Beneficial Role of Hydrogen Sulfide in Renal Ischemia Reperfusion Injury in Rats

Abstract

Purpose: Hydrogen sulfide (H₂S) is an endogenous gaseous molecule with important physiological roles. It is synthesized from cysteine by cystathionine γ-lyase (CGL) and cystathionine β-synthase (CBS). The present study examined the benefits of exogenous H₂S on renal ischemia reperfusion (IR) injury, as well as the effects of CGL or CBS inhibition. Furthermore, we elucidated the mechanism underlying the action of H₂S in the kidneys.

Materials and methods: Thirty male Sprague-Dawley rats were randomly assigned to five groups: a sham, renal IR control, sodium hydrosulfide (NaHS) treatment, H₂S donor, and CGL or CBS inhibitor administration group. Levels of blood urea nitrogen (BUN), serum creatinine (Cr), renal tissue malondialdehyde (MDA), and superoxide dismutase (SOD) were estimated. Histological changes, apoptosis, and expression of mitogen-activated protein kinase (MAPK) family members (extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38) were also evaluated.

Results: NaHS attenuated serum BUN and Cr levels, as well as histological damage caused by renal IR injury. Administration of NaHS also reduced oxidative stress as evident from decreased MDA, preserved SOD, and reduced apoptotic cells. Additionally, NaHS prevented renal IR-induced MAPK phosphorylation. The CGL or CBS group showed increased MAPK family activity; however, there was no significant difference in the IR control group.

Conclusion: Exogenous H₂S can mitigate IR injury-led renal damage. The proposed beneficial effect of H₂S is, in part, because of the anti-oxidative stress associated with modulation of the MAPK signaling pathways.

Keywords: Hydrogen sulfide; cystathionine β-synthase; cystathionine γ-lyase; ischemia-reperfusion injury.

Fig. 1. Changes in renal function. (A and B) The IR control group showed a significant increase in serum Cr and BUN levels, compared to those in the sham group. Rats treated with NaHS showed lower serum Cr and BUN levels, compared with those in the IR control group. The results are expressed as a mean±SEM (n=5 rats/group). ^*p<0.05 vs. the sham group, ^†p<0.05 vs. the IR control group. IR, ischemia reperfusion; Cr, creatinine; BUN, blood urea nitrogen; NaHS, sodium hydrosulfide; AOAA, amino-oxyacetic acid; PAG, D,L-propargylglycine.

Fig. 2. Changes in renal oxidative stress. (A) The IR group showed a significant increase in MDA levels, compared to those in the sham group. MDA levels in the NaHS group were attenuated, compared with those in the IR control group. (B) The IR group showed a significant decrease in SOD levels, compared to those in the sham group. SOD levels in the NaHS group were preserved, compared with those in the IR group. SOD levels were also preserved in the AOAA and PAG groups, compared to those in the IR control group. The results are expressed as a mean±SEM (n=5 rats/group). ^*p<0.05 vs. the sham group, ^†p<0.05 vs. the IR control group. IR, ischemia reperfuion; MDA, malondialdehyde; SOD, superoxide dismutase; NaHS, sodium hydrosulfide; AOAA, amino-oxyacetic acid; PAG, D,L-propargylglycine.

Fig. 3. Changes in renal MAPKs. Western blotting was performed to measure (A) phosphorylated ERK, (B) phosphorylated JNK, and (C) phosphorylated p38 expression levels in kidney cell homogenates. Renal IR injury promoted phosphorylation of ERK, JNK, and p38. NaHS significantly inhibited renal IR-induced activation of MAPK pathways. The results are expressed as a mean±SEM (n=5 rats/group). ^*p<0.05 vs. the sham group, ^†p<0.05 vs. the IR control group. MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IR, ischemia reperfusion; NaHS, sodium hydrosulfide; AOAA, amino-oxyacetic acid; PAG, D,L-propargylglycine.

Fig. 4. Histopathological examination of the renal tissues from the experimental groups (A) and damage score (B). Rats in the IR control group exhibited tubular dilatation and luminal congestion (thick arrow) along with tubular cell atrophy (thin arrow). In the NaHS group, kidney structures were preserved, with mild tubular dilatation and luminal congestion. Kidney structures in the AOAA and PAG group appeared better, compared with those from the IR control group. Tissue sections were stained with periodic acid Schiff stain and visualized at magnification ×200. The results are expressed as a mean±SEM (n=5 rats/group). ^*p<0.05 vs. the sham group, ^†p<0.05 vs. the IR control group. IR, ischemia reperfusion; NaHS, sodium hydrosulfide; AOAA, amino-oxyacetic acid; PAG, D,L-propargylglycine.

Fig. 5. Apoptosis evaluation (representative images and quantitative analyses of TUNEL-positive cells) in renal tissue from the experimental groups. (A and B) Kidney sections from the IR control group showed increased TUNEL-positive cells, compared with those from the sham group. The degree of apoptosis in the NaHS group was significantly decreased, compared with those in the IR control group. Rats in the AOAA and PAG group exhibited decreased TUNEL-positive cells, compared with those in the IR control group. The results are expressed as a mean±SEM (n=5 rats/group, magnification ×400). ^*p<0.05 vs. the sham group, ^†p<0.05 vs. the IR control group. TUNEL, transferase dUTP nick-end labeling; IR, ischemia reperfusion; NaHS, sodium hydrosulfide; AOAA, amino-oxyacetic acid; PAG, D,L-propargylglycine.