Fig 1.
Experimental protocol and grouping.
Rats were randomly divided into four groups: Control, saline+Gly, SnCl2+Gly, and SnCl2+SnMP+Gly. Rats in the control group were untreated. Rats in the other three group were treated with saline or SnCl2 (s.c.) and then deprived of water for 24 h. Rats in the SnCl2+SnMP+Gly group received an additional treatment of SnMP (i.v.) 1 h before Gly injection. 24 h after saline or SnCl2 treatment, rats were treated with Gly (i.m.); afterward, at five time points (1, 3, 6, 12, and 24 h) and their blood and kidneys collected.
Fig 2.
Changes in renal HO-1 protein expression after Gly treatment.
Rats pretreated with saline or SnCl2 (s.c.) were sacrificed 1, 3, and 6 h after Gly treatment (10 mL/kg of 50% glycerol, i.m.), and renal tissues were collected for western blot analysis. Control, untreated control rats; saline+Gly, rats pretreated with saline before Gly injection; SnCl2+Gly, rats pretreated with SnCl₂ before Gly injection. The densitometric ratio between HO-1 and GAPDH was calculated. Data are expressed as the mean ± SD. *p < 0.05 vs control group. #p < 0.05 vs saline+Gly group.
Fig 3.
Expression of renal HO-1 mRNA 6 h after Gly treatment.
Rats pretreated with saline or SnCl2 (s.c.) were sacrificed 6 h after Gly treatment (10 mL/kg of 50% glycerol, i.m.) and renal tissues were collected for northern blot analysis. Control; untreated control rats, saline+Gly; saline-treated before Gly-treated rats, SnCl2+Gly; SnCl2-treated before Gly-treated rats. Ethidium bromide staining of the same RNA is shown as a loading control. Closed arrowhead, 28S ribosomal RNA; open arrowhead, 18S ribosomal RNA. HO-1 mRNA expression levels are expressed as arbitrary densitometric units. Data are expressed as the mean ± SD. *p < 0.05 vs saline+Gly group.
Fig 4.
Effect of SnCl2 pretreatment on changes in serum BUN and Cre levels after glycerol treatment.
Rats pretreated with saline (s.c.) or SnCl2 (s.c.) or SnCl₂ and SnMP (i.v.) 24 h before Gly treatment (10 mL/kg of 50% glycerol, i.m.) were sacrificed at 1, 3, 6, 12, and 24 h after Gly injection. Serum BUN and Cre levels were measured at each time after treatment. Control, untreated control rats; saline+Gly, saline-treated before Gly-treated rats; SnCl2+Gly, SnCl2-treated before Gly-treated rats; SnCl2+SnMP+Gly, SnCl2 and SnMP-treated before Gly-treated rats. Data are expressed as the mean ± SD (n = 3–7). * p < 0.05 vs control group; # p < 0.05 vs SnCl2+Gly group. BUN, blood urea nitrogen; Cre, creatinine.
Fig 5.
Effect of SnCl2 pretreatment on renal histopathological changes at 24 h after Gly treatment.
Renal tissues were stained with HE and assessed using a light microscope. Representative images 24 h after Gly treatment (HE staining, original magnification × 200, scale bar = 100 μm). The severity of histopathological changes was evaluated using a tubular injury score. A total of 40 areas from a renal section of each group were assessed. The tubular injury was defined as tubular epithelial cell swelling, vacuolar degeneration, necrosis, and desquamation. Tubular injury score was graded by estimating the percentage of injured tubules as follows: 0, areas of injured tubules < 5%; 1, areas of injured tubules 5–25%; 2, areas of injured tubules 26–50%; 3, areas of injured tubules 51–75%; and 4, areas of injured tubules > 75%. The mean score for each kidney is provided, followed by the mean score for the group. Data are expressed as the mean ± SD (n = 4). * p < 0.05 vs control group; # p < 0.05 vs SnCl2+Gly group.
Fig 6.
Effect of SnCl2 pretreatment on the renal HO activity at 24 h after Gly treatment.
Renal tissues were collected 24 h after Gly treatment, and HO activity was assessed as described in Material and Methods. Data are expressed as the mean ± SD (n = 3–4). *p < 0.05 vs control group; #p < 0.05 vs saline+Gly group.
Fig 7.
Expression of renal ALAS1 mRNA levels 3 h after Gly treatment.
Rats pretreated with saline or SnCl2 (s.c.) were sacrificed 3 h after Gly treatment (10 mL/kg of 50% glycerol, i.m.), and renal tissues were collected for northern blot analysis. Control, untreated control rats; saline+Gly, saline-treated before Gly-treated rats; SnCl2+Gly, SnCl2-treated before Gly-treated rats. Ethidium bromide staining of the same RNA is shown as a loading control. Closed arrowhead, 28S ribosomal RNA; open arrowhead, 18S ribosomal RNA. ALAS1 mRNA expression levels are expressed as arbitrary densitometric units. Data are expressed as the mean ± SD. *p < 0.05 vs control group.
Fig 8.
Renal nuclear Bach1 protein expression levels 3 h after Gly treatment.
Rats pretreated with saline or SnCl2 (s.c.) were sacrificed 3 h after Gly treatment (10 mL/kg of 50% glycerol, i.m.), and renal tissues were collected for western blot analysis. Control, untreated control rats; saline+Gly, saline-treated before Gly-treated rats; SnCl2+Gly, SnCl₂-treated before Gly-treated rats. The densitometric ratio between Bach1 and β-actin was calculated. Data are expressed as the mean ± SD. *p < 0.05 vs control group.