Construction method and application of kidney stone induced acute kidney injury mouse model

Abstract

The invention belongs to the technical field of animal models, and provides a construction method and application of a kidney stone induced acute kidney injury mouse model. The construction method of the kidney stone induced acute kidney injury mouse model comprises the following steps: firstly, fasting and depriving water of a mouse for 12 hours, then injecting 10mg / kg sodium oxalate solution into the body of the mouse in an intraperitoneal injection manner, and meanwhile, replacing drinking water with 3wt% sodium oxalate solution, so that the kidney stone induced acute kidney injury mouse model can be obtained 24 hours later. The sodium oxalate solution is used as the lithigenic agent, the effect is obvious and reliable, the action is strong, the stability and the repeatability are high, the obtained acute kidney injury mouse model is used for subsequent AKI pathogenesis and AKI generation experiment research or drug screening, the observation is obvious, and the method is simple.

Description

Technical Field

[0001] The invention belongs to the technical field of animal models and relates to a method for constructing a mouse model of acute renal injury induced by kidney stones and application thereof. Background Art

[0002] Acute kidney injury (AKI), also known as "acute renal failure", is characterized by a rapid decline in renal function within hours or days. The pathological characteristics of AKI are dilation of the renal tubular lumen and thinning of the tubular wall; necrosis of renal tubular epithelial cells; foreign bodies in the tubular lumen; and occasionally interstitial edema. Clinically, changes in renal function are generally assessed by serum creatinine and urine volume. Due to its high mortality and morbidity, AKI has become a major problem affecting human health worldwide. Existing studies have shown that excessive intake of exogenous oxalate or increased intestinal absorption can lead to accumulation of calcium oxalate in the kidneys, leading to hyperoxaluria, and further leading to acute kidney injury. Existing AKI modeling methods include ischemia-reperfusion induction, cisplatin or contrast agent induction, and infection sepsis induction. In the process of these model studies, the professional requirements of ischemia-reperfusion induction surgery are high and subject to certain limitations, and drugs usually bring many other effects to mice, such as gastrointestinal toxicity and neurotoxicity, which may not only cause pain to mice but also affect the experimental results. Therefore, there is an urgent need for a modeling method that is simple to operate and does not cause other effects on mice. Summary of the invention

[0003] The present invention aims to provide a method for constructing a mouse model of acute renal injury induced by kidney stones and its application.

[0004] To achieve the above purpose, the technical solution adopted by the present invention is: a method for constructing a mouse model of acute kidney injury induced by kidney stones, the construction method comprising: administering sodium oxalate to C57BL / 6J mice, and obtaining a mouse model of acute kidney injury induced by kidney stones after 24 hours.

[0005] The inventors of the present application have found through a large number of experimental studies that excessive intake of exogenous oxalate or increased intestinal absorption can lead to accumulation of calcium oxalate in the kidneys, resulting in hyperoxaluria and further leading to acute kidney injury. Therefore, the inventors of the present application administered sodium oxalate to mice to establish a mouse model of kidney stone-induced acute kidney injury.

[0006] As a preferred embodiment of the method for constructing a mouse model of acute kidney injury induced by kidney stones according to the present invention, the mice are mice that have been fasted for 12 hours.

[0007] As a preferred embodiment of the method for constructing a mouse model of acute renal injury induced by kidney stones of the present invention, the sodium oxalate administration method includes intraperitoneal injection.

[0008] As a preferred embodiment of the method for constructing a mouse model of acute renal injury induced by kidney stones of the present invention, the sodium oxalate administration method also includes drinking.

[0009] As a preferred embodiment of the method for constructing a mouse model of acute renal injury induced by kidney stones of the present invention, the intraperitoneal injection dosage is 10 mg / kg.

[0010] As a preferred embodiment of the method for constructing a mouse model of acute renal injury induced by kidney stones according to the present invention, the drinking is drinking a 3 wt % sodium oxalate solution.

[0011] The present invention also provides a mouse model of kidney stone-induced acute kidney injury constructed by the construction method.

[0012] The present invention also provides the application of the method for constructing the kidney stone-induced acute kidney injury mouse model in screening or preparing candidate drugs for treating kidney stone-induced acute kidney injury disease or studying the occurrence and development mechanism of kidney stone-induced acute kidney injury disease.

[0013] The present invention also provides the use of sodium oxalate in the method for constructing the above-mentioned kidney stone-induced acute renal injury mouse model.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention provides a method for constructing a mouse model of acute kidney injury induced by kidney stones. The present invention constructs a mouse model of acute kidney injury induced by kidney stones by applying a certain dose of sodium oxalate to mice. Compared with the control group, the pathological staining of the kidneys of the mice in the sodium oxalate injection group showed dilation of the renal tubular lumen and thinning of the tubular wall; necrosis of renal tubular epithelial cells; AKI characteristics of foreign bodies in the lumen. And calcium oxalate staining can show that there are a large number of positive calcium oxalate crystals deposited in the kidneys of the sodium oxalate injection group, indicating that the model effect is obvious and reliable, and the effect is strong, and the stability and repeatability are high, which can be used for the development and research of the cause of AKI, the occurrence and development mechanism of AKI and related therapeutic drugs. The method for constructing the mouse model of acute kidney injury induced by kidney stones of the present invention is simple to operate, has a high success rate of construction, and a short construction cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The statistical graphs of serum urea nitrogen level (A) and serum creatinine level (B) of two groups of mice are shown;

[0017] Figure 2 CaOx staining photos of kidney sections of two groups of mice (A) and statistical graphs of stone area percentage (B) are shown;

[0018] Figure 3 HE staining photos of kidney sections of two groups of mice (A) and injury score statistics (B) are shown. DETAILED DESCRIPTION

[0019] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0020] Experimental Materials:

[0021] (1) Experimental animals: 8-week-old male C57BL / 6J mice, with an average body weight of about 22 g, were provided by the Experimental Animal Center of Nantong University and were raised in the Experimental Animal Center of Nantong University.

[0022] (2) Experimental reagents: sodium oxalate powder (Thermo Fisher Scientific), normal saline, double distilled water, serum urea nitrogen detection kit (Shanghai Sabo Biotechnology Co., Ltd.), creatinine detection kit (Shanghai Sabo Biotechnology Co., Ltd.), Avertin (Nantong Feiyu Biotechnology Co., Ltd.), 4% paraformaldehyde (biosharp).

[0023] Example 1: Construction of kidney stone-induced acute renal injury model

[0024] C57BL / 6J mice were randomly divided into 2 groups, 6 mice in each group: control group and sodium oxalate-treated stone-inducing group.

[0025] The mice in the sodium oxalate-treated stone-inducing group were deprived of water and food at 10:00 p.m. on the first day, while the mice in the control group were not treated. At 10:00 a.m. on the second day, 10 mg / kg body weight of sodium oxalate solution was intraperitoneally injected into the mice, and the water source was replaced with 3 wt% sodium oxalate solution. The mice in the control group were intraperitoneally injected with normal saline and given normal drinking water. After 24 hours, the mice in the sodium oxalate-treated group obtained a kidney stone-induced acute renal injury model.

[0026] Example 2: Serum urea nitrogen and creatinine level detection

[0027] After the model was established, blood was collected from the control group and the sodium oxalate treatment group through the retro-orbital venous plexus, and the ELISA method was used to detect the levels of serum urea nitrogen and creatinine in mice. The specific steps were as follows:

[0028] (1) The mice in the two groups were weighed and anesthetized with Avertin (250 mg / kg, intraperitoneal injection) until the animals showed anesthesia reactions such as loss of flexor reflex and flaccid paralysis.

[0029] (2) Peripheral blood was collected through the tail vein, and 10 μl EDTA (0.5 M, pH 8) was added to a 1.5 ml EP tube for anticoagulation.

[0030] (3) When blood flow is fast, 8-9 drops are enough.

[0031] (4) Place the centrifuge tube containing peripheral blood in a centrifuge and centrifuge at 8000 rpm for 6 minutes at room temperature. Aspirate the supernatant into a new centrifuge tube and store the collected plasma in an ice box.

[0032] (5) ELISA test was performed on mouse plasma to detect urea nitrogen and creatinine levels (ELISA kit was purchased from Shanghai Sabo Biotechnology Co., Ltd.), and the ELISA operation was performed according to the requirements of the kit.

[0033] The serum urea nitrogen level and creatinine level in the sodium oxalate-treated stone-inducing group were significantly higher than those in the control group. Figure 1 .

[0034] Example 3: Pathological detection of mouse kidneys

[0035] (1) After the mouse model obtained in Example 1 is established, it is weighed and anesthetized. After the mouse shows anesthesia reactions such as loss of corneal reflex, decreased muscle strength, and flaccid paralysis, it is fixed in a prone position on a mouse board.

[0036] (2) Kidney sampling: The kidneys were found on both sides of the lumbar spine outside the peritoneum. They were smooth in surface, slightly flattened dorsally and ventrally, and shaped like broad beans. After cutting open the abdominal cavity, both kidneys were removed, and the surrounding fascia and skin were removed. The kidneys were placed in a culture dish filled with sterile saline to wash and remove blood stains. The kidneys were then fixed in 4% paraformaldehyde for 24 hours before making tissue wax blocks.

[0037] (3) Preparation of paraffin sections: Remove the tissue from the fixative, trim it into a suitable shape with a scalpel, place it in a dehydration frame, mark it, and rinse the tissue block thoroughly with running water to remove the paraformaldehyde fixative. Dehydrate the tissue using alcohol from low to high concentrations, make the dehydrated tissue transparent, dip it in wax, embed it, and slice it.

[0038] (4) Tissue calcium oxalate staining: Paraffin sections were routinely dewaxed to water, washed with distilled water for 1 min, and completely immersed in Von Kossa silver solution and irradiated with strong light for 15-60 min, then washed with distilled water for 1 min, treated with Hypo solution for 2 min, and then counterstained with nuclear fast red staining solution for 5 min. After routine dehydration and transparency, the sections were sealed with neutral resin, and the formation of calcium oxalate in the rat kidneys was observed under a light microscope and photographed. The formation of calcium oxalate stones in the kidneys was analyzed using ImageJ software.

[0039] Figure 2 The CaOx staining photos of the kidney sections of the two groups of mice (A) and the stone area percentage statistics (B) are shown. The percentage of calcium oxalate positive staining area was analyzed by ImageJ software, and it was found that the sodium oxalate injection group had obvious calcium oxalate stone formation.

[0040] Oxalate ions enter the blood circulation through intraperitoneal injection. In the kidneys, oxalate ions combine with calcium ions to form calcium oxalate stones, which further lead to acute kidney injury. Figure 2 The number of calcium oxalate stones in the sodium oxalate-treated group in A was significantly higher than that in the control group. Figure 2 The area of ​​calcium oxalate stones was statistically analyzed, which showed that the stones in the sodium oxalate treatment group were significantly different from those in the control group, with more stones formed.

[0041] (5) HE staining of tissue: paraffin sections were routinely dewaxed to water, and the tissue was completely immersed in hematoxylin for 3-5 min until the nucleus was obviously stained. The sections were thoroughly rinsed in PBS buffer and counterstained with eosin. The degree of staining was observed under a microscope. After the sections were dried, they were sealed with neutral resin. The morphological changes of the rat kidney were observed under a light microscope and photographed.

[0042] HE staining renal injury score includes five indicators (renal tubular dilatation, tubular foreign body, tubular cell necrosis, brush border shedding, and interstitial edema). Each indicator is scored as 0 points if the damage is less than 10%, 1 point if 10-30%, 2 points if 30-50%, 3 points if 50-70%, 4 points if 70-90%, and 5 points if greater than 90%. The calcium oxalate staining evaluation method is: take a picture and use ImageJ software to analyze the percentage of the area occupied by positive staining. The study was conducted in a randomized and double-blind manner. GraphPad Prism 8 software was used for statistical analysis. All data are expressed as mean ± standard error. In all statistical analyses, P < 0.05 was considered statistically significant.

[0043] Figure 3 HE staining photos of kidney sections of two groups of mice (A) and injury score statistics (B) are shown.

[0044] Figure 3 In A, after sodium oxalate treatment, the mouse renal epithelial cells showed obvious interstitial edema, the renal tubular wall was dilated, there were obvious sloughing and foreign matter in the renal tubular lumen, and the renal tubular epithelial cells were necrotic. We used HE staining of the kidneys of mice treated with sodium oxalate to score renal injury, and the statistical graph is shown in the figure below. Figure 3 As shown, the renal injury scores of mice treated with sodium oxalate were significantly higher than those of control mice.

[0045] In summary, the mouse model of acute kidney injury caused by kidney stones constructed by the scheme of the present invention showed dilatation of the renal tubular wall, obvious detached matter and foreign matter in the lumen of the renal tubules, and AKI characteristics of necrosis of renal tubular epithelial cells. It can be seen that this embodiment successfully constructed a mouse model of acute kidney injury caused by kidney stones, which has obvious and reliable effects, strong effects, high stability and repeatability, and obtained an acute kidney injury mouse model for subsequent AKI etiology, AKI occurrence experimental research or drug screening, with obvious observation and simple method.

[0046] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for constructing a mouse model of acute kidney injury induced by kidney stones. It is characterized in that The construction method is: sodium oxalate is injected into C57BL / 6J mice, and a mouse model of kidney stone-induced acute kidney injury can be obtained 24 hours later.

2. The construction method according to claim 1, It is characterized in that The mice were fasted for 12 hours.

3. The construction method according to claim 1, It is characterized in that The sodium oxalate administration method includes intraperitoneal injection.

4. The construction method according to claim 3, It is characterized in that The sodium oxalate administration method also includes drinking.

5. The construction method according to claim 4, It is characterized in that The intraperitoneal injection dosage is 10 mg / kg.

6. The construction method according to claim 4, It is characterized in that The drinking is drinking 3wt% sodium oxalate solution.

7. Use of the method for constructing a mouse model of kidney stone-induced acute kidney injury according to any one of claims 1 to 6 in screening or preparing candidate drugs for treating kidney stone-induced acute kidney injury or studying the occurrence and development mechanism of kidney stone-induced acute kidney injury.

8. Use of sodium oxalate in the method for constructing a mouse model of acute kidney injury induced by kidney stones as claimed in claim 1.

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