N-acetylcysteine suppresses colistimethate sodium-induced nephrotoxicity via activation of SOD2, eNOS, and MMP3 protein expressions

Figures & data

Table 1. Comparison of pretreatment and post-treatment serum creatinine levels.

	Pretreatment creatinine (mg/dl)	Post-treatment creatinine (mg/dl)	p
Sterile saline group	0.76 ± 0.22	0.86 ± 0.005	.371
N-acetylcysteine group	0.88 ± 0.17	1.01 ± 0.18	.117
Colistimethate sodium group	0.72 ± 0.08	1.25 ± 0.28	.001
Colistimethate sodium + N-acetylcysteine group	0.74 ± 0.13	0.82 ± 0.15	.273

Figure 1. Comparison of urine N-acetyl-β-d-glucosaminidase levels on day 1, 5, and 10 of treatment (^ap = .001 when colistimethate sodium group and sterile saline group were compared on day 10; ^bp = .0001 when colistimethate sodium group and N-acetylcysteine group were compared on day 10; ^cp = .001 when colistimethate sodium group and colistimethate sodium + N-acetylcysteine group were compared on day 10; ^dp = .001 when the 10th day of the treatment was compared with the baseline in the colistimethate sodium group; ^ep = .0001 when the 10th day of the treatment was compared with the first day in the colistimethate sodium group).

Figure 2. Histological findings of the study: normal kidney structure of the sterile saline group (A), Grade 1 renal histological lesion in N-acetylcysteine group (B), Grade 2 renal histological lesion in colistimethate sodium group (C), Grade 1 renal histological lesion in colistimethate sodium + N-acetylcysteine group (D). Tubular dilatation (asterisk), tubular casts (hollow arrow), and tubular cell necrosis (black arrow) (original magnification ×200).

Table 2. TUNEL results and renal histological damage scoring in all experimental groups.

	Sterile saline group	N-acetylcysteine group	Colistimethate sodium group	Colistimethate sodium + N-acetylcysteine group
Rate of apoptosis^a	45.31 ± 30.49	29.90 ± 27.89	122.62 ± 52.14^b^,c,d	66.58 ± 33.32
Semiquantitative score for renal histological changes	0 (0–1)	2 (0–6)	7 (3–10)^e^,f,g	2 (2–7)^h

^a Number of apoptotic cells under each – 400 magnifications of light microscopy.

^b p = .006 as compared with the sterile saline group.

^c p = .001 as compared with the N-acetylcysteine group.

^d p = .046 as compared with the colistimethate sodium–N-acetylcysteine group.

^e p = .001 as compared with the sterile saline group.

^f p = .001 as compared with the N-acetylcysteine group.

^g p = .007 as compared with the colistimethate sodium–N-acetylcysteine group.

^h p = .001 as compared with the sterile saline group.

Table 3. The values of renal histological changes among experimental groups.

	The score related to the grade of renal histology^a	The score related to the size of the affected area in kidney^b	Semiquantitative score for renal histological changes^c
Sterile saline group	1	0	1
	0	0	0
	0	0	0
	0	0	0
	0	0	0
	0	0	0
N-acetylcysteine group	1	1	2
	1	1	2
	1	1	2
	1	1	2
	1	1	2
	0	0	0
Colistimethate sodium group	4	3	7
	4	6	10
	4	3	7
	4	3	7
	1	2	3
	1	2	3
	1	2	3
Colistimethate sodium + N-acetylcysteine group	1	1	2
	1	1	2
	1	1	2
	1	1	2
	1	1	2
	1	1	2
	4	3	7

^a Grade 1: mild acute tubular damage (tubular dilatation, evident nucleus, very few and pale tubular casts); Grade 2: severe acute tubular damage (tubular cell necrosis and many tubular casts); Grade 3: acute cortical necrosis with or without papillary necrosis. 1 score for Grade 1 change, 4 score for Grade 2 change, 10 score for Grade 3 change.

^b The following scores were given according to the percentage of kidney’s affected area: <1%=0 score; 1–4%=1 score; 5–9%=2 score; 10–19%=3 score; 20–29%=4 score; 30–39%=5 score; and ≥40%=6 score.

^c Score found by adding the score related to the size of the affected area in kidney and the score related to the degree of renal histology.

Figure 3. The results of Western blotting analysis in the renal tissue in the sterile saline group, N-acetylcysteine group, colistimethate sodium group, and colistimethate sodium + N-acetylcysteine group: (A) the expression levels of endothelial nitric oxide synthase (a: p = .03, compared with the sterile saline group; b: p = .0001, compared with the N-acetylcysteine group; c: p = .0001, compared with the colistimethate sodium + N-acetylcysteine group; d: p = .0001, when N-acetylcysteine group and the sterile saline group were compared; e: p = .001, when colistimethate sodium group + N-acetylcysteine group and the sterile saline group were compared); (B) the expression levels of superoxide dismutase 2 (a: p = .0001, compared with the sterile saline group; b: p = .0001, compared with the N-acetylcysteine group; c: p = .002, compared with the colistimethate sodium + N-acetylcysteine group; d: p = .001, when N-acetylcysteine group and the sterile saline group were compared); (C) the expression levels of matrix metalloproteinase 3 (a: p = .007, compared with the sterile saline group; b: p = .0001, compared with the N-acetylcysteine group; c: p = .0001, compared with the colistimethate sodium + N-acetylcysteine group; d: p = .011, when N-acetylcysteine group and the sterile saline group were compared; e: p = .001, when colistimethate sodium group + N-acetylcysteine group and the sterile saline group were compared).

Table 4. Total oxidant and antioxidant status and determination of oxidative stress index levels in renal tissue of the sterile saline, N-acetylcysteine, colistimethate sodium, and N-acetylcysteine + colistimethate sodium-treated groups.

	Sterile saline group	N-acetylcysteine group	Colistimethate sodium group	Colistimethate sodium + N-acetylcysteine group	p
Total oxidant status	3.14 (2.99–3.28)	2.95 (2.91–2.95)	3.10 (2.85–3.43)	3.09 (2.97–3.2)	.240
Total antioxidant status	7.20 (4.65–15.09)	6.88 (2.57–7.58)	4.26 (0.78–6.95)	4.57 (0.05–7.92)	.336
Oxidative stress index	0.24 (0.15–0.46)	0.24 (0.09–0.26)	0.14 (0.003–0.20)	0.15 (0.10–0.26)	.27

Figure 4. The roles of superoxide dismutase, nitric oxide synthetase, and glutathione in the neutralization of reactive oxygen species formed in the body (O₂⁻: superoxide; SOD: superoxide dismutase; GSH-Px: glutathione peroxidases; NO: nitric oxide; ONOO⁻: peroxynitrite; H₂O₂: hydrogen peroxide; eNOS: endothelial nitric oxide synthase; GSSG: oxidized glutathione; GSSH: reduced glutathione).