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. 2023 Jan 5:13:1074488.
doi: 10.3389/fimmu.2022.1074488. eCollection 2022.

A novel model of urosepsis in rats developed by injection of Escherichia coli into the renal pelvis

Yuanfei Cao  1 , Can Bai  1 , Penghui Si  1 , Xin Yan  1 , Peng Zhang  2 , Zuhaer Yisha  1 , Peixiang Lu  1 , Kuerban Tuoheti  1 , Linfa Guo  1 , Zhao Chen  1 , Xiaojie Bai  1 , Tongzu Liu  1
Affiliations

A novel model of urosepsis in rats developed by injection of Escherichia coli into the renal pelvis

Yuanfei Cao et al. Front Immunol. .

Abstract

Despite extensive research, urosepsis remains a life-threatening, high-mortality disease. Currently, animal models of urosepsis widely accepted by investigators are very scarce. This study aimed to establish a standardized and reproducible model of urosepsis in rats. Forty adult Wistar rats were randomly divided into four groups according to the concentration of injected E. coli suspensions: Sham, Sep 3×, Sep 6×, and Sep 12×. Because the ureter is so thin and fragile, no conventional needle can be inserted into the ureter, which is probably why rats are rarely used to develop models of urosepsis. To solve this problem, the left ureter was ligated in the first procedure. After 24 hours, the left ureter above the ligation was significantly dilated, then saline or different concentrations of E. coli at 3 ml/kg were injected into the left renal pelvis using a 30G needle. The left ureter was subsequently ligated again at a distance of 1 cm from the renal hilum to maintain high pressure in the renal pelvis. Following injection of E. coli or saline for 24 h, three rats from each group were sacrificed and their organs (lung, liver, and right kidney) were collected. In contrast, the remaining seven rats continued to be observed for survival. At 10 days after E. coli injection, rats in the sep12× group had a higher mortality rate (100%) compared to the sep3× group (28.6%) or the sep6× group (71.4%). The significant changes in peripheral blood WBC count, serum IL-6 and TNF-α levels were also in the sep12× group. In addition, rats in the sepsis group showed multi-organ dysfunction, including damage to the lungs, liver, and kidneys. The establishment of a standardized rat model of urosepsis may be of great value for studying the pathophysiological of urosepsis.

Keywords: Escherichia coli; animal model; pathophysiology; rats; upper urinary tract obstruction; urosepsis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The experimental procedure and critical steps in the process of urinary sepsis model in rats.(A) Schematic diagram of the experimental procedure. (B) The ureter prior to ligation. (C) Dilated ureter after ligation for 24h. (D) 30G syringe needle.
Figure 2
Figure 2
Survival analysis and Blood cultures after ureter ligation and inoculation of E coli. (A) Survival analysis after ureter ligation and inoculation of E coli. (B) Blood specimens were incubated on MacConkey agar at 37°C after 24h inoculation with E coli.
Figure 3
Figure 3
Changes in WBC (A), serum IL-6 (B), and serum TNF-α (C) in rats at different E coli concentrations and intervals. Values were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P <0.001, and ****P < 0.0001 vs sham group.
Figure 4
Figure 4
Lung injury in the rat model of urinary sepsis. (A) Representative images of hematoxylin and eosin (H&E) staining of lung tissue from the sham and sepsis groups, immunohistochemical analysis of IL-6 and TNF-α in lung tissue. (B) The relative mRNA levels of IL-6 and TNF-α in the lung tissue. (C) Expressions of IL-6 and TNF-α in lung tissue were tested by western blot, and β-actin was used as a loading control. (D, E) Quantitative data of the levels of IL-6 and TNF-α. The sep 6× group was selected as a representative of the sepsis groups. Values were shown as mean ± SD. *P < 0.05, vs sham group.
Figure 5
Figure 5
Liver injury in the rat model of urinary sepsis (A) Representative images of hematoxylin and eosin (H&E) staining of liver tissue from the sham and sepsis groups, immunohistochemical analysis of IL-6 and TNF-α in liver tissue (magnification, 200×; bar). (B, C) Serum AST and ALT levels in rats 24 hours after E. coli or saline inoculation of the renal pelvis. (D) The relative mRNA levels of IL-6 and TNF-α in the lung tissue. (E) Expressions of IL-6 and TNF-α in liver tissue were tested by western blot, and β-actin was used as a loading control. (F, G) Quantitative data of the levels of IL-6 and TNF-α. The sep 6× group was selected as a representative of the sepsis groups. Values were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P <0.001, and ****P < 0.0001 vs sham group. "ns" means "not signifcant".
Figure 6
Figure 6
Kidney injury in the rat model of urinary sepsis (A) Representative images of hematoxylin and eosin (H&E) staining of kidney tissue from the sham and sepsis groups, immunohistochemical analysis of IL-6 and TNF-α in kidney tissue (magnification, 200×; bar, 50 μm). (B, C) Serum BUN and Cre levels in rats 24 hours after E. coli or saline inoculation of the renal pelvis. (D) The relative mRNA levels of IL-6 and TNF-α in kidney tissue. (E) Expressions of IL-6 and TNF-α in kidney tissue were tested by western blot, and β-actin was used as a loading control. (F, G) Quantitative data of the levels of IL-6 and TNF-α. The sep 6× group was selected as a representative of the sepsis groups. Values were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P <0.001, and ****P < 0.0001 vs sham group. "ns" means "not signifcant".
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