Research on mechanism of sildenafil protecting renal fibrosis in mice through AKT/GSK-3β signaling pathway / 医学研究生学报

ABSTRACT

ObjectiveRenal fibrosis is the basic pathological process of chronic kidney disease. To explore the protective effect of sildenafil (Sil) on renal fibrosis in mice, and provide experimental evidence for the clinical application of sildenafil in the treatment of renal fibrosis.Methods90 Kunming mice were randomly divided into three groups. Sham operation group (n=30), the mice only had ureteral separation, no ligation and ureteral clipping, which was subcutaneously injected with 0.9% NaCl solution in dose of 1 mL/(10 g·d); UUO model group (n=30), the UUO model was prepared by separating and ligating ureters in mice, and those were given subcutaneous injection with 0.9% NaCl solution in 1 mL/(10 g·d); UUO+Sil medicated group (n=30), mice were subcutaneously injected with sildena in 12 mg/(kg·d) at the same time every day for 14 days from the 1st day of UUO model. On the 3rd, 7th and 14th day, 10 mice whoes blood from eyeball was collected to determine serum creatinine and urea nitrogen were randomly selected from each group. HE and Masson staining were performed on the left kidney tissue to observe the pathological changes of the kidney tissue. The levels of Akt and GSK-3β protein and its phosphorylation in renal tissue were determined by western blot.ResultsAfter 3 days in UUO model, the contents of serum creatinine (63.10±2.90mol/L ) and urea nitrogen (12.87±0.40mmol/L) in the UUO group were significantly higher than those in the sham group [(26.00±3.70) mol/L, (8.07±0.60) mmol/L] (P<0.05). The contents of serum creatinine and urea nitrogen [(64.39±2.50) mol/L, (13.59±0.30) mmol/L] on the 7th day were higher than those in the sham group [(29.18±3.50) mol/L, (9.14±0.50) mmol/L] (P<0.05). The contents of serum creatinine and urea nitrogen [(64.39±2.50) mol/L, (15.03±0.50) mmol/L] on the 14th day were also significantly higher than those in the sham group [(29.74±2.50) mol/L, (9.90±0.20) mmol/L] (P<0.05). Compared with UUO group, the creatinine of mice on the 3rd, 7th and 14th day in the medicated group was lower (P<0.05). Compared with UUO group , urea nitrogen on the 3rd, 7th and 14th day in the medicated group was decreased (P<0.05). Compared with the sham operation group, the expression levels of p-AKT /Akt and p-GSK-3β/GSK-3β in the UUO group were significantly decreased (P<0.05), while the protein expression levels of p-AKT /Akt and p-GSK-3β/GSK-3β in the medicated group were significantly increased compared with the UUO group (P<0.05). On the 7th day in UUO model, there were many changes included atrophic renal tubular epithelial cells, dilated lumen, widened interstitium and more infiltrated inflammatory cells. On the 14th day in UUO, the above changes were more obvious, interstitial fibroblast hyperplasia and interstitial fibrosis, and the above pathological changes were reduced in the medicated group compared with the UUO model group. The collagen fibers in the UUO model group increased gradually with time. On the 14th day in UUO, the collagen fibers in the interstitium increased significantly, the tubular epithelium was damaged, and the red staining cells became lighter. These findings were less severe in the medicated group than in the UUO model group.ConclusionSildenafil can alleviate renal damage caused by renal fibrosis. Sildenafil inhibited renal fibrosis in UUO model, and its mechanism may be related to up-regulation of Akt/GSK3β pathway.