Effects Of Contralateral Nephrectomy Timing And Ischemic Conditions On Kidney Fibrosis After Unilateral Kidney Ischemia-reperfusion Injury
Oct 27, 2023
Introduction
The incidence of acute kidney injury (AKI) has increased dramatically worldwide in recent years [1]. AKI can be caused by organ transplantation, postoperative underperfusion, bleeding, dehydration, shock and sepsis in clinical practice [2]. Recent epidemiological and experimental studies have shown that the severity, frequency, and duration of AKI are closely associated with the subsequent incidence of chronic kidney disease (CKD). Kidney fibrosis after AKI can lead to CKD or even end-stage kidney disease (ESKD) [3–5], implying that AKI and CKD are linked syndromes [6]. However, the mechanisms underlying the transition from AKI to CKD remain unknown despite numerous experimental and clinical studies. Animal models are important tools for investigating the mechanisms by which AKI leads to CKD. Kidney ischemia-reperfusion injury (IRI) is one of the major causes of AKI, and several animal models of IRI-induced AKI have been developed [7], including the bilateral IRI (bIRI) model, the unilateral IRI model with intact contralateral kidney, the uIRI model with simultaneous contralateral nephrectomy, and the uIRI model with delayed contralateral nephrectomy.
The bIRI model is less consistent because when the ischemic injury of both kidneys is too mild, there are no significant fibrotic changes in the long term. When AKI is too severe, mice may die in the acute injury phase. Furthermore, small differences in the injury of the two kidneys may lead to significant differences in their chronic kidney pathology after a few weeks [8,9]. The uIRI model with simultaneous contralateral nephrotomy is similar to the bIRI model as only the injured kidney remains due to the other side nephrectomy [10]. In the IRI model with an intact contralateral kidney, the fibrosis of the ischemic kidney is severe [11,12]. This model is very consistent and reliable for long-term observation by researchers. However, due to the presence of a contralateral undamaged kidney, it cannot be used for the evaluation of estimated glomerular filtration rate (eGFR), serum creatinine (SCr), and blood urea nitrogen (BUN) [11–14]. Compared with the two uIRI models mentioned above, the uIRI model with delayed contralateral nephrectomy overcomes these problems and can be used for both long-term observations after AKI and kidney function monitoring. Therefore, this model is suitable for studying AKI-CKD transition [15].
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Nevertheless, different ischemic conditions and the interval between contralateral nephrectomy and uIRI may have significant impacts on the progression and severity of CKD after AKI. Previous studies have focused on the effect of the ischemic duration on post-AKI kidney fibrosis and have not paid much attention to the core body temperature during ischemia. For example, Skrypnyk et al. [12] controlled the ischemic temperature at 38 C by a water bath system, with an ischemic duration of 30 min, and the contralateral intact kidney was removed 8 days after uIRI. The result showed that the ischemic kidney had significant fibrotic changes 28 days after uIRI. With the same ischemic duration, Xiao et al. [8] showed that significant fibrotic changes happened 11 days after uIRI when the ischemic temperature was controlled at 37 C 38 C and the time interval between uIRI and contralateral nephrectomy was 10 days. Some researchers, such as Colombaro et al. [14] and Li et al. [16] did not mention the ischemic temperature in their studies.
Meanwhile, few people researched the effect of the time interval between contralateral nephrectomy and uIRI in this model, resulting in widely varying results among different laboratories, which also dramatically affected subsequent experiments. In this study, we investigated the effects of the main factors affecting kidney fibrosis after AKI, including the time interval between contralateral nephrectomy and uIRI, the core body temperature during ischemia, and the ischemic duration. We aimed to provide a reference to make an appropriate animal model for studying the AKICKD transition
Materials and methods Animal models
Male C57BL/6J mice (8–10 weeks of age, approximately 22 g of weight; GemPharmatech Co., Ltd., Nanjing, China) were provided with free access to food and water in the Animal Experiment Center of Shanxi Medical University. The breeding conditions were as follows: humidity 40–70%, temperature 23 C, air cleanliness class 7, controlled 12-h light/dark cycle under pathogen-free conditions, feed Co60 irradiation, and purified water. All procedures for this study were approved by the Animal Ethics Committee of the Second Hospital of Shanxi Medical University (ethics number: DW2022028).
Protocols
Considering that ischemic durations, core body temperatures during ischemia, and resection timings of the contralateral uninjured kidney may all have impacts on the severity of AKI and subsequent kidney fibrosis, we investigated the effects of the above three factors individually, with the other two fixed. Three experiments were conducted as follows.
Protocol 1: To observe the effect of the interval between contralateral nephrectomy and uIRI on the kidney function and post-AKI fibrosis, the ischemic time of the left kidney was fixed at 24 min and the core body temperature was fixed at 37 C during the kidney ischemia meanwhile. The right kidney resection was conducted on day 7, day 10, and day 14, respectively (n ¼ 8 per group) after the left kidney IRI. We determined the duration of ischemia and kidney resection based on the results of previous studies [8,10,12,15] and our pre-experimental results. See Figure 1(A–C) for the study protocol.
Protocol 2: To observe the effect of core body temperature during ischemia on the AKI-CKD transition, the ischemic time of the left kidney was fixed at 24 min, and the right kidney was removed 14 days after the left kidney IRI. The core body temperature was controlled at 36 C, 36.5 C, 37 C, 37.5 C, and 38 C, respectively (n ¼ 8 per group). See Figure 1(D) for the study protocol.
Protocol 3: To observe the effect of kidney ischemic durations on the transition from AKI to CKD, the core body temperature was fixed at 37 C during ischemia, and the right kidney was removed 14 days after the left kidney IRI. We chose this time point was because kidney fibrosis, which was the typical change after AKI to CKD, was very obvious according to the reports [8,10,12,15] and our pre-experimental results. The ischemic time of the left kidney was controlled at 21 min, 24 min, 27 min and 30 min, respectively (n ¼ 8 per group). See Figure 1(E) for the study protocol.
All mice were euthanized at 8 weeks after IRI of the left kidney. Part of the left kidney tissue was stored at 70 C for the subsequent RT-PCR and western blot analysis, and the other part was fixed in neutral formalin for histopathological analysis. Blood samples were collected via the retro-orbital sinus for biochemical testing.
Figure 1. Protocols of the unilateral ischemia-reperfusion injury (uIRI) with delayed contralateral nephrectomy. (A–C): The left kidney was subjected to IRI on day 0, and right kidney resection was conducted at different time points (day 7, day 10, and day14) with the ischemic duration fixed at 24 min with the core body temperature fixed at 37 C during kidney ischemia. (D): The left kidney was subjected to IRI on day 0, and right kidney resection was conducted under different core body temperatures (36 C, 36.5 C, 37 C, 37.5 C, and 38 C) with the ischemic time of the left kidney fixed at 24 min, and the right kidney was removed 14 days after the left kidney IRI. (E): The left kidney was subjected to IRI on day 0, and right kidney resection was conducted at different ischemic durations (21 min, 24 min, 27 min, and 30 min) with the core body temperature fixed at 37 C during kidney ischemia, and the right kidney was removed 14 days after the left kidney IRI. Kidney weight, kidney function, kidney histological injury, and kidney fibrosis were assessed until day 56.
Surgical procedure
Before surgery, all mice fasted for 12 h with free access to water and were injected with 3% pentobarbital sodium 50 mg/kg intraperitoneally. During anesthesia, the rectal temperature was maintained at 36 C, 36.5 C, 37 C, 37.5 C, or 38 C, respectively, in different experimental groups. To achieve this, the mouse was placed with its back on a heating pad (CW-26, BARBAROUS GROWTH, Zhejiang, China) in a position with its head and neck extended to ensure that its airway remained unobstructed, and the eyes were covered with warm saline gauze to avoid drying during the procedure. After anesthesia, the hair on the left back of the mouse was shaved with the hair clipper. The skin in the surgical area was then wiped clean with a 75% alcohol swab. Generally, the body temperature of the mouse drops after hair shaving. The surgical procedure was carried out when the core body temperature became constant, which could take about 15 min. The left kidney could be seen by cutting the left dorsal skin 0.5 cm next to the midline and 0.5 cm at the lower edge of the rib cage of the mouse. Then we squeezed the left abdomen with one hand to make the left kidney out of the body cavity, lifted the kidney with blunt curved forceps (taking care to avoid damaging the kidney), detached the kidney pedicle and clamped it quickly, and placed the clamped kidney under the skin to keep it warm and moisturized. Successful ischemia was characterized by a gradual color change of the kidney from red to dark purple, which lasted 21 min, 24 min, 27 min and 30 min respectively, in different experimental groups. The kidney color changed back to red within 2–5 min after releasing the clamp. Then we closed the abdominal cavity with layered sutures. The right dorsal skin and muscle were incised on day 7, day 10, and day 14 after uIRI, respectively, in different experimental groups, and the right kidney was resected. Then close the wound with standard sutures. After each surgery, 500 lL saline was injected intraperitoneally to compensate for the fluid loss during surgery. At the same time, the mouse was kept on a heating pad for about 2–3 h until it gained full consciousness before being transferred to the housing cage. The sham group did not clamp the kidney pedicle, and the rest of the surgical operations were the same as those of the uIRI group. All surgical operations were performed by the same person with more than 2 years of experience in the operation of this model.
Kidney function assay
SCr levels were measured by the Creatinine Kit (Jaffe Method, 100020170, Biosino Biotechnology and Science). BUN levels were measured by the Urea Kit (Urease-Glutamate Dehydrogenase Method, Biosino Biotechnology and Science).
Kidney weight assay
After urination, the body weights of mice were measured by a precision electronic scale. Fresh kidneys were rinsed three times in pre-chilled saline, then excess water on the kidneys was blotted off with filter paper. The kidney capsules were peeled off on the ice and the kidneys were weighed immediately. The ratio of kidney weight to body weight was calculated to evaluate corrected kidney weight in mg/g.
Hematoxylin-eosin (HE) staining and kidney injury score
Neutral formalin-fixed kidney tissues were routinely dehydrated, embedded in paraffin, sectioned (3 lm), and stained with HE to observe the damage of kidney tissues. Ten randomly selected fields of the kidney cortex of each section were captured with light microscopy at 400 magnification. Two pathologists scored the injury to the glomerulus and kidney tubules separately. The tubular injury was scored on a scale of 0–4 using the following criteria [17]: 0, no injury; 1, affecting 1–25% of the kidney area; 2, affecting 26–50% of the kidney area; 3, affecting 51–75% of the kidney area; and 4, affecting 75–100% of the kidney area. The degree of tubular injury was evaluated semi-quantitatively by calculating the area of kidney tubular injury. The glomerular injury was evaluated and scored on a scale of 0–4 according to the percentage area of mesangial expansion or sclerosis using the following criteria [17]: 0, normal glomeruli; 1, 1–25% of area injured; 2, 26–50% of area injured; 3, 51–75% of area injured; and 4, 76–100% of area injured. The final glomerular damage score ¼ 0 (% grade 0 glomeruli) þ 1 (% grade 1 glomeruli) þ 2 (% grade 2 glomeruli) þ 3 (% grade 3 glomeruli) þ 4 (% grade 4 glomeruli). More than 50 glomeruli were scored per mouse.
Masson’s trichrome staining
Neutral formalin-fixed kidney tissues were routinely dehydrated, embedded in paraffin, sectioned (3 lm), and stained with Masson’s trichrome to observe kidney fibrosis. In Masson’s trichrome-stained slices, twenty non-overlapping fields of kidney cortex were captured under 200 magnification. Image-Pro Plus Image Analysis Software was used for automatic measurement and analysis. The percentage of the fibrotic area was used to figure out the level of kidney interstitial fibrosis [8].
RT-PCR
Total mRNA was extracted from the kidney tissue of each group by Trizol and its concentration was measured, then it was converted to cDNA (Taka Reverse Transcription Kit). The Taqman fluorescence method (TB Green Premix Ex Taq II Real-time RT-PCR) was used to quantify fibronectin (FN) and collagen I (COL-I) RNA expressions in kidney tissues. Specific primers were used for FN (forward: 50 -GGAGTGGCACTGTCAACCTC-30 and reverse: 50 -ACTGGATGGGGTGGGAAT-30 ), and COL-I (forward: 50 -ACATGTTCAGCTTTGTGGACC-30 and reverse: 50 -TAGGCCATTGTGTATGCAGC-30 ) [18] (provided by Sangon Biotech Co., Ltd., Shanghai, China). The amplification conditions are as follows: 95 C for 30 s, 95 C for 5 s, 60 C for 30 s, 40 cycles; 95 C for 10 s, 65 C for 5 s, and 95 C/5 C. After analyzing the amplification curves and solubility curves of the products, the relative mRNA levels were determined by the comparative CT method (2-DDCt) with 18SrRNA (forward: 50 - CGGACACGGACAG GATTGACAG-30 and reverse: 50 -AATCGCTCCACCAACT AAGAACGG-30 ) as the internal reference.
Western blot
Radio-immuno-precitation-assay (RIPA) lysis buffer was used to lyse the kidney tissues and the concentration of protein was determined by the BCA method. Gradient gel electrophoresis was used to separate the proteins from 50 lg of samples. Then the proteins were transferred to a nitrocellulose membrane. After blocking with 5% nonfat milk for 2 h, the membranes were blotted overnight at 4 C with rabbit anti-mouse monoclonal antibody against a-smooth muscle actin (a-SMA) (1:1000, No.19245, Cell Signaling Technology), FN (1:1000, No.ab2413, ABCam), or COL-I (1:1000, No.72026, Cell Signaling Technology), respectively. On the next day, the membranes were rewarmed at room temperature for 0.5 h. After being washed with TBST, the membranes were incubated with HRP Conjugated AffiniPure Goat Anti-rabbit IgG(H þ L)(1:1000, No.BA1054, BOSTER Biological Technology) for 2 h. After being washed with TBST again, they were stained with ultra-high-sensitivity ECL chemiluminescence reagent to visualize the immunoblots. b-actin was measured on the same membrane as the internal reference. Protein expressions were determined by quantifying the relative expressions of target proteins versus b-actin.
Data and statistical analysis
All data examined are expressed as mean ± standard deviation. Comparisons between the two groups were accessed by an independent t-test. Comparisons between multiple groups were accessed by one-way ANOVA when the data distribution is normal, and the variance is homogeneous, Welch ANOVA was used to assess statistical significance when the variance was heterogeneous, followed by Tukey’s post hoc test for multiple comparisons. All statistical analyses were conducted by the GraphPad Prism 8.0 software. p < 0.05 was considered to be statistically significant.
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