Protective effects of hydrogen sulfide on chronic kidney disease by reducing oxidative stress, inflammation and apoptosis

Abstract

The current study aimed to examine the renoprotective effects of long-term treatment with sodium hydrosulfide (NaHS), a prominent hydrogen sulfide donor, in 5/6 nephrectomy animal model. Twenty-four rats were randomly divided into 3 groups including sham-operated group (Sham), 5/6-nephrectomized group (5/6 Nx), and NaHS-treated group (5/6Nx+NaHS). NaHS (30 micromol/l) was added twice daily into the drinking water and renal failure was induced by 5/6 nephrectomy. Twelve weeks after surgical procedure, blood pressure, creatinine clearance (CCr), urine concentration of neutrophil gelatinase associated lipocalin (NGAL) and tissue concentration of malondialdehyde (MDA), superoxide dismutase (SOD), as well as renal morphological changes, apoptosis (cleaved caspase-3) and inflammation (p-NF-κB) were measured. Five-sixth nephrectomy induced severe renal damage as indicated by renal dysfunction, hypertension and significant histopathological injury which were associated with increased NGAL and MDA levels, oxidant/antioxidant imbalance, decreased SOD activity and CCr and also overexpression of p-NF-κB and cleaved caspase-3 proteins. Instead, NaHS treatment attenuated renal dysfunction through reduction of NGAL concentration, hypertension, CCr, oxidant/antioxidant imbalance, inflammation and apoptosis. These findings suggest that long term NaHS treatment can be useful in preventing the progression of CKD by improving oxidant/antioxidant balance and reducing inflammation and apoptosis in the kidney.

Keywords: apoptosis; blood pressure; chronic kidney disease; creatinine clearance; hydrogen sulfide; inflammation; oxidative stress.

Table 1. Effect of NaHS on systolic blood pressure and body weight in 5/6 Nx rats. The data are presented as mean ± SEM (n=8). *p < 0.05 vs sham group. ^#p < 0.05 vs 5/6 Nx group

Figure 1. Effect of NaHS on (a) creatinine clearance and (b) urine lipocalin-2 (NGAL) concentration in 5/6 Nx rats. The data are presented as mean ± SEM (n=8). *p<0.05 vs sham group and ^#p<0.05 vs 5/6 Nx group

Figure 2. Effect of NaHS on (a) malondialdehyde (MDA) content and (b) superoxide dismutase (SOD) activity in 5/6 Nx rats. The data are presented as mean ± SEM (n=8).*p<0.05 vs sham group and ^#p<0.05 vs 5/6 Nx group

Figure 3. Renal histological changes in the (a) sham, (b) 5/6 nephrectomy (5/6 Nx) and (c) 5/6 Nx+ NaHS groups. The histological changes observed 12 weeks after surgery (a) there was no obvious damage to the glomeruli or tubules in the kidneys of sham group. (b) In the 5/6 Nx group, the most noticeable change was the Bowman's space widening which was present in the glomeruli of the kidneys. There were also extensive tubular dilations which were present in all proximal and distal tubules. Tubular debris was present and leukocytes infiltrations were also frequent in these tissues. (c) In the Nx + NaHS kidneys the degree of Bowman's space widening was lower and also less tubular casts was present comparing to 5/6 Nx group. Magnification ×400; Hematoxylin and Eosin staining. Scale bar is 200 µm. The black thin arrow indicates cast formation, the white arrow shows widening of the Bowman's space. The black thick arrow indicates tubular obstruction. (d) Summary of the histological damage. Histological damage was scored as follows: 0: minimal or no lesions; 1: < 25 % of tubules involved; 2: 25 %-50 % of tubules involved; 3: > 50 % of tubules involved. Data are presented as the mean and range (n=8). *p<0.05 vs sham group and ^#p<0.05 vs 5/6 Nx group

Figure 4. Double stained fluorescence microscopy image of p-NF-κB and cleaved caspase-3 related antigen from paraffin-embedded kidney rat 12 week after sham operation or 5/6 nephrectomy. Five-sixth nephrectomy resulted in overexpression of cleaved caspase-3 and p-NF-κB, whereas treatment with NaHS resulted in milder expression of cleaved caspase-3 and p-NF-κB in comparison with 5/6 Nx subjects. Left side of this figure shows the fluorescence signals obtained with antibodies against p-NF-κB (green) or cleaved caspase-3 (red) and DAPI staining of the DNA (blue). Scale bar is 25 µm. (A-C) nuclei were stained with DAPI (blue). (D) p-NF-κB was stained with secondary antibodies, no p-NF-κB was found in the kidney of rat after sham operation (n = 5-6). (E) p-NF-κB -positive cells (in green) were found to be positive in 5/6 Nx. (F) p-NF-κB -positive cells (in green) were found to be positive in 5/6 Nx+ NaHS rats. (G) No cleaved caspase-3-positive cells were found in the kidney of rat after sham operation (n = 5-6). (H) Cleaved caspase-3-positive cells were found to be positive in 5/6 Nx rats. (I) Cleaved caspase-3-positive cells (in red) were found to be positive in 5/6 Nx+ NaHS rats. (J-L) Double stained fluorescence microcopy image of p-NF-κB and cleaved caspase-3 was obtained. The right side of this figure shows the effects of H₂S on cleaved caspase-3 and NF-κB expression in kidney tissue 12 weeks after surgery. The data are presented as mean ± SEM (n=8). *p<0.05 vs sham group and ^#p<0.05 vs 5/6 Nx group