A method for constructing an animal model of kidney injury caused by uric acid crystallization

Abstract

The application discloses a method for constructing an animal model of kidney injury caused by uric acid crystallization, which comprises the following steps: administering a urate crystal suspension to experimental animals, wherein the dosage of the urate crystal suspension is 0.5-100 mg / kg. Further, the urate crystal suspension is administered to the experimental animals by injection, especially by injection into the renal cortex and renal parenchyma. The method for constructing the animal model of kidney injury caused by urate crystal injection into the renal cortex and renal parenchyma is stable, reproducible, simple, objective, and easy to operate, and other possible interference factors are reduced, thereby providing necessary experimental basis for studying the process of kidney injury caused by urate crystal.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology, specifically relating to a method for constructing an animal model of kidney damage caused by uric acid crystals. Background Technology

[0002] Hyperuricemia nephropathy (HN) is a serious threat to public health. Studies have shown a significant impact of hyperuricemia on the progression of chronic kidney disease, although the underlying mechanisms remain unclear. However, researchers agree that the kidneys, as the main tissues responsible for uric acid excretion and reabsorption, lead to kidney damage due to the elevated uric acid levels and accumulation of sodium urate (MSU) crystals in the kidneys following hyperuricemia.

[0003] However, most animal models used in basic research on hyperuricemic nephropathy currently employ methods that increase serum uric acid concentration. Theoretically, this represents kidney damage caused by serum uric acid, and cannot investigate the effects of sodium urate crystals on the kidneys. Furthermore, the formation and observation of sodium urate in vivo presents significant technical challenges. Therefore, elucidating the mechanism by which sodium urate crystals cause kidney damage is of great importance.

[0004] The latest research indicates that hyperuricemic nephropathy is caused by kidney damage triggered by sodium urate crystals, rather than by elevated blood uric acid levels. This differs from previous animal models of hyperuricemic nephropathy.

[0005] Therefore, developing a stable animal model of sodium urate-induced kidney injury is particularly important. Current research on models of hyperuricemic nephropathy mainly includes: (1) increasing uric acid sources; (2) inhibiting uric acid excretion; and (3) inhibiting uric acid metabolism, as detailed below:

[0006] (1) Increasing uric acid sources: Increasing uric acid sources can be achieved by directly supplementing uric acid or uric acid precursors, mainly including adenine, hypoxanthine, fructose, and yeast extract. In most studies, the above-mentioned drugs were administered by gavage to establish the model.

[0007] (2) Uric acid excretion inhibition method: Anti-tuberculosis drugs, such as ethambutol and pyrazinamide, can inhibit uric acid excretion and cause an increase in blood uric acid. Most studies use the method of gavage administration of the above drugs to establish the model. However, there are few studies on the modeling of ethambutol alone. Usually, it is used in combination with other chemical drugs.

[0008] (3) Uric acid metabolism inhibition method: Potassium oxonate, as a uricase inhibitor, can inhibit uricase activity and highly simulate the human condition without uricase. Its single administration can induce the HUA model in a short time, but it is excreted quickly and cannot be used alone for long-term modeling. Therefore, it is often used in combination with other drugs to create the model.

[0009] The three models described above simulate elevated blood uric acid concentrations in different ways, observing the effects of high uric acid levels on the kidneys by extending the modeling time. However, none of these three models can stably deposit sodium urate crystals in the kidneys, thus failing to investigate the effects of sodium urate crystals on the kidneys. Therefore, there is an urgent need for a method to construct and evaluate an animal model that can simulate sodium urate crystal deposition in the kidneys and simultaneously induce kidney damage. Summary of the Invention

[0010] The purpose of this invention is to provide a method for constructing an animal model of kidney injury caused by uric acid crystals. This method can simply and stably establish an animal model of kidney injury caused by urate crystals in the kidneys.

[0011] The above-mentioned objective of the present invention can be achieved by the following technical solution: a method for constructing an animal model of kidney injury caused by uric acid crystals, comprising the following steps: administering a urate crystal suspension to experimental animals, wherein the amount of the urate crystal suspension is 0.5 to 100 mg / kg.

[0012] Preferably, the urate crystal suspension is administered to experimental animals by injection, wherein the amount of the urate crystal suspension is 0.5–100 mg / kg.

[0013] More preferably: the experimental animals are modeled by intrarenal injection, and a urate crystal suspension is administered, wherein the amount of the urate crystal suspension is 0.5 to 100 mg / kg.

[0014] Specifically, the urate crystal suspension is administered via intrarenal injection through the renal cortex.

[0015] Preferably, the injection depth is 1 / 3 to 2 / 3 of the total width of the kidney of the experimental animal.

[0016] Taking rats as an example, the syringe needle is inserted to a depth of about 1 / 3 to 2 / 3 of the total width of the rat kidney, and the injection depth is about 5-10 mm, which can reach the corticomedullary junction of the rat kidney tissue.

[0017] Preferably, the amount of the urate crystal suspension used is 50-80 mg / kg.

[0018] Preferably, the urate crystal suspension is a sodium urate crystal suspension.

[0019] Preferably, the experimental animal is selected from at least one of rats, dogs, monkeys, pigs, and cattle.

[0020] More preferably, the experimental animal is a rat.

[0021] Furthermore, the construction method also includes verifying the pathological morphology, serum uric acid value, and serum creatinine value of the experimental animals after they are injected with urate crystal suspension.

[0022] HE detection results showed that the pathological features of the HN rat model constructed by the method of the present invention were characterized by focal pathological damage at the site of urate crystals.

[0023] MASSON results showed that the pathological features of the HN rat model constructed by the method of the present invention were characterized by focal renal fibrosis at the site of urate crystals.

[0024] In this invention, there was no significant difference in serum uric acid levels between the model group and the control group in HN rats. This indicates that the pathological damage induced by this modeling method is not due to elevated serum uric acid levels.

[0025] The experimental verification index for serum uric acid value is that there is no significant change in serum uric acid value between rats in the urate crystal injection group and rats in the control group. If this condition is met, the animal model of hyperuricemic nephropathy (HN) is successfully established.

[0026] Compared with the control group, the serum creatinine level of HN rats in the model group of this invention was significantly increased. This indicates that this modeling method can affect the renal function of rats in the fourth week. The rat model of renal injury and renal fibrosis prepared by the method of this invention exhibits manifestations of renal function impairment in rats.

[0027] Therefore, this invention proposes an animal model of kidney damage caused by uric acid crystals, which is constructed using the above method.

[0028] The present invention has the following advantages:

[0029] (1) In order to reduce the harm to animal models, this invention uses the intrarenal injection method to construct an animal model of kidney injury caused by urate crystals. The animal model of kidney injury caused by urate crystals has been successfully established. It has good stability and repeatability, is simple and objective, and is highly operable. It reduces other possible interfering factors and provides a necessary experimental animal model for studying the process of kidney injury caused by urate crystals.

[0030] (2) The evaluation method provided by this invention is highly reliable, accurate and universally applicable for evaluating the constructed animal model of kidney injury caused by urate crystals. Attached Figure Description

[0031] Figure 1 This is a flowchart of the experiment in Embodiment 1 of the present invention;

[0032] Figure 2 This is a schematic diagram of intrarenal injection of urate crystals into rats in Example 1 of the present invention;

[0033] Figure 3 This is a histopathological examination result of the renal corticomedullary junction in the rat model of Example 2 of the present invention;

[0034] Figure 4 This is a diagram showing the detection results of renal cortical fibrosis in the rat model of Example 2 of the present invention;

[0035] Figure 5 This is a graph showing the detection results of serum uric acid levels in a rat model in Example 2 of the present invention;

[0036] Figure 6 This is a graph showing the detection results of serum uric acid levels in a rat model in Example 2 of the present invention. Detailed Implementation

[0037] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the implementation of the present invention.

[0038] Experimental animals: 7-week-old male SD rats, with an average weight of about 200g, were provided by Shanghai Jihui Experimental Animal Breeding Co., Ltd., and were raised at the Animal Research Center of Guangdong Provincial Academy of Traditional Chinese Medicine.

[0039] Preferred but not limited experimental materials include: alcohol, iodine solution, cotton pads, drapes, syringes, physiological saline, 1mL syringes, animal shaving clippers, surgical scissors (curved and straight tips), hemostatic forceps (curved and straight tips), tweezers (curved and straight tips), sutures (8-0 and 5-0), heating plates, chloral hydrate (purchased from the Animal Research Center of Guangdong Provincial Academy of Traditional Chinese Medicine); uric acid (purchased from Sigma); rat serum ELISA uric acid antibody (purchased from Nanjing Jiancheng Biotechnology Co., Ltd.); 4% paraformaldehyde (purchased from Biosharp); tissue embedding cassettes (purchased from Beyotime Biotechnology Research Institute); hematoxylin and eosin staining kit (purchased from Beyotime Biotechnology Research Institute); and Masson's staining kit (purchased from Beijing Regen Biotechnology Co., Ltd.).

[0040] Example 1

[0041] This embodiment provides a method for constructing an animal model of kidney injury caused by uric acid crystals, as shown in the flowchart below. Figure 1 As shown, it includes the following steps:

[0042] (1) Selection of experimental animals:

[0043] Seven-week-old male SD rats with an average weight of about 200g were selected and placed in a clean cage at a temperature of 22±2℃ and a relative humidity of 40%-45%. The rats were kept in a dark environment for 12 hours each. They were fed with standard rat feed and had free access to water.

[0044] (2) Preparation of urate crystallization injection:

[0045] ① Dissolve 400mg Sigma uric acid in 80mL of pure water and heat it to 60℃ in a constant temperature water bath;

[0046] ② Dissolve uric acid in 3M NaOH (about 2.8 mL) until completely dissolved;

[0047] ③ Adjust the pH to 8.9 with NaOH and glacial acetic acid, and crystallize at room temperature for 2 days;

[0048] ④ Collect the crystals on filter paper, wash them three times with anhydrous ethanol, and then dry them in a microwave oven on high.

[0049] ⑤ Collect sodium urate (MSU) crystals in centrifuge tubes and sterilize them at 180℃ for 2 hours;

[0050] ⑥ Prepare MSU crystal suspension using sterile PBS;

[0051] ⑦ Draw up the MSU crystal suspension with a syringe;

[0052] Sterilization of molding supplies: Surgical scissors (bent and straight), hemostatic forceps (bent and straight), tweezers (bent and straight), and cotton pads are sterilized by high temperature and high pressure.

[0053] (3) Urate crystal injection surgery: After one week of acclimatization feeding, the rats were weighed and anesthetized with chloral hydrate (350 mg / kg, ip) via intraperitoneal injection. After anesthesia, the hair below the sternum on the back of the rats was removed and disinfected with povidone-iodine. After disinfection, the iodine was removed with physiological saline. When the rats collapsed, became weak, and lost their reflexes, it indicated that the rats had reached a suitable state of anesthesia. The rats were placed in a prone position on a sterile operating table. Using surgical scissors, the epidermis and muscle layers of the rats were cut open, making the opening as small as possible. After the kidneys were visible, the abdomen of the rats was gently squeezed to expel the kidneys from the abdominal cavity through the surgical opening on the back.

[0054] Insert the syringe needle into the cortical-medullary junction at the upper pole of the rat kidney (injection depth approximately 1 / 3-2 / 3 of the rat kidney width, i.e., 5-10 mm), and slowly inject the drug. The injection volume is 10 mg per kidney (50 mg / kg, based on rat body weight). Figure 2 As shown. The lower pole of the kidney was not injected, serving as a self-control for the injected group rats, indicating the absence of urate crystals in the kidneys. After injection, the needle was withdrawn, the kidney was returned to the abdominal cavity, and 1 mL of physiological saline was administered. The rat muscle and epidermal layers were sutured together. After suturing, blood was wiped from the wound with saline-soaked cotton pads, and the rat was placed on a heating plate. Once the rat awoke, it was returned to its cage. This established a rat model of hyperuricemic nephropathy (HN) induced by urate crystals.

[0055] Control group rats did not undergo urate crystal injection surgery.

[0056] Example 2

[0057] 1. Pathological examination of rat kidneys

[0058] (1) The rat model prepared in Example 1 was weighed after modeling (after intrarenal injection of urate crystals), and anesthetized with chloral hydrate (350mg / kg, ip). After the animals showed anesthesia reactions such as loss of corneal reflex, decreased muscle strength, and flaccid paralysis, they were fixed in a prone position on a rat board.

[0059] (2) Kidney perfusion: Cut open the pleura and peritoneum, insert the syringe into the left ventricle of the mouse, and slowly push in physiological saline until the kidney turns white, indicating that the red blood cells in the kidney have been washed away. Then stop the perfusion.

[0060] (3) Kidney sampling: The kidneys were located on both sides of the spine in the extraperitoneal lumbar region. They were smooth, slightly flattened dorsoventrally, and bean-shaped. They were white in color. The renal hilum on both sides was cut off with scissors, and the kidneys were removed. The renal capsule was removed, and the kidneys were placed in a culture dish containing sterile physiological saline. The left kidney was transversely cut at the middle renal hilum and fixed in 4% paraformaldehyde. After 24 hours of fixation, tissue paraffin blocks were prepared.

[0061] (4) Paraffin section preparation: Remove the tissue from the fixative, trim it into a suitable shape with a scalpel, place it in a dehydration frame and label it. Rinse the tissue block thoroughly with running water to remove the paraformaldehyde fixative. Dehydrate the tissue using alcohol from low to high concentrations. Clear, embed, and section the dehydrated tissue.

[0062] (5) HE staining of tissue: Paraffin sections were dewaxed routinely and then placed in water. The tissue was completely immersed in hematoxylin for 15-20 minutes until the cell nuclei were clearly stained. The sections were thoroughly rinsed in PBS buffer, counterstained with eosin, and the degree of staining was observed under a microscope. After the sections dried, they were mounted with neutral resin and the morphological changes of rat kidneys were observed and photographed under a light microscope.

[0063] (6) HE test results are as follows Figure 3As shown in the figure, the renal tubules of the control group rats were well-developed, with no obvious apoptosis or inflammatory cell infiltration. In the kidney tissue of rats with urate crystal injection model, urate crystals were observed in the upper pole of the kidney where urate crystals were injected. Pathologically, the area around the urate crystals showed vacuolar degeneration and necrosis of the renal tubular epithelium, renal tubular dilation, loss of the brush border of the proximal convoluted tubule, and an increased number of inflammatory cells. The kidney injury score and the number of inflammatory cells infiltrated in this model were higher than those in the control group. In contrast, in the kidney tissue of rats with urate crystal injection model, no urate crystals were found in the lower pole of the kidney where no urate crystals were injected, and no pathological damage was observed in the upper pole; the tissue condition was the same as that of the control group rats. This indicates that urate crystals in kidney tissue can cause pathological damage to the surrounding tissues. Other models characterized by elevated uric acid showed diffuse kidney pathological damage. The results indicate that the pathological characteristics of the HN rat model established by the present invention are characterized by focal pathological damage at the site of urate crystals.

[0064] (7) MASSON staining: Paraffin sections were routinely dewaxed and placed in water. The sections were then stained with Bouin's solution at 37°C for 2.5 hours. The sections were rinsed with running water until the yellow color disappeared. Azurite blue staining solution was applied for 3 minutes, followed by a slight rinse. Mayer's hematoxylin staining solution was applied for 3 minutes, followed by a slight rinse. The sections were stained with a separating solution for 15 seconds and rinsed with running water. Ponceau S staining solution was applied for 10 minutes, followed by a slight rinse with distilled water. The sections were treated with phosphomolybdic acid solution for 10 minutes. Aniline blue staining solution was applied directly to the sections for 4 minutes. The sections were treated with a weak acid solution for 2 minutes. The sections were dehydrated three times with anhydrous ethanol for 10 seconds each time. The sections were cleared three times with xylene for 2 minutes each time, then mounted with neutral resin. The degree of fibrosis in the rat kidney tissue was observed under a microscope.

[0065] (8) MASSON test results are as follows Figure 4 As shown in the figure, fibrin expression in the renal tubules and interstitium of the control group rats was uniform and low. In the kidney tissue of rats with urate crystal injection model, urate crystals were observed in the upper pole of the kidney where urate crystals were injected. The renal tubules around the urate crystals were damaged, the interstitial area was enlarged, and the area of ​​fibrin-positive interstitium in the interstitium was significantly higher than that in the control group rat model. However, in the kidney tissue of rats with urate crystal injection model, no urate crystals were found in the lower pole of the kidney where no urate crystals were injected, and there was no increase in the area of ​​fibrosis in the upper pole; the fibrosis in the lower pole was the same as in the control group rats. This indicates that urate crystals induce fibrosis in the surrounding tissues of the kidney. Other models characterized by elevated uric acid showed diffuse renal fibrosis. The results indicate that the pathological features of the HN rat model established by this invention are characterized by focal renal fibrosis at the site of urate crystals.

[0066] Overall pathological results indicate that the rat model of kidney injury and fibrosis prepared by the present invention was obtained.

[0067] 2. Serum uric acid test

[0068] After the rat HN model was prepared in Example 1, blood was collected from the inferior vena cava, and serum uric acid was detected by ELISA. The specific steps were as follows:

[0069] (1) After weighing, anesthetize with chloral hydrate (350mg / kg, ip) until the animal shows anesthetic reactions such as loss of corneal reflex, decreased muscle strength, and flaccid paralysis.

[0070] (2) Cut open the skin and muscle layers of the rat's lower abdomen to expose the intestinal tissue. After gently moving the intestinal tissue to one side, the inferior vena cava can be seen.

[0071] (3) Insert the blood collection needle into the inferior vena cava. After finding blood in the blood collection needle connecting tube, connect the blood collection tube on the other side.

[0072] (4) After the collected blood was left at room temperature for 2 hours, it was centrifuged at 2000 rpm for 15 minutes to separate the serum.

[0073] (5) Rat serum was used to detect uric acid by ELISA (ELISA kit purchased from Nanjing Jiancheng Biotechnology Co., Ltd., C012-21). The ELISA procedure was performed according to the kit requirements.

[0074] Serum uric acid data were analyzed and statistically plotted using GraphPad Prism 6.0, as shown below. Figure 5 As shown. For normally distributed measurement data, comparisons between groups were performed using t-tests or analysis of variance (ANOVA); for non-normally distributed data, nonparametric rank-sum tests were used. P < 0.05 was considered statistically significant. A significant increase in serum uric acid levels in the model group compared to the control group indicates that this animal model is the same as other methods for inducing elevated uric acid. No significant change in serum uric acid levels in the model group compared to the control group indicates that this animal model differs from other methods for inducing elevated uric acid, and that the pathological damage and fibrosis induced by this model are not caused by an increase in serum uric acid levels.

[0075] In this invention, the serum uric acid levels of HN rats in the model group (successfully modeled) were not significantly different from those in the control group. This indicates that the pathological damage induced by this modeling method is not due to elevated serum uric acid levels.

[0076] Therefore, the present invention has prepared a rat model of kidney injury and renal fibrosis. The above pathological changes are not caused by the increase of blood uric acid concentration, but by focal lesions caused by urate crystals injected into the kidney.

[0077] 3. Serum creatinine test

[0078] After the rat HN model was prepared in Example 1, blood was collected from the inferior vena cava, and serum creatinine was detected by ELISA. The specific steps were as follows:

[0079] (1) The serum collection procedure is the same as step 2 above.

[0080] (2) Rat serum was used to detect creatinine by ELISA (ELISA kit purchased from Nanjing Jiancheng Biotechnology Co., Ltd., C011-2-1). The ELISA procedure was performed according to the kit requirements.

[0081] Serum creatinine data were analyzed and statistically plotted using GraphPad Prism 6.0, as shown below. Figure 6 As shown. For normally distributed measurement data, comparisons between groups were performed using t-tests or analysis of variance (ANOVA); for non-normally distributed data, nonparametric rank-sum tests were used. P < 0.05 was considered statistically significant. A significant increase in serum creatinine levels in the model group compared to the control group indicates that the animal model can impair rat renal function. No significant change in serum urinary creatinine levels in the model group compared to the control group indicates that the animal model does not affect rat renal function in the fourth week.

[0082] Compared with the control group, the serum creatinine level of HN rats in the model group of this invention was significantly increased. This indicates that this modeling method can impair renal function in rats by the fourth week.

[0083] The results show that the rat model of kidney injury and renal fibrosis prepared by the present invention exhibits signs of impaired kidney function in rats.

[0084] In summary, the animal model of hyperuricemia-induced kidney injury prepared by the method of this invention can simulate the formation of urate crystals in the kidneys when uric acid levels are elevated. It also addresses kidney damage caused by the urate crystals present in the kidneys after the blood uric acid concentration decreases. The modeling method of this invention clearly demonstrates the presence of urate crystals in the kidneys and their induction of kidney disease, providing a direct and consistent animal model for HN research that aligns with urate crystal-induced kidney disease. Furthermore, this model also provides a valuable tool for drug testing.

[0085] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for constructing an animal model of kidney injury induced by uric acid crystals, characterized in that... Includes the following steps: The model was established in experimental animals by intrarenal injection of urate crystal suspension; The dosage of the urate crystal suspension is 50~80 mg / kg; The injection depth is 1 / 3 to 2 / 3 of the total width of the kidney of the experimental animal; The experimental animal was a rat; The urate crystal suspension is a sodium urate crystal suspension; The sodium urate crystal suspension is prepared as follows: ① Dissolve 400mg Sigma uric acid in 80mL of pure water and heat it to 60℃ in a constant temperature water bath; ② Dissolve uric acid in 2.8 mL of 3M NaOH until completely dissolved; ③ Adjust the pH to 8.9 with NaOH and glacial acetic acid, and crystallize at room temperature for 2 days; ④ Collect the crystals on filter paper, wash them three times with anhydrous ethanol, and then dry them in a microwave oven on high. ⑤ Collect sodium urate (MSU) crystals in centrifuge tubes and sterilize at 180°C for 2 hours; ⑥ Prepare sodium urate MSU crystal suspension using sterile PBS.

2. The method for constructing an animal model of kidney injury induced by uric acid crystals according to claim 1, characterized in that: The urate crystal suspension was administered via intrarenal injection through the renal cortex.

3. The method for constructing an animal model of kidney injury induced by uric acid crystals according to claim 1 or 2, characterized in that: The construction method also includes verifying the pathological morphology, serum uric acid value, and serum creatinine value of the experimental animals after they are injected with urate crystal suspension.

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