A modified relief of unilateral ureteral obstruction model

Abstract

Objectives: To improve the mouse model of relief for unilateral ureteral obstruction (RUUO) and explore the pathological process of renal fibrosis after the obstruction was relieved. Methods: C57BL/6 mice in model group were randomly divided into RUUO group, improved RUUO group, and UUO group. After leaving Unilateral Ureteral Obstruction (UUO) for 3 days, the obstruction was released by reimplantation way in RUUO group and in reimplantation + catheter way in improved RUUO group. C57BL/6 mice in observation group were randomly divided into 1d RUUO group, 3d RUUO group, 7d RUUO group, and 14d RUUO group. Three days after UUO, the obstruction was released by reimplantation + catheter in four groups. We detected the renal volume, H&E, Masson staining, and immunohistochemistry of kidney pathology on the seventh day after RUUO in model group and on the 1st, 3rd, 7th, and 14th day after RUUO in observation group. Results: Comparing with mice in RUUO group, mice in improved RUUO group had lower renal volume, tubular damage score, and collagen area percentage. After the obstruction was relieved, the renal volume decreased gradually within 2 weeks. The tubular damage score in 7d RUUO group was lower than that in 1d RUUO and 3d RUUO group. However, the tubular damage score in 14d RUUO group was higher than that in 7d RUUO group. The tendency of collagen area percentage and α-SMA IOD value were consistent with the tubular damage score. Conclusions: Using the method of reimplantation + catheter, a reliable mice model of RUUO can be got. After RUUO, the de-obstructed kidneys are still in damage and fibrosis state.

Keywords: Relief of unilateral ureteral obstruction; catheter; pathology; renal fibrosis.

Figure 1.

Surgery pictures. (A) The end of the left enlarged ureter was found and isolated under the identification of the black 7/0 silk tie used to tie the left ureter in the first operation. The solid arrow indicates the enlarged ureter. The dashed arrow indicates the filled bladder. (B) A 20-mL syringe was pushed through into the bladder from the left-hand side, into its cavity, and out again through the right-hand side. The solid arrow indicates the enlarged ureter. The dashed arrow indicates the bladder. (C) A vascular clamp was taken to secure the distal end of the ureter and prevent it from slipping back into the bladder cavity. Using 10/0 monocryl wire, four or five evenly spaced interrupted sutures were placed to stitch the ureter to the wall of the bladder on the left. The solid arrow indicates the enlarged ureter. The dashed arrow indicates a vascular clamp. (D) Under the guidance of the insulin needle, a diameter of 6–9 mm PTFE tube was pushed into the ureteral cavity with tube mouth exposed 1–2 mm. The solid arrow indicates the PTFE tube. The dashed arrow indicates the ureteral wall. (E) The details of the figure (D) under the microscope. (F) The anterior wall of the bladder was sutured. Arrows indicate the anterior wall of the bladder. (G, P) The comparison of kidney at 10 days post-UUO (figure right) and CUK (figure left). (H, Q) The comparison of kidney at 7 days post RUUO by reimplantation way (figure right) and CUK (figure left). (I, R) The comparison of kidney at 7 days post RUUO by reimplantation + catheter way (figure right) and CUK (figure left). (J) The kidney at 7 days post RUUO by reimplantation way. The solid arrow indicates hydronephrosis. The dashed arrow indicates the bladder. (K) The details of the figure (J) under the microscope. (L) The catheterization section of the ureter was surrounded by connective tissue, when the bladder is cut open. There was no dye flow out of the ureter when the brown dye was injected into the renal pelvis. The arrow indicates the segment of the ureter. (M) The kidney at 7 days post RUUO by reimplantation + catheter way. The solid arrow indicates the no hydronephrosis. The dashed arrow indicates the bladder. (N) The catheter was seen in the bladder wall and smoothly, when the bladder in figure (M) was cut open. The arrow indicates the mouth of the catheter. (O) The details of the figure (M) under the microscope. The arrow indicates the mouth of the catheter.

Figure 2.

Renal volume, tubular damage score and collagen area percentage in different groups. (A) The length of the kidney L, the width T, and the depth A were measured by the vernier caliper. The renal volume is estimated according to the following equation: V = πLTA/6. The renal volume in RUUO group was smaller than in UUO group (139.793 ± 2.78 vs.412.536 ± 31.12 mm³, p < 0.05), which was larger than that in improved RUUO group (139.793 ± 2.78 vs. 85.019 ± 12.36 mm³, p < 0.05). *p < 0.05 for the indicated comparison. (B) The tubular damage score in RUUO and improved RUUO group was 4.38 ± 0.74 (n = 8) and 3.14 ± 0.69 (n = 7). *p < 0.05 for the indicated comparison. (C) The proportion of collagen area in RUUO and improved RUUO group was 0.55 ± 0.19% (n = 8) and 0.26 ± 0.11% (n = 7). *p < 0.05 for the indicated comparison.

Figure 3.

Representative histological sections (magnification: 200×). De-obstructed kidneys at 7 days post-RUUO showed renal tubular disorder, brush border loss, lumen expansion, tube formation in RUUO group, and the damage and fibrosis still can be observed. The renal histopathology in improved RUUO group was better than that in RUUO group, which was still more serious than the CUK.

Figure 4.

Appearance of kidneys and renal volume in four groups. (A) 1d RUUO: The comparison of RUUO kidney on 1st day post RUUO (figure right) and CUK (figure left). 3d RUUO: The comparison of RUUO kidney on 3rd day post RUUO (figure right) and CUK (figure left). 7d RUUO: The comparison of RUUO kidney on 7th day post RUUO (figure right) and CUK (figure left). 14d RUUO: The comparison of RUUO kidney on 14th day post RUUO (figure right) and CUK (figure left). (B) The renal volume in 1d RUUO, 3d RUUO, 7d RUUO, and 14d RUUO group was 135.525 ± 2.054 mm³ (n = 8), 100.174 ± 2.209 mm³ (n = 7), 85.530 ± 1.534 mm³ (n = 8), and 43.204 ± 1.208 mm³ (n = 8) respectively. *p < 0.05 for the indicated comparison.

Figure 5.

Representative histological sections of four groups. The pathology of kidneys undergoing 3 days UUO was considerable tubular injury after 1 day of RUUO, as manifested by tubular dilatation and epithelial cell flattening. As the time of de-obstruction was prolonged, the degree of renal fibrosis was gradually reduced, accompanied by the recovery of normal histological appearance within a week. At day 14 post-RUUO, the degree of kidney damage was aggravated, manifested as inflammation, tubular atrophy, and interstitial fibrosis. Masson staining showed less and less blue areas within a week, while at 14th day post-RUUO, the blue-stained areas increased significantly. Immunohistochemistry showed that with prolong of de-obstruction time, the expression of α-SMA decreased. At 14th day post-RUUO, the expression of α-SMA increased. The immunohistochemical results of collagen-I were consistent with the results of Masson staining.

Figure 6.

The tubular damage score by H&E staining. The tubular damage score in 1d RUUO, 3d RUUO, 7d RUUO, and 14d RUUO group was 4.88 ± 0.35 (n = 8), 4.43 ± 0.79 (n = 7), 3.25 ± 0.71 (n = 8), and 4.75 ± 0.47 (n = 8) respectively. *p < 0.05 for the indicated comparison.

Figure 7.

The proportion of collagen area by Masson staining and Immunohistochemistry. (A) The proportion of collagen area of contralateral kidneys and de-obstructed kidneys in 1d RUUO, 3d RUUO, 7d RUUO, and 14d RUUO group was 0.14 ± 0.13% vs 1.13 ± 0.84% (n = 8), 0.05 ± 0.03% vs 0.53 ± 0.21% (n = 7), 0.17 ± 0.27% vs 0.30 ± 0.21% (n = 8), and 0.05 ± 0.04% vs 0.78 ± 0.79% (n = 8). *p < 0.05 for the indicated comparison. (B) The proportion of collagen-I area of contralateral kidneys and de-obstructed kidneys in 1d RUUO, 3d RUUO, 7d RUUO, and 14d RUUO group was 0.06 ± 0.01% vs 0.53 ± 0.12% (n = 8), 0.04 ± 0.01% vs 0.33 ± 0.07% (n = 7), 0.06 ± 0.01% vs 0.07 ± 0.03% (n = 8), and 0.04 ± 0.01% vs 0.44 ± 0.06% (n = 8). *p < 0.05 for the indicated comparison.

Figure 8.

The average optical density of α-SMA of contralateral kidneys and de-obstructed kidneys in 1d RUUO, 3d RUUO, 7d RUUO, and 14d RUUO group was 16582.90 ± 4114.84 vs 44499.41 ± 7928.23 (n = 8), 22892.77 ± 14315.49 vs 38907.00 ± 7337.67 (n = 7), 17585.28 ± 7203.37 vs 19644.91 ± 8942.41 (n = 8), and 17793.53 ± 10832.55 vs 59355.14 ± 14360.67 (n = 8). *p < 0.05 for the indicated comparison.