHS against acute tubular necrosis (Table 1).

Table 1. Effects of GEN alone and its combination with H₂S on plasma urea, creatinine, Na, K and 24-h urine volume levels in rats.

Parameters	Control (Group 1)	GEN (Group 2)	GEN + Ve (Group 3)	GEN + NaHS (Group 4)
Urea (mg/dl)	33 ± 7.8	107 ± 13.9^a	105 ± 13.4	42.8 ± 6.9^b
Creatinine (mg/dl)	0.42 ± 0.1	2.08 ± 0.4^a	1.97 ± 0.4	0.98 ± 0.09^b
Na (mmol/l)	138.9 ± 1.3	139.9 ± 2.5	139.3 ± 2.2	137.8 ± 1.9
K (mmol/l)	3.88 ± 0.2	4.1 ± 0.5	4.04 ± 0.4	3.94 ± 0.28
24-h urine  volume (ml)	8.9 ± 1.2	21.2 ± 4.1^a	20.1 ± 3.2	10.8 ± 2.4^b

Notes: Values are expressed as mean ± SD for six rats in each group.

Groups: Control, GEN (gentamicin), GEN + Ve (gentamicin + vehicle), GEN + NaHS (gentamicin + sodium hydrogen sulfide).

^aSignificantly different from control.

^bSignificantly different from gentamicin group (p < 0.001).

Biochemical variables in plasma and tissue

Na⁺ and K⁺ concentrations among the four groups were similar. Serum urea and creatinine levels were significantly higher in rats treated with GEN alone than the rats in control and GEN + NaHS groups (p < 0.01). Administration of NaHS to the GEN-treated rats caused decrease in serum urea and creatinine levels (Table 1). The GSH levels in renal tissue of only GEN-treated rats were significantly lower than those in control group (p < 0.05), and administration of NaHS to GEN-treated rats significantly increased the level of GSH (p < 0.05) (Table 2 and Figure 1).

Figure 1. (a) Normal tubulus and glomerules in kidney cortex H&E × 100 (control group). (b) Severe tubular necrosis, tubular degeneration and epithelial vacuolization in the proximal tubules ×H&E 100 (GEN-treated group). (c) Moderate tubular necrosis, tubular degeneration and epithelial vacuolization in the proximal tubules H&E × 200 (GEN + vehicle-treated group). (d) Mild epithelial granulovacuolization in the proximal tubules and normal glomerules H&E × 100 (GEN + NaHS-treated group).

Table 2. Effects of H₂S on rat kidney NO, MDA and GSH levels.

Parameters	Control (Group 1)	GEN (Group 2)	GEN + Ve (Group 3)	GEN + NaHS (Group 4)
NO (nmol/g  wet tissue)	29.8 ± 8.5	47.6 ± 7.2^a	44.9 ± 5.7	32.8 ± 6.4^b
MDA (nmol/g  wet tissue)	2.6 ± 0.7	4.2 ± 1.1^a	3.9 ± 1.1	2.8 ± 1.6^b
GSH (μmol/g  wet tissue)	2.4 ± 0.9	1.4 ± 0.5^a	1.3 ± 0.6	2.3 ± 0.6^b

Notes: Values are expressed as mean ± SD for six rats in each group.

NO, nitric oxide; MDA, malondialdehyde; GSH, glutathione.

^aSignificantly different from control.

^bSignificantly different from gentamicin group (p < 0.05).

The group gave GEN and NaHS had significantly lower MDA levels in kidney cortex tissue than those given GEN alone. There was high level of NO in GEN-treated group; however, NO levels in group treated with GEN + NaHS were significantly lower than GEN-treated group (Table 2 and Figure 1).

Histopathologic examinations results

Histopathologic examination of kidney showed that there were no pathologic findings in control group (Figure 1a). In rats treated with GEN and GEN + vehicle, there were mild and severe tubular necrosis, tubular degeneration, and epithelial vacuolization in the proximal tubules, and parietal cell hyperplasia compared to control group (Figure 1b and c). In rats treated with GEN + NaHS, despite the presence of mild tubular degeneration and epithelial vacuolization in the proximal tubules, parietal cell hyperplasia, and tubular necrosis are less severe, and glomeruli maintained a better morphology when compared with GEN group (Figure 2b). These changes are summarized in Table 3. After staining with Masson trichrome, no statistical difference was found between groups in kidney fibrosis scores (Table 3 and Figure 3a–c).

Figure 2. (a) Severe mononuclear leukocyte infiltration in the cortex of GEN-treated group. (hematoxylin & eosin, ×400). (b) Leukocyte infiltration was reduced in the NaHS-treated group (hematoxylin & eosin, ×400).

Figure 3. (a) Normal kidney morphology in a control group (masson & trichrome ×200). (b) Severe fibrosis was observed in the peritubular interstitium of the GEN-treated group (masson & trichrome ×400). (c) Mild fibrosis was reduced in the NaHS-treated group (masson & trichrome ×400).

Table 3. Semiquantitative analysis of tubular necrosis, interstitiel fibrosis, leukocte infiltration in control, GEN, GEN + Ve and GEN + NaHS group.

		Tubular necrosis				Interstitial fibrosis				Mononuclear cell infiltration
	n	0	1	2	3	0	1	2	3	0	1	2	3
Control	6	6	0	0	0	6	0	0	0	5	1	0	0
GEN^a	6	0	0	4	2	0	0	4	2	0	1	2	3
Gen + Ve	6	0	0	3	3	0	1	3	2	0	1	3	2
GEN + NaHS^b	6	1	3	2	0	2	3	1	0	1	1	4	0

Notes: Score 0, no degeneration; 1, mild degeneration; 2, moderate degeneration; 3, severe degeneration.

^aStatistical significant difference from the control group.

^bStatistical significant difference from the GEN and GEN + Ve group and p < 0.05.

Discussion

The main finding of this study is that hydrogen sulfide can protect renal tubule cells against gentamicin. To the best of our knowledge, this report is the first to show that H₂S prevent functional, histological kidney injury by gentamicin. These effects are caused by the antioxidative, anti-inflammatory and anti-apoptotic activities of hydrogen sulfide.

Gentamicin was considered to be an important drug against life-threatening infections, although the mechanism for the nephrotoxic adverse effects is still not very well elucidated.24 Some studies reported that gentamicin-induced reactive oxygen species, which has a major role in the mechanism of nephrotoxicity of gentamicin.25 Gentamicin is still one of the main antibiotics in use against Gram-negative bacteria. Therefore, we are still concerned about the strategies to ameliorate its nephrotoxicity.

The understanding of aminoglycoside nephrotoxicity is clinically important, such as nephrotoxicity is typically associated with non-oliguric acute renal failure, that is, azotemia in the presence of 1–2 l/day urine output. In present study, the 24-h urine volume in the GEN group was significantly higher than in the control group, indicating the presence of GEN-induced polyuria, whereas in GEN + NaHS group, it was not different from that of the control group, suggesting the protective role of H₂S against acute tubular necrosis. Also, increased serum urea and creatinine levels in GEN-treated rats reflect the renal damage. In contrast to previous studies, serum K⁺ levels were similar between group GEN-induced nephrotoxicity and control group. Previous studies show that K⁺ levels were decreased in rats with GEN-induced nephrotoxicity. It is well documented that GEN nephrotoxicity in experimental animals causes acute renal failure and reduction in serum K⁺ levels. These results could be attributed to the fact that GEN-induced oxidative stress can promote the formation of a variety of vasoactive mediators that can affect renal function directly by causing renal vasoconstriction or decreasing the glomerular capillary ultrafiltration coefficient and thus reducing glomerular filtration rate (GFR). ROS generated by GEN may also impair the expression of endothelial nitric oxide synthase (eNOS), whereas may scavenge nitric oxide (NO), thereby reducing the amount of the endogenous vasodilator in the vasculature.26,27 Alternatively, the GEN-induced tubular necrosis could lead to a decrease in the number of functioning nephrons, with a subsequent decline in GFR. This effect may trigger multiple adaptive processes in the hyper-functioning remaining nephrons, most notably the alteration in a variety of tubular transport functions including augmented rates of Na⁺ reabsorption or K⁺ secretion. The present biochemical finding is confirmed by histopathological examination of GEN-intoxicated kidneys that revealed diffused renal tubular necrosis, associated with periglomerular and perivascular inflammatory cells infiltration, as well as atrophy in the tuft of the glomeruli with thickening in the Bowman’s capsule.2,26–28

Endogenous H₂S has been proposed as a novel cytoprotective mediator,29 and there is growing evidence of direct and indirect antioxidant effects of H₂S. In cell culture experiments, H₂S/HS⁻ generated from NaHS has been shown to “scavenge” detrimental pro-inflammatory oxidants, such as H₂O₂,30 ClO⁻,31 superoxide,32 ONOO⁻³³ and NO,32 and also can inhibit cell death induced by these mediators as well as prevent oxidative modification of intracellular proteins31,33 and LDL (low-density lipoprotein).34 In neuronal cells, NaHS inhibited cell death induced by β-amyloid, mediated at least in part via antioxidant effects35 and up-regulating intracellular glutathione synthesis through increasing cysteine uptake and elevating γ-glutamylcysteine synthetase activity.36 NaHS is also reported to degrade lipid peroxides,35 inhibit the expression and activity of NADPH oxidase37 and up-regulate thioredoxin-1 expression in vascular endothelial cells.38 Increased hepatic GSH synthesis and decreased lipid peroxidation are also observed with NaHS treatment in a murine hepatic ischemia/reperfusion injury model.39

Increased lipid peroxidation (LPO) has been reported in renal cortexes by the induction of excessive ROS in renal ischemic reperfusion.40 MDA is the product in the LPO process and is widely used as a reliable marker of tissue damage. In the present study, we found increased MDA levels in GEN and GEN + Ve group, and also as protective effect of H₂S lower MDA levels in group determined by GEN + NaHS. The GSH antioxidant system is considered the most notable cellular protective mechanism. GSH has a very important role in protecting against oxygen free radical damage by providing reducing equivalents for several enzymes, as well as scavenging hydroxyl radicals and singlet oxygen. Its depletion is a common consequence of increased formation of ROS41 like GEN-induced nephrotoxicity. In group given GEN + NaHS, we found increased GSH levels. However, our study shown that H₂S effect NO levels protectively in similar to some previous studies with different antioxidant agents.42,43 H₂S can inhibit NO production and NF-kappa B activation in LPS-stimulated macrophages through a mechanism that involves the action of HO-1/CO.44 Because of that, in our study we found decreased NO levels in GEN + NaHS group compared to UUO group. These findings strongly indicate that H₂S is important in protecting the kidney from GEN-induced injury through improvement in oxidant status.

In conclusion, the results reported here indicate that H₂S exerts a preventative effect on GEN-induced kidney damage in rats by reducing oxidative stress. One limitation in our study was that the underlying molecular mechanisms that are responsible for the positive effects of NaHS are yet to be determined. We therefore propose that H₂S supplementation therapy can be used for kidney protection in patients with GEN-induced nephrotoxicity. However, further animal and clinical studies are needed to confirm our suggestion.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.