A Modified Rat Model of Cecal Ligation and Puncture for Adjusting the Severity of Septic Acute Kidney Injury

Ning Zhao; Jiaquan Chen; Wei Peng; Rong Jiang; Zhiyong Peng; Fen Liu

doi:10.4103/jtccm.jtccm_29_21

ORIGINAL ARTICLE

Year : 2021 | Volume : 3 | Issue : 1 | Page : 15

A Modified Rat Model of Cecal Ligation and Puncture for Adjusting the Severity of Septic Acute Kidney Injury

Ning Zhao¹, Jiaquan Chen¹, Wei Peng¹, Rong Jiang², Zhiyong Peng³, Fen Liu¹
¹ Department of Critical Care Medicine, The First Affiliated Hospital of Nanchang University; Medical Innovation Center, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China
² Department of Critical Care Medicine, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China
³ Department of Critical Care Medicine, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi; Department of Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, China

Date of Submission	03-Sep-2021
Date of Acceptance	21-Oct-2021
Date of Web Publication	06-Dec-2021

Correspondence Address:
Dr. Fen Liu
17, YongwaiZheng Street, Nanchang, Jiangxi
China
Dr. Zhiyong Peng
169, East Lake Road, Wuchang District, Wuhan, Hubei
China

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jtccm.jtccm_29_21

Abstract

Background: We aimed to establish a modified cecal ligation and puncture (CLP) rat model with omentectomy for simulating moderate-to-severe septic kidney injury (AKI). Subjects and Methods: Rats were randomly divided into four groups subjected to: Traditional CLP (n = 40); the modified CLP procedure (n = 40); sham surgery (abdominal incision only) (n = 6); and abdominal incision and omentectomy only (n = 6). We evaluated the severity of renal injury according to serum creatinine levels at 0, 24, and 48 h postoperatively and cystatin C and neutrophil gelatinase-associated lipocalin (NGAL) levels at 12 h postoperatively, graded the severity of sepsis, monitored the degree of peritonitis, and examined animal survival. Results: Compared with the traditional CLP group, the modified CLP group developed more severe sepsis at postoperative 48 h, had more severe renal pathology, and developed more severe AKI with high homogeneity. Most rats in the traditional CLP group exhibited complete wrapping of the puncture site by omentum with limited peritonitis, while partial wrapping with pus spreading was observed in some. All rats in the modified CLP group developed puncture site necrosis, with pus spreading without formation of an encapsulated abscess, and a consistent degree of peritonitis. The 7-day survival rates in the traditional and modified CLP groups were 65.0% and 50.0%, respectively. Conclusion: The traditional CLP approach was associated with an even distribution of different AKI severities among treated animals, whereas the modified CLP approach established a consistently more severe rat model of septic AKI, constituting an important advance for optimizing septic AKI animal models.

Keywords: Acute kidney injury, cecal ligation and puncture, omentum, sepsis

How to cite this article:
Zhao N, Chen J, Peng W, Jiang R, Peng Z, Liu F. A Modified Rat Model of Cecal Ligation and Puncture for Adjusting the Severity of Septic Acute Kidney Injury. J Transl Crit Care Med 2021;3:15

How to cite this URL:
Zhao N, Chen J, Peng W, Jiang R, Peng Z, Liu F. A Modified Rat Model of Cecal Ligation and Puncture for Adjusting the Severity of Septic Acute Kidney Injury. J Transl Crit Care Med [serial online] 2021 [cited 2023 Mar 31];3:15. Available from: http://www.tccmjournal.com/text.asp?2021/3/1/15/331844

Introduction

Septic acute kidney injury (AKI) is common among critically ill patients, with an estimated incidence of 47.9% among those receiving treatment in intensive care units. The severity of AKI is positively correlated with overall mortality; those with milder AKI have a relatively lower mortality, while those with moderate-to-severe AKI sustain a substantially higher mortality despite aggressive interventions.^{[1],[2],[3],[4]} For this reason, it is imperative that an animal model of moderate-to-severe septic AKI be established to facilitate a better understanding of septic AKI and the design of effective treatments.

Existing animal models of septic AKI are generated by exogenous administration of lipopolysaccharide (LPS), live pathogens, or inflammatory mediators such as tumor necrosis factor-α, but these models are not popular because they cannot simulate the process of continuous release of bacterial toxins during sepsis.^[5],[6],[7] Cecal ligation and puncture (CLP) is now recognized as the golden standard for animal models of sepsis.^[8] This experimental procedure causes sepsis due to the leakage of cecal content into the peritoneal cavity, which leads to peritoneal infection, sepsis, and multi-organ injury. Compared with the model of exogenous LPS administration, CLP simulates the pathophysiology of sepsis-induced organ injury more closely and is more commonly used for approximating septic AKI experimentally.^[9],[10]

However, the application of CLP as a septic AKI model still has limitations, especially for moderate-to-severe AKI, due to model inconsistent AKI severity. Our previous experience also indicated that in some animals treated with CLP, the puncture site is covered by omentum, leading to mild and limited peritonitis with minor or even absent AKI. On the other hand, other animals without omental wrapping may have severe and diffuse peritonitis with prominent AKI. Establishing an animal model of moderate-to-severe septic AKI with a consistent severity has been a difficult task, and we presumed that omental wrapping around the cecal puncture site might be the main reason for the heterogeneity of AKI. In the present study, we aimed to create a modified CLP model by including omentectomy to address this issue and monitored survival, sepsis severity, and the extent of renal injury among the treated animals.

Subjects and Methods

Experimental animals

The animal experiment protocol for this study followed the regulations outlined by the National Institute of Health guidelines for the use of experimental animals and was approved by the Nanchang University experimental animal ethics committee (NDSYDWIL-201792). We selected healthy adult male-specific pathogen-free Sprague–Dawley rats aged 24–28 weeks with a body weight between 400 and 600 g from the Experimental Animal Center of Jiangxi Chinese Medical University (SCXK-2018-0003) and kept them in the Nanchang University Experimental Animal Center (SYXK-2015-0001). Animals were housed at a temperature between 22°C and 25°C with a humidity between 45% and 70% and a light-dark cycle of 12 h per day with liberal food and water available. The animals were acclimatized for 1 week before the experiment commenced.

Establishment of animal model and categorization

Rats were randomly allocated to four groups using a random number table: The CLP group (n = 40), treated with the traditional CLP procedure; the modified CLP group (n = 40), treated with the traditional procedure in combination with omentectomy; the sham group (n = 6) that received the abdominal incision only; and the omentectomy group (without CLP, n = 6).

The traditional CLP procedure was performed as previously described.^[11] In brief, rats were fasted for 8 h before the operation, and water intake was prohibited for 4 h before the operation. After being anesthetized with intraperitoneal 1.5% pentobarbital (40 mg/kg), rats were placed in supine position, and the hair was shaved for disinfection of the skin. A midline incision of 3–4 cm was created, and the cecum and ileocecal mesentery were identified, followed by ligation at the site one-fourth of the distance upstream of the cecal end and puncture using an 18-gauge needle twice through the ligation site. We squeezed out some stool from the punctured cecum and transferred it to the peritoneal cavity, closed the incision wound layer-by-layer, and administered lactated ringer solution (20 mL/kg) subcutaneously after the operation. After operation, we injected 20 mL/kg sodium lactate Ringer solution and 125 mg/Kg ampicillin and sulbactam sodium subcutaneously in rats of both the traditional CLP group and the modified CLP group. The rats were re-housed in the cage with liberal food and water intake. Buprenorphine (0.5 mg/kg) subcutaneous injection every 12 h for 72 h was given postoperatively for analgesia. After recovery from anesthesia, rats could eat and drink normally and were given a 10% glucose solution prepared in advance.

For the modified CLP procedure, an abdominal incision was made and the omentum freed. The omentum remained attached to the greater curvature and spleen. We used 2-0 thread to ligate the omental root and excised omentum. The rest of the procedure remained the same as in the traditional CLP procedure.

Monitoring septic acute kidney injury in the modified rat model

Forty-eight hours after the CLP procedure, we scored the severity of sepsis and the clinical manifestations based on the appearance, consciousness, activity, response to stimuli, eye activity, respiratory rate, and respiratory quality of rats.^[12] For euthanasia, we filled a container with 70% carbon dioxide and placed the rats in the container. After 15–20 min, the rats lost consciousness and reflex, and then we performed cervical dislocation. The peritoneal cavity was opened for examination of the severity of peritonitis. We also estimated the severity of AKI according to the serum creatinine (Scr) level based on the RIFLE classification; that is, we defined no AKI (stage O), Risk (stage R), Injury (stage I), and Failure (stage F) according to increases in the serum creatinine level by <150%, 150%–200%, 200%–300%, and >300% from the baseline value, respectively.^[13]

We collected the rats that died within 48 h for pathological examination. If the rats survived 48 h after CLP, they were euthanized for the collection of kidneys. For rats whose death was not caused by sepsis, but rather causes such as anesthesia accident, postoperative hemorrhage, or heart rupture, additional rats were included to obtain additional data. We collected the left kidneys from the rats and immersed them in 10% neutral formalin followed by paraffin embedment, dehydration, and sectioning in 4 μm thickness. These specimens were stained with hematoxylin and eosin and periodic acid–Schiff. Two pathologists independently reviewed 10 high-power-field (×200) images of each section and defined renal injury according to the following criteria: Tubular vacuolization, tubular dilatation, brush border sloughing, and inflammatory cell infiltration. They scored the extent of tubular injury in a double-blind fashion, using the following scheme: No injury, 0; ≤10%, 1; 11%–25%, 2; 26%–45%, 3; 46%–75%, 4; and ≥76%, 5.^[14]

We collected 0.8-mL blood samples at 0 h after operation and at 12, 24, and 48 h after operation using for the measurement of AKI biomarkers. We used central venous catheterization with catheter fixation subcutaneously. About 0.8–1 mL blood was drawn from the catheter.^[15] Blood samples were placed at room temperature for 1 h and centrifuged at 3000 rpm for 15 min to obtain serum samples, which were stored at-80°C. We used the enzymatic method to measure Scr (BioVision Technologies, Mountain View, CA) and measured neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C (Cyc) levels using enzyme-linked immunosorbent assays (for NGAL, BioPorto Diagnostics, Gentofte, Denmark; for Cyc, AXXORA, Lörrach, Germany).

Monitoring animal survival

A separate group of rats was divided into the same four treatment groups randomly: A CLP group (n = 40); a modified CLP group (n = 40); a sham group (n = 6); and an omentectomy group (n = 6). We monitored the survival of these rats and constructed survival curves based on these findings.

Statistical analysis

We used SPSS version 17 software to perform the statistical analysis and GraphPad Prism 5 to graph the data. Normally distributed continuous data are presented as mean ± standard deviation. For the analysis of variance, we used the test of homogeneity of variance. If the variance was homogeneous, we performed an ANOVA and applied the least significant difference t-test for comparisons between groups. If the variance was not homogeneous, we used the Welch test and the Dunnett's T3 test for comparisons. We compared nonnormally distributed data using the Mann–Whitney U-test and compared the proportions of each group in the entire cohort using the Chi-square test. Survival analysis was done using the Kaplan–Meier technique, and the results compared with a log-rank test. A two-tailed P < 0.05 was considered statistically significant.

Results

Changes in serum acute kidney injury biomarkers

We examined Scr levels at 0, 24, and 48 h postoperatively in rats of all four groups [Figure 1]a. We observed no significant difference in Scr at 24 and 48 h between the sham and omentectomy groups; however, the Scr level at 24 h postoperatively was significantly higher in the traditional CLP group than in the sham group (31.67 ± 8.13 vs. 19.26 ± 2.56 μmol/L, P = 0.000), and the difference persisted to postoperative 48 h (47.55 ± 19.89 vs. 19.45 ± 1.22 μmol/L, P = 0.000). Scr in the modified CLP group was significantly higher at postoperative 24 h than that in the omentectomy group (42.68 ± 15.43 vs. 21.4 ± 1.25 μmol/L, P = 0.000), and the difference also persisted to postoperative 48 h (65.18 ± 28.44 vs. 22.58 ± 3.22 μmol/L, P = 0.000). Furthermore, the Scr levels in the modified CLP group were even higher than those in the traditional CLP group at 24 h (42.68 ± 15.43 vs. 31.67 ± 8.13 μmol/L, P = 0.001) and 48 h (65.18 ± 28.44 vs. 47.55 ± 19.89 μmol/L, P = 0.013). We subsequently used the RIFLE classification to categorize AKI severity based on the maximal Scr levels within 48 h postoperatively compared with baseline levels and analyzed the distribution of each RIFLE stage [Figure 1]b. We found that the proportions of rats with AKI within each RIFLE stage were similar in the traditional CLP group (R, 35%; I, 27.5%; and F, 25%), whereas the proportions in each RIFLE stage differed in the modified CLP group (R, 10%; I, 42.5%; and F, 47.5%, P = 0.001). The proportions of rats with stage I and F AKI in the modified CLP group (90%) were significantly higher than those in the traditional CLP group [52.5%, P = 0.000, [Figure 1]c].

Figure 1: Changes in serum creatinine in rats with septic acute kidney injury and the RIFLE categories. Sham group (n = 6), omentectomy group (n = 6), traditional cecal ligation and puncture group (n = 40), and modified cecal ligation and puncture group (n = 40). (a) Scr levels (μmol/L). (b) Comparison of RIFLE categories between groups. (c) Comparison of proportions of rats with moderate-to-severe acute kidney injury between groups. ^nsP > 0.05 compared with the sham group; ^αP < 0.05 compared with the sham group; ^βP < 0.05 compared with the omentectomy group; ^δP < 0.05 compared with the traditional cecal ligation and puncture group

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To better evaluate early changes in renal function, we also examined serum Cyc and NGAL levels at postoperative 12 h in each group. The mean Cyc levels did not differ between the omentectomy and sham groups, whereas the mean Cyc level in the traditional CLP group was significantly higher than that in the sham group (2028.77 ± 457.48 vs. 1183.66 ± 176.99 pg/mL, P = 0.000). In addition, the mean Cyc level in the modified CLP group was significantly higher than that in the omentectomy group (2515.17 ± 574.43 vs. 1197.33 ± 92.53 pg/mL, P = 0.000) and that in the traditional CLP group [2515.17 ± 574.43 vs. 2028.77 ± 457.48 pg/mL, P = 0.001; [Figure 2]a]. Similarly, the NGAL level did not differ between the omentectomy and sham groups but was significantly higher in the traditional CLP group than in the sham group (3003.57 ± 984.95 vs. 49.83 ± 8.35 IU/mL, P = 0.000). The mean NGAL level in the modified CLP group was significantly higher than that in the omentectomy group (3672.75 ± 788.23 vs. 55.66 ± 14.25 IU/mL, P = 0.000) and that in the traditional CLP group [3672.75 ± 788.23 vs. 3003.57 ± 984.95 IU/mL, P = 0.009; [Figure 2]b].

Figure 2: Changes in biomarkers of early acute kidney injury. Sham group (n = 6), omentectomy group (n = 6), traditional cecal ligation and puncture group (n = 40), and modified cecal ligation and puncture group (n = 40). (a) Cyc levels (pg/mL). (b) NGAL levels (IU/mL). ^nsP > 0.05 compared with the sham group; ^αP < 0.05 compared with the sham group; ^βP < 0.05 compared with the omentectomy group; ^δP < 0.05 compared with the traditional cecal ligation and puncture group

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Murine sepsis score

Sepsis severity estimation was done at 48 h after operation, and the results are shown in [Figure 3]a. The proportions of the different severities of sepsis among the traditional CLP group were similar, while in the modified CLP group, they were as follows: 0–7, 5%; 8–14, 12.5%; 15–21, 40%; and 22–28, 42.5% (P = 0.001). Thus, the majority of septic animals in the modified CLP group had scores of 15–21 and 22–28.

Figure 3: Murine sepsis scores in rats 48 h after surgery. Traditional cecal ligation and puncture group (n = 40) and modified cecal ligation and puncture group (n = 40). (a) Distribution of different murine sepsis severity score categories in the two groups. (b) Distribution of murine sepsis severity scores in the two groups. (c) Comparisons of the proportion of rats with moderate-to-severe sepsis between groups. P < 0.05 for the traditional cecal ligation and puncture group versus the modified cecal ligation and puncture group

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Further analysis showed that the median sepsis score in the traditional CLP group was 13.5 (interquartile range [IQR] 8.25–19), whereas that of the modified CLP group was 20 [IQR 16.25–24, P = 0.000; [Figure 3]b]. In addition, the distribution of animals with a sepsis score of 15–28 (82.5%) in the modified CLP group was significantly higher than that in the traditional group [45%, P = 0.02; [Figure 3]c].

Gross pathology of peritonitis

The procedures of omentectomy are shown in [Figure 4]a, [Figure 4]b, [Figure 4]c. Forty-eight hours after the operation, no cases of intraperitoneal bleeding, necrosis, or purulent ascites were observed in the sham and omentectomy groups grossly, and the bowel color was red without dilatation [Figure 4]d and [Figure 4]e. In the traditional CLP group, two types of peritonitis were noted with differing severities. Most rats had puncture site necrosis, and the necrotic sites were completely wrapped by the omentum, forming an encapsulated abscess accompanied by adjacent tissues and the peritoneal wall. Other intestinal segments appeared red without dilation or necrosis [Figure 4]f. A few rats in the traditional CLP group had puncture site necrosis but with only partial wrapping by omentum and pus spreading, leading to prominent bowel dilatation, edema, pallor, necrosis, and purulent ascites [Figure 4]g. In the modified CLP group, all rats had puncture site necrosis, peritoneal wall adhesion, and pus leakage without encapsulated abscess formation; their bowel walls appeared dilated, edematous, and pale with focal necrosis with visible purulent ascites [Figure 4]h. Thus, the rats of the modified CLP group exhibited consistent peritonitis of uniformly high severity.

Figure 4: Gross pathology of the peritoneal cavity in the experimental rats. Sham group (n = 6), omentectomy group (n = 6), traditional cecal ligation and puncture group (n = 40), and modified cecal ligation and puncture group (n = 40). (a-c) Procedures of omentectomy. (a) Peritoneal cavity entry and omentum identification. (b) Omental root ligation. (c) Omentum excision). (d-h) Peritoneal examination at postoperative 48 h. (d) Sham group. (e) Omentectomy group. (f) Complete wrapping of puncture sites by omentum in the traditional cecal ligation and puncture group. (g) Partial wrapping. (h) The modified cecal ligation and puncture group)

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Renal pathological findings

We defined the renal pathological findings according to the extent of tubule injury under microscopic examination [for scores 0, 1, 2, 3, 4, and 5, [Figure 5]a, [Figure 5]b, [Figure 5]c, [Figure 5]d, [Figure 5]e, [Figure 5]f, respectively]. We found no tubule injury in rats of the sham and omentectomy groups. The proportions of different severities of tubule injury among the traditional CLP group were similar, while those in the modified CLP group were as follows: 0–1, 5%; 1.1–2, 7.5%; 2.1–3.1, 15%; 3.1–4, 35%; and 4.1–5, 37.5% (P = 0.005). Thus, most rats in the modified CLP group had tubule injury of a severity between 3.1–4 and 4.1–5 [Figure 5]g. Further analyses showed that the median tubule injury score for the traditional CLP group was 2.3 (IQR 1.53–3.5), whereas that for the modified CLP group was 3.7 [IQR 2.5–4.5, P = 0.000; [Figure 5]h]. Using a cut-off score for tubule injury of 2, the proportion of rats with a score of 2.1–5 (87.5%) in the modified CLP group was significantly higher than that in the traditional CLP group [60%, P = 0.000; [Figure 5]i].

Figure 5: Microscopic findings of renal pathology (bar 100 μm, ×200). Traditional cecal ligation and puncture group (n = 40) and modified cecal ligation and puncture group (n = 40). (a) Tubular injury score 0. (b) Score 1. (c) Score 2. (d) Score 3. (e) Score 4. (f) Score 5. (g) Comparison of tubular injury score categories. (h) Distribution of renal pathological tubular injury scores in the two groups. (i) Comparison of proportions of rats with moderate-to-severe tubular injury between groups. P <0.05 for the traditional cecal ligation and puncture group versus the modified cecal ligation and puncture group

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Survival analysis

The survival status of animals in each group is shown in [Figure 6]. None of the animals in the sham and omentectomy groups died, whereas the 7-day survival rates in the traditional CLP group and modified CLP group were 65% and 50%, respectively.

Figure 6: Seven-day survival rates of rats with septic acute kidney injury, based on results of Kaplan–Meier analysis. Sham group (n = 6), omentectomy group (n = 6), traditional cecal ligation and puncture group (n = 40), and modified cecal ligation and puncture group (n = 40). ^nsP > 0.05 for the traditional cecal ligation and puncture group vs. the modified cecal ligation and puncture group

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Discussion

The severity of septic AKI is closely associated with that of sepsis per SE Stool leakage during the procedure of CLP leads to intraperitoneal infection and subsequent sepsis. It is acknowledged that the severity of CLP-induced sepsis is mainly affected by the length of ligation and the size and number of puncture holes, which correlate with the amount of stool in the cecum.^[8] Studies have suggested that with a higher position of cecum ligation, more severe sepsis will develop,^[16] which is consistent with our findings. However, we discovered that the severity of sepsis in the traditional CLP group was variable, with wide variation in the extent of peritonitis and murine sepsis severity scores, whereas rats in the modified CLP group developed predominantly moderate-to-severe sepsis. Rats with lower sepsis severity had complete wrapping of the punctured cecum by omentum accompanied by the formation of an encapsulating abscess along the adjacent tissues, whereas rats of the modified CLP group had more consistent and more severe peritonitis, manifesting as necrotic cecum at the puncture site without encapsulation and the development of purulent ascites. We believe that the extent and severity of CLP in the rat model are associated not only with the length of ligation and the size and number of puncture holes but also with the ability of the omentum to enwrap the puncture holes. Our results show that omentectomy during CLP can prominently increase the severity of sepsis and promote sepsis consistency.

Previous studies have found that the omentum, as an integral part of the immune system, plays an important role in maintaining the immunologic homeostasis of the abdominal cavity. On the one hand, the omentum contains cells with phagocytosis function. On the other hand, when the viscera in the abdominal cavity are inflamed or the gastrointestinal tract is perforated, the omentum tends to repair the foci or seal the perforation by wrapping up the foci or creating adhesions, in order to prevent the formation of diffuse peritonitis. In this study, the immunologic characteristics of the omentum were used to refine the traditional CLP approach. We showed that cecal ligation and perforation combined with omentum resection overcame the disadvantage of variation in the severity of renal injury with traditional CLP, by creating a model of sepsis-related renal injury with a more homogeneously moderate-to-severe insult. This refined approach provides a better way to study sepsis using animal models. In addition, since only the omentum was removed, no celiac hemorrhage or necrotic lesions were noted, nor were any abnormalities in renal pathology, any increase in serum markers of renal injury, or deaths of rats; these findings suggest that it could be safe to adopt this model for studying sepsis-related renal injury.

The Scr level is an important measure for evaluating the success rate of a septic AKI model in rats.^[15] Our study revealed that the Scr level in the traditional CLP group was significantly lower than that in the modified CLP group. We also found that the proportion of rats with stages I and F AKI in the traditional CLP group was 52.5%, consistent with results from other reports, whereas that with stages I and F in the modified CLP group was 90%. This suggests that omentectomy during CLP not only aggravates the severity of AKI in rats but also raises the consistency of AKI severity. In previous studies, researchers attempted to increase the severity of septic AKI in rats receiving CLP by placing stents into the cecum,^[17],[18] but the heterogeneity of this approach remained high due to the presence of omental wrapping of stents. In addition, the complexity of this stent approach is high, reducing its popularity. It is worth noting that the distribution of rats with RIFLE-classified AKI matched well with the distributions of renal pathology and sepsis severity, lending support to the validity of our model and the pathophysiology of septic AKI.

Literature reports suggest that changes in Scr cannot reflect early renal damage, and other biomarkers, such as Cyc, can be more important surrogates. Cyc is a more sensitive indicator of the decline in the glomerular filtration rate. NGAL is also found to increase rapidly, within 2 h after renal injury occurs, and has been considered an early and sensitive AKI biomarker.^[19],[20] In addition, higher levels of serum Cyc and NGAL suggest more severe AKI. In this study, the serum Cyc and NGAL levels in the CLP group and the modified CLP group were higher than those in the sham group at 12 h after operation, suggesting that renal injury was successfully induced in the CLP group and the modified CLP group. The serum Cyc and NGAL levels in the modified CLP group were higher than those in the CLP group, suggesting that the severity of renal injury in the modified CLP group was higher. However, this study only measured serum Cyc and NGAL levels at 12 h after operation; thus, whether the modified CLP group experienced renal injury earlier than the CLP group needs further study. Simple omental resection without CLP does not induce AKI; when omental resection coexists with CLP, the perforated and necrotic bowels cannot be wrapped, thus aggravating the inflammatory response and precipitating subsequent AKI.

The 7-day survival in animal models of septic AKI is another important measure of model validity.^[21] In the present study, the 7-day survival rate of rats in the traditional CLP group was 65%, whereas that in the modified CLP group was 50%, compatible with the model requirement of moderate-to-severe AKI. However, if the survival rate in the modified CLP group can be increased further, this model will be more useful. Prior studies used fluid supplementation and antibiotic treatment to increase the 7-day survival of septic animals,^{[22],[23],[24]} which was not done in the present study and constitutes a limitation of our study. In this study, we administered a single bolus of fluid supplement and one dose of antibiotic, without persistent fluid resuscitation or continuous antibiotic treatments.

In this study, we did not detect the level of inflammatory factors in AKI model, which is the limitation of our study.

Conclusion

Animal models of septic AKI established using the traditional CLP approach have high heterogeneity, and a modified CLP approach using omentectomy can establish a more stable model of moderate-to-severe septic AKI in rats.

Ethics approval

This animal experiment was approved by the Nanchan University experimental animal ethics committee (NDSYDWIL-201792).

Financial support and sponsorship

This study was financially supported in part by grants from the National Natural Science Foundation of China (grant No. 81460292 to Dr. Fen Liu, and grant No. 81560131 and No. 81772046 to Dr. Zhiyong Peng).

Conflicts of interest

There are no conflicts of interest.

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