Ulinastatin inhibits renal tubular epithelial apoptosis and interstitial fibrosis in rats with unilateral ureteral obstruction

Abstract

The effect of ulinastatin (UTI) on renal tubular epithelial apoptosis and interstitial fibrosis in rats with unilateral ureteral obstruction (UUO) was investigated. A total of 18 male Wistar rats were randomly divided into the following 3 groups: The Sham group (n=6), the UUO group (n=6), and the UTI group (n=6). In the UUO and UTI groups, the left ureter was ligated to establish a UUO model. Starting from day 1 after surgery, an intervention treatment was performed using normal saline (1 ml/kg/d) and UTI (40,000 unit/kg/d). On day 7 after surgery, 6 rats from each group were sacrificed. In the Sham group, the left ureter was only freed, not ligated; after 7 days of abdominal closure, all of the rats were sacrificed. Blood samples were collected prior to sacrificing the animals to measure the blood urea nitrogen (BUN) and serum creatinine (Scr). The incidence of renal interstitial lesions on the obstruction side was observed by hematoxylin and eosin, and Masson staining. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and immunohistochemical detection of apoptosis regulator Bax (Bax), apoptosis regulator Bcl‑2 (Bcl‑2) and caspase‑3 were performed to observe the presence of renal tubular epithelial cell apoptosis. The UTI did not have a significant influence on the mouse BUN and Scr levels in any of the groups (P>0.05). Compared with that in the Sham group, renal tissue injury in the UUO group was significantly aggravated with renal tubular dilation, epithelial cell atrophy, renal interstitial inflammatory cell infiltration and fibrous tissue hyperplasia (P<0.01). Furthermore, the renal tubular epithelial TUNEL+ cell number and Bax and caspase‑3 levels were increased, and the expression of Bcl‑2 was decreased (P<0.01). Following the UTI treatment, the renal interstitial injury at the obstruction side was significantly attenuated (P<0.05), the renal tubular epithelial TUNEL+ cell number, and Bax and caspase‑3 levels significantly decreased, and the expression of Bcl‑2 was restored (P<0.05). UTI inhibited renal tubular epithelial apoptosis and interstitial fibrosis in UUO rats.

Figure 1.

The rat (A) Scr and (B) BUN levels in the Sham, UUO and UTI groups were all within the normal ranges. Comparison among the 3 groups did not show any significant difference (P>0.05). Scr, serum creatinine; BUN, blood urea nitrogen; UUO, unilateral ureteral obstruction; UTI, ulinastatin.

Figure 2.

UTI attenuated renal pathological changes of tubulointerstitial injury after ureteral obstruction. (A) H&E staining results in the Sham, UUO and UTI groups (magnification, ×100). (B) Masson staining results in the Sham, UUO and UTI groups (magnification, ×100). (C) Statistical graph showing the semi-quantitative analysis of the tubulointerstitial injury results. (D) The statistical graph showing the semi-quantitative analysis of the tubulointerstitial fibrosis results. All data are expressed as the mean ± standard deviation. Each group comprised 6 rats. ^#P<0.01 for the UUO and UTI groups compared to the Sham group; *P<0.05 for the UTI group compared to the UUO group. H&E, hematoxylin and eosin; UUO, unilateral ureteral obstruction; UTI, ulinastatin.

Figure 3.

UTI decreased the number of apoptotic cells in the UUO model. (A) The numbers of apoptotic renal tubular epithelial cells in the Sham, UUO and UTI groups (magnification, ×400). (B) Statistical graph showing the statistical analysis results of the numbers of apoptotic cells in the 3 groups. All data are expressed as the mean ± standard deviation. Each group comprised 6 rats. ^#P<0.01 for the UUO and UTI groups compared to the Sham group; *P<0.05 for the UTI group compared to the UUO group. UUO, unilateral ureteral obstruction; UTI, ulinastatin.

Figure 4.

Bax expression decreased and Bcl-2 expression increased in the UUO model after UTI intervention. (A) Expression of Bax in the Sham, UUO and UTI groups (magnification, ×200). (B) Expression of Bcl-2 in the Sham, UUO and UTI groups (magnification, ×200). (C) Statistical graph showing the semi-quantitative analysis of Bax expression in the 3 groups. (D) Statistical graph showing the semi-quantitative analysis of Bcl-2 expression in the 3 groups. All data are expressed as the mean ± standard deviation. Each group comprised 6 rats. ^#P<0.01 for the UUO and UTI groups compared to the Sham group; *P<0.05 for the UTI group compared to the UUO group. UUO, unilateral ureteral obstruction; UTI, ulinastatin; Bax, apoptosis regulator Bax; Bcl-2, apoptosis regulator Bcl-2.

Figure 5.

UTI decreased caspase-3 expression in the UUO model. (A) Expression of caspase-3 in the Sham, UUO and UTI groups (magnification, ×200). (B) Statistical graph showing the semi-quantitative analysis of caspase-3 expression in the 3 groups. All data are expressed as the mean ± standard deviation. Each group comprised 6 rats. ^#P<0.01 for the UUO and UTI groups compared to the Sham group; *P<0.05 for the UTI group compared to the UUO group. UUO, unilateral ureteral obstruction; UTI, ulinastatin.