Uric acid crystal and soluble uric acid combined renal injury animal model and construction method thereof

Abstract

The invention discloses a method for constructing a renal injury animal model caused by combination of uric acid crystals and soluble uric acid, which comprises the following steps: applying a urate crystal suspension and a mixed solution of oteracil potassium and uric acid to an experimental animal, the dosage of the urate crystal suspension being 0.5-100mg / kg; the dosage of oteracil potassium per day in the mixed solution of oteracil potassium and uric acid is 750 mg / kg, the dosage of uric acid per day is 300 mg / kg, the dosage of oteracil potassium per day and the dosage of uric acid per day are both calculated according to the weight of the experimental animal, and the application time of the mixed solution of oteracil potassium and uric acid is 3-4 weeks. The invention also discloses the uric acid crystal and soluble uric acid combined renal injury animal model constructed by the method. The method can stably establish an animal kidney injury model caused by increase of soluble uric acid in the kidney and coexistence of urate crystals. And the established model is high in credibility, high in accuracy and universal.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal model construction, and specifically relates to an animal model of renal injury caused by the combination of uric acid crystals and soluble uric acid and a construction method thereof. Background Art

[0002] Hyperuricemia nephropathy (HN) is one of the diseases that seriously threaten public health. Studies have shown the significant impact of hyperuricemia in promoting the progression of chronic kidney disease, although its underlying mechanism remains unclear. At the same time, researchers agree that the kidney is the main tissue responsible for uric acid excretion and reabsorption. After hyperuricemia, uric acid increases in the kidney and accumulates to form sodium urate (MSU) crystals, which lead to kidney damage. Soluble uric acid (sUA) is also believed to trigger renal inflammatory response, immune cell dysregulation and kidney damage. However, the environment of uric acid in the human body is too complex. In addition to the increase in renal soluble uric acid (sUA) caused by hyperuricemia, there are also MSU crystals deposited after sUA increases, and there are also cases where both exist at the same time. The interaction between sUA and MSU is still unclear. Different studies have shown that the relationship between the two may be antagonistic or functionally pathogenic. This shows that exploring the relationship between the two is of great significance for the pathogenesis and treatment of HN.

[0003] However, most animal models used in basic research on hyperuricemia nephropathy currently use methods that increase serum uric acid concentrations, that is, to simulate the effects of elevated sUA on the kidneys, including (1) increasing the source of uric acid; (2) inhibiting uric acid excretion; and (3) inhibiting uric acid metabolism. The team of the inventor of this application previously invented a method for intrarenal injection of MSU crystals to simulate the effects of MSU crystals on the kidneys.

[0004] The above four models include using different methods to simulate the increase of blood uric acid concentration, and observing the effect of high uric acid levels on the kidneys by prolonging the modeling time. And exogenous injection of MSU crystals into the kidneys to simulate the effect of MSU on the kidneys in the absence of sUA. However, these four models cannot stably and controllably simulate the effects of sUA and MSU on the kidneys at the same time, and explore the relationship between the two when used alone.

[0005] Therefore, the present invention uses a new combined modeling method to explore the simultaneous effects of sUA and MSU on the kidneys, as well as the pathogenic relationship between the two. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing an animal model of renal injury caused by the combined presence of uric acid crystals and soluble uric acid, which can stably establish an animal renal injury model caused by the coexistence of increased soluble uric acid and urate crystals in the kidney.

[0007] The present invention also aims to provide an animal model of renal injury caused by the combination of uric acid crystals and soluble uric acid constructed by the above method.

[0008] The above-mentioned first purpose of the present invention can be achieved by the following technical scheme: a method for constructing an animal model of renal injury caused by a combination of uric acid crystals and soluble uric acid, comprising the following steps: administering a urate crystal suspension and a mixture of potassium oxonate and uric acid to experimental animals, wherein the dosage of the urate crystal suspension is 0.5-100 mg / kg; the daily dosage of potassium oxonate in the mixture of potassium oxonate and uric acid is 750 mg / kg, and the daily dosage of uric acid is 300 mg / kg, both based on the body weight of the experimental animals, and the potassium oxonate and uric acid mixture is administered for 3 to 4 weeks.

[0009] Preferably, the urate crystal suspension is administered to the experimental animals by injection, and the potassium oxonate and uric acid mixture is administered by oral gavage.

[0010] More preferably, the urate crystal suspension is administered to the experimental animal by intrarenal injection.

[0011] Furthermore, the urate crystal suspension was administered to the experimental animals by intrarenal injection through the renal cortex.

[0012] Preferably, the injection depth is 1 / 3 to 2 / 3 of the total width of the kidney of the experimental animal.

[0013] Taking rats as an example, the depth of insertion of the syringe needle is about 1 / 3 to 2 / 3 of the total width of the rat kidney, that is, the injection depth is about 5 to 10 mm, which can reach the cortical-medullary junction of the rat kidney tissue.

[0014] Preferably, the dosage of the urate crystal suspension is 50-80 mg / kg, based on the body weight of the experimental animal.

[0015] Preferably, the urate crystal suspension is a sodium urate crystal suspension.

[0016] Preferably, the experimental animal is selected from at least one of mice, dogs, monkeys, pigs and cattle.

[0017] More preferably, the experimental animal is a rat.

[0018] Furthermore, the construction method also includes verifying the pathological morphology, serum uric acid value and serum creatinine value of the experimental animal after the experimental animal is injected with a urate crystal suspension and gavaged with a mixture of potassium oxonic acid and uric acid.

[0019] The pathological morphology of the experimental animals includes:

[0020] The results of H&E staining showed that the pathological characteristics of the rat model constructed by the method of the present invention were characterized by diffuse pathological damage to the kidney and aggravated focal pathological damage at the site where urate crystals were located. This indicates that soluble uric acid and MSU showed a synergistic damaging effect in affecting kidney pathology.

[0021] The immunohistochemical staining results of F4 / 80 and NLRP3 showed that the pathological characteristics of the rat model constructed by the method of the present invention were diffusely increased macrophage infiltration and inflammasome activation. The pathological characteristics were further aggravated in the area where MSU crystals existed. This indicates that soluble uric acid and MSU showed a synergistic damaging effect in affecting the inflammatory response of the kidney.

[0022] The results of MASSON staining and immunohistochemical staining of α-SMA and Vimentin showed that the pathological characteristics of the rat model constructed by the method of the present invention were characterized by diffuse renal fibrosis and aggravated focal renal fibrosis at the site where urate crystals were located. This indicates that soluble uric acid and MSU showed a synergistic damaging effect in affecting renal fibrosis.

[0023] Compared with the normal control group and the MSU crystal injection group, the serum uric acid value of rats in the combined modeling group (UA+MSU group) of the present invention was significantly increased, and compared with the combined oral administration group of potassium oxonate (PO) and uric acid, the serum uric acid value of rats in the combined modeling group was further significantly increased, indicating that this modeling method more seriously damaged the renal function related to uric acid metabolism.

[0024] Compared with the control group, the serum creatinine value of the modeling group (UA+MSU group) of the present invention was significantly increased, indicating that this modeling method can damage the renal function of rats.

[0025] Therefore, compared with the rats in the control group, the serum creatinine value of the rats in the modeling group of the present invention was significantly increased, and urate crystals appeared in the kidneys of the rats in the modeling group; if this condition is met, the animal model of renal damage caused by the combination of uric acid crystals and soluble uric acid is successfully modeled.

[0026] The above-mentioned second object of the present invention can be achieved by the following technical scheme: an animal model of renal damage caused by the combination of uric acid crystals and soluble uric acid is constructed by the above-mentioned method.

[0027] The present invention has the following advantages:

[0028] (1) While reducing the harm caused by the animal model, the present invention uses an intrarenal injection method to construct an animal urate crystal-induced animal renal injury model and combines it with a method of gavage of a mixture of potassium oxonate and uric acid to successfully establish an animal model of renal injury caused by uric acid crystals and soluble uric acid. The model has good stability and repeatability, is objective, and has strong operability, reducing other possible interference factors, and providing a necessary experimental animal model for studying the renal injury process caused by the simultaneous presence of urate crystals and soluble uric acid;

[0029] (2) The present invention evaluates the constructed animal model of renal injury caused by the combination of uric acid crystals and soluble uric acid by verifying the pathological morphology (such as the number of nephritic cells) and renal function indicators (such as serum uric acid and serum creatinine values) of the experimental animals. The model has high credibility, high accuracy and universal applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is an experimental flow chart of Example 1 of the present invention;

[0031] Figure 2 This is a schematic diagram of the intrarenal injection of urate crystals in rats in Example 1 of the present invention;

[0032] Figure 3 This is a diagram showing the HE detection results of the rat model in Example 2 of the present invention;

[0033] Figure 4 This is a diagram showing the results of NLRP3, F4 / 80 multiplex recombinant immunofluorescence detection of the rat model in Example 2 of the present invention;

[0034] Figure 5 The MASSON staining and immunohistochemical staining of the tissue in Example 2 of the present invention are shown in Figures A and B, respectively, and the α-SMA and Vimentin detection results are shown in Figures C to F;

[0035] Figure 6 This is a graph showing the test results of serum uric acid (sUA) in the rat model in Example 2 of the present invention;

[0036] Figure 7 This is a graph showing the test results of the creatinine value (sCr) of the rat model in Example 2 of the present invention. DETAILED DESCRIPTION

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

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

[0039] Experimental materials are preferred but not limited to: alcohol, iodine, cotton pads, towels, syringes, saline, 1 mL syringes, animal shaving tweezers, surgical scissors (elbow, straight), hemostatic forceps (elbow, straight), tweezers (elbow, straight), sutures (8-0, 5-0), heating plate, chloral hydrate (purchased from the Animal Research Center of Guangdong Provincial Academy of Traditional Chinese Medicine); uric acid (purchased from sigma); uric acid test kit (purchased from Nanjing Jiancheng Biotechnology Co., Ltd.); creatinine test kit (purchased from Nanjing Jiancheng Biotechnology Co., Ltd.); urea nitrogen test kit (purchased from Nanjing Jiancheng Biotechnology Co., Ltd.); 4% paraformaldehyde (purchased from biosharp); tissue embedding box (purchased from Beyotime Biotechnology Research Institute); hematoxylin and eosin staining kit (purchased from Beyotime Biotechnology Research Institute); Masson staining kit purchased from (Beijing Regen Biotechnology Co., Ltd.); potassium oxonate (purchased from Sigma); F4 / 80 antibody (purchased from Abcam), NLRP3 antibody (purchased from Abmart)

[0040] Example 1

[0041] The method for constructing an animal model of renal injury induced by uric acid crystals and soluble uric acid provided in this embodiment is as follows: Figure 1 As shown, the following steps are included:

[0042] (1) Selection of experimental animals:

[0043] Seven-week-old male SD rats with an average body weight of about 200 g were placed in a clean cage at a temperature of 22±2°C and a relative humidity of 40%-45%. The rats were kept on a light-on and dark-off cycle of 12 h each and fed with standard rat feed with free access to water.

[0044] (2) Animal grouping:

[0045] The rats in the control group did not undergo urate crystal injection surgery and were not gavaged with a mixture of potassium oxonate and uric acid.

[0046] UA group (soluble uric acid-induced renal injury): oral administration of a mixture of potassium oxonate and uric acid once a day;

[0047] MSU group (uric acid crystals causing renal injury): MSU crystals were injected into the upper pole of the kidney, and an equal volume of PBS solution was injected into the lower pole;

[0048] UA+MSU combined group (renal injury caused by uric acid crystals and soluble uric acid): MSU injection surgery was performed one day before oral gavage, MSU crystals were injected into the upper pole of the kidney, and an equal volume of PBS solution was injected into the lower pole of the kidney. After that, a mixture of potassium oxonate and uric acid was gavaged once a day.

[0049] (3) Preparation of urate crystal injection:

[0050] ① Dissolve 400 mg of Sigma uric acid in 80 mL of pure water and heat the thermostatic water bath to 60°C;

[0051] ②Dissolve uric acid with 3M NaOH (about 2.8 mL) until it is completely dissolved;

[0052] ③ Adjust the pH to 8.9 with NaOH and glacial acetic acid and crystallize at room temperature for 2 days;

[0053] ④ Collect the crystals on filter paper, wash with anhydrous ethanol three times, and accelerate drying in a microwave oven at high heat;

[0054] ⑤ Collect sodium urate (MSU) crystals in centrifuge tubes and sterilize at 180℃ for 2h;

[0055] ⑥Use sterile PBS to prepare MSU crystal suspension:

[0056] ⑦ Draw out the MSU crystal suspension with a syringe;

[0057] Sterilization of modeling supplies: surgical scissors (elbow, straight), hemostatic forceps (elbow, straight), tweezers (elbow, straight) and cotton pads are sterilized with high temperature and high pressure.

[0058] (4) Urate crystal injection surgery

[0059] After 1 week of adaptive feeding, the rats were weighed and anesthetized by intraperitoneal injection of chloral hydrate anesthesia (350 mg / kg, ip). After anesthesia, the hair below the sternum on the back of the rat was removed and disinfected with iodine. After disinfection, deiodine was removed with normal saline. When the rat fell down, the whole body was weak and the reaction disappeared, indicating that the rat had reached a suitable anesthesia state. The rat was placed in a prone position on a sterile operating table. Using surgical scissors, cut the epidermis and muscle layer of the rat, making the opening as small as possible. After the kidneys can be seen, gently squeeze the abdomen of the rat to squeeze the kidney out of the abdominal cavity and out of the surgical port on the back.

[0060] Insert the syringe needle into the cortical-medullary junction of the rat kidney at the upper pole (the injection depth is about 1 / 3-2 / 3 of the width of the rat kidney, i.e. 5-10 mm), and slowly push the drug in. The injection volume is 10 mg MSU per kidney (50 mg / kg, based on the rat's body weight). Figure 2 The lower pole of the kidney was not injected, which served as a self-control for the absence of urate crystals in the kidneys of the injected rats.

[0061] After the injection, the needle was withdrawn, the kidney was placed back into the abdominal cavity, and 1 mL of saline was added. The muscle layer and epidermis of the rat were sutured with sutures. After suturing, the blood was wiped off the wound with saline cotton pads, and the rat was placed on a heating plate. After the rat woke up, it was placed back into the cage.

[0062] (5) Oral administration of a mixture of potassium oxonate and uric acid

[0063] Add 750 mg of potassium oxonate and 300 mg of uric acid to every 1 L of normal saline. Gavage at a concentration of 750 mg / kg (based on rat body weight) potassium oxonate and 300 mg / kg (based on rat body weight) uric acid every day for 21 or 28 days.

[0064] (6) Thus, a rat model of renal injury induced by uric acid crystals and soluble uric acid was obtained.

[0065] Example 2

[0066] 1. Pathological detection of rat kidney

[0067] (1) After the rat model prepared in Example 1 was established (after intrarenal injection of urate crystals, rats were gavaged with a mixture of potassium oxonate and uric acid), the rats were weighed and anesthetized with chloral hydrate (350 mg / 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 mouse board.

[0068] (2) Renal perfusion

[0069] Cut the thoracic peritoneum, insert the syringe into the left ventricle of the mouse, and slowly push in normal saline until the kidney turns white, indicating that the red blood cells in the kidney are flushed clean, then stop the perfusion.

[0070] (3) Kidney sampling

[0071] 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 bean-shaped. They were white in color. The bilateral renal hilums were cut with scissors, and the bilateral kidneys were removed. The renal capsules were removed and placed in a culture dish filled with sterile saline. The left kidney was cut horizontally at the middle renal hilum and fixed in 4% paraformaldehyde. After fixation for 24 hours, tissue wax blocks were made.

[0072] (4) Preparation of paraffin sections

[0073] Remove the tissue from the fixative, trim it into a suitable shape with a scalpel, put it into a dehydration frame and mark it, and rinse the tissue block with running water to remove the paraformaldehyde fixative. Dehydrate the tissue using alcohol from low to high concentrations, and make the dehydrated tissue transparent, wax-impregnated, tissue embedded and sliced.

[0074] (5) Tissue HE staining

[0075] Paraffin sections were routinely dewaxed and placed in water after dewaxing. The tissue was completely immersed in hematoxylin for 15-20 minutes until the nucleus was stained obviously. The sections were fully washed 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 rat kidneys were observed under a light microscope and photographed.

[0076] HE test results are as follows Figure 3 As shown, from Figure 3 It can be seen that:

[0077] The renal tubules of rats in the control group were well developed, with no obvious apoptosis and inflammatory cell infiltration;

[0078] In the renal tissue of rats in the UA group, diffuse vacuolar degeneration and necrosis of the renal tubular epithelium, tubular dilatation, shedding of the brush border of the proximal tubules, and an increase in the number of infiltrating inflammatory cells were observed.

[0079] In the renal tissue of MSU rats, urate crystals could be observed in the upper pole tissue of the kidney injected with urate crystals. Pathologically, tubular epithelial vacuolar degeneration, necrosis, tubular dilatation, proximal tubular brush border shedding, and increased number of inflammatory cell infiltration were observed around the urate crystals. The renal injury score and number of inflammatory cell infiltration were higher than those in the control group rat model. In the renal tissue of rats injected with urate crystals, there were no urate crystals in the lower pole tissue of the kidney that was not injected with urate crystals, and no pathological damage to the upper pole occurred. The tissue condition was the same as that of the control group rats.

[0080] The rats in the UA+MSU group showed diffuse tubular epithelial vacuolar degeneration, necrosis, and tubular dilatation in the upper and lower poles of the kidney, shedding of the brush border of the proximal tubules, increased infiltration of inflammatory cells, and further aggravated pathological damage around the site of MSU crystal injection in the upper pole of the kidney. The kidney injury score and the number of inflammatory cell infiltration in the upper pole of the kidney were higher than those in the control group, UA group, and UA+MSU group, and the statistical difference between the upper and lower poles of the kidney and the control group was further increased.

[0081] This indicates that both urate crystals and soluble uric acid can cause pathological damage to the kidneys. Uric acid crystals cause focal damage to the kidneys, while soluble uric acid causes diffuse damage to the kidneys. The coexistence of the two further aggravates the pathological damage to the kidneys.

[0082] The results showed that the pathological characteristics of the rat model prepared by the scheme of the present invention were diffuse pathological damage and further aggravated at the site where MSU crystals existed. This indicates that soluble uric acid and MSU showed a synergistic damaging effect in affecting kidney pathology.

[0083] (6) Tissue Immunofluorescence Staining

[0084] Paraffin sections were routinely dewaxed, placed in water, and heat-fixed with citrate for 5 minutes. Primary antibody (NLRP3, F4 / 80) was incubated overnight, sections were washed with PBS, and incubated with 488 and 647 fluorescent secondary antibodies for 1 hour. DAPI was used to seal the sections, and the morphological changes of rat kidneys were observed under a fluorescent microscope and photographed.

[0085] Immunofluorescence test results Figure 4 As shown, from Figure 4 It can be seen that:

[0086] There was a small amount of macrophage infiltration (F4 / 80 red) and a small amount of inflammasome activation (NLRP3) in the renal interstitium of rats in the control group.

[0087] The rats in the UA group had diffusely increased macrophage infiltration in the kidney tissue, activated inflammasomes, and the number was higher than that in the control group.

[0088] In the kidney tissue of MSU rats, increased macrophage infiltration and activated inflammasomes were observed in the upper pole tissue of the kidney injected with urate crystals, and the number of macrophages was higher than that in the control group rat model; while in the kidney tissue of rats injected with urate crystals, the macrophage infiltration and inflammasome activation in the lower pole tissue of the kidney that was not injected with urate crystals were the same as those in the control group rats.

[0089] The rats in the UA+MSU group had diffuse macrophage infiltration and inflammasome activation in the upper and lower poles of the kidneys. In addition, the macrophage infiltration and inflammasome activation in the upper pole of the kidney were further aggravated around the site of MSU crystal injection, and the number was higher than that in the lower pole of the kidney in the control group and UA+MSU group.

[0090] This indicates that both urate crystals and soluble uric acid can lead to increased macrophage infiltration in the kidney and activate macrophage inflammatory bodies. Uric acid crystals show focal induction of increased macrophage infiltration, while soluble uric acid shows diffuse induction of increased macrophage infiltration. When both are present at the same time, the number of macrophage infiltration in the kidney is further aggravated.

[0091] The results showed that the pathological characteristics of the rat model prepared by the present invention were diffusely increased macrophage infiltration and inflammasome activation. The pathological characteristics were further aggravated in the area where MSU crystals were present. This indicates that soluble uric acid and MSU showed a synergistic damaging effect in affecting the inflammatory response of the kidney.

[0092] (7) Tissue MASSON staining

[0093] Paraffin sections were routinely dewaxed, placed in water after dewaxing, and stained with Bouin solution in a 37-degree incubator for 2.5 hours. Rinse with running water until the yellow color on the sections disappears, and then drip with lapis lazuli blue staining solution for 3 minutes, and rinse with water. Mayer hematoxylin staining solution was dripped for 3 minutes, and then rinsed with water. Color separation solution was stained for 15 seconds, and then rinsed with running water. Ponceau red staining solution was dripped for 10 minutes, and then rinsed with distilled water. Phosphomolybdic acid solution was used for 10 minutes. The sections were directly dripped with aniline blue staining solution for 4 minutes. Weak acid solution was used for 2 minutes. Dehydrated with anhydrous ethanol 3 times, 10 seconds each time. Xylene was transparent 3 times, 2 minutes each time, and neutral gum was used for sealing. The degree of fibrosis of rat kidney tissue was observed under a microscope.

[0094] (8) Tissue immunohistochemical staining

[0095] Paraffin sections were routinely dewaxed, placed in water, and heat-fixed with citrate for 5 minutes. Primary antibodies (α-SMA, Vimentin) were incubated overnight, sections were washed with PBS, and incubated with general histochemical staining secondary antibodies for 1 hour. DAB was used for color development, hematoxylin was used for counterstaining of cell nuclei, and neutral resin was used for sealing. The positive area in the rat kidney was observed under a light microscope and photographed.

[0096] The results of MASSON test are as follows Figure 5 As shown in Figures A and B, the results of α-SMA and Vimentin detection are as follows Figure 5 As shown in Figures C to F, Figure 5 It can be seen that:

[0097] At the 3rd and 4th weeks, the fibrinogen in the tubulointerstitium of the control group rats was uniform and the expression level was low.

[0098] Interstitial fibrosis was diffusely present in the renal tissues of rats in the UA group, and the area was significantly higher than that in the control group.

[0099] In the renal tissue of the rats in the MSU group, urate crystals could be observed in the upper pole tissue of the kidney injected with urate crystals, the renal tubules around the urate crystals were damaged, the interstitial area became larger, and the fibrin-positive area in the interstitium was significantly higher than that in the rat model of the control group; while in the renal tissue of the rats in the MSU group, there were no urate crystals in the lower pole tissue of the kidney that was not injected with urate crystals, and there was no increase in the fibrosis area of ​​the upper pole, and the fibrosis of the lower pole tissue was the same as that of the rats in the control group.

[0100] Interstitial fibrosis was diffusely present in the renal tissue of rats in the UA+MSU group, and urate crystals could be observed in the upper pole renal tissue injected with urate crystals. The renal tubules around the urate crystals were damaged, the interstitial area became larger, and the fibrin-positive area in the interstitium was significantly higher than that in the rat model of the control group. In the lower pole renal tissue that was not injected with MSU crystals, there were no urate crystals, but interstitial fibrosis occurred, which was significantly higher than that in the control group.

[0101] This indicates that both urate crystals and soluble uric acid can cause renal interstitial fibrosis, with uric acid crystals inducing renal interstitial fibrosis focally and soluble uric acid inducing renal interstitial fibrosis diffusely. The coexistence of the two can further aggravate the pathological damage to the kidney.

[0102] The results showed that the pathological characteristics of the rat model prepared by the present invention were characterized by diffuse induction of renal interstitial fibrosis and further aggravation of fibrosis at the site where MSU crystals were present. This indicates that soluble uric acid and MSU showed a synergistic damaging effect in affecting renal fibrosis.

[0103] The overall pathological results showed that the renal injury and fibrosis rat model was prepared by the scheme of the present invention.

[0104] 2. Serum uric acid test

[0105] After the rat HN model was prepared in Example 1, blood was collected through the inferior vena artery, and the ELISA method was used to detect the serum uric acid of the rats. The specific steps were as follows:

[0106] (1) After weighing, the animals were anesthetized with chloral hydrate (350 mg / kg, ip) until the animals showed signs of anesthetic reactions such as loss of corneal reflex, decreased muscle strength, and flaccid paralysis.

[0107] (2) Cut open the skin and muscle layers of the rat's lower abdomen to expose the intestinal tissue. Gently move the intestinal tissue to one side to reveal the inferior vena artery.

[0108] (3) Insert the blood collection needle into the inferior vena artery. When blood is found in the blood collection needle connecting tube, connect the blood collection tube on the other side.

[0109] (4) After the collected blood was placed at room temperature for 2 hours, it was centrifuged at 2000 rpm for 15 minutes to separate the serum.

[0110] (5) Rat serum was subjected to ELISA test to detect uric acid (ELISA kit was purchased from Nanjing Jiancheng Biotechnology Co., Ltd., C012-21). The ELISA operation was performed according to the requirements of the kit.

[0111] GraphPad Prism 6.0 was used to analyze the serum uric acid data and statistically plot them. Figure 6As shown. The inter-group comparison of quantitative data that obeyed the normal distribution was performed using the t-test or analysis of variance (ANOVA), and the non-parametric rank sum test was used when it did not obey the normal distribution. P<0.05 was considered statistically significant. Compared with the control group, the serum uric acid level of rats in the modeling group increased significantly, indicating that the animal model affects the kidney by causing an increase in sUA. Compared with the control group, the serum uric acid level of rats in the modeling group did not change significantly, indicating that the animal model did not affect the kidney by causing an increase in uric acid.

[0112] At the 3rd and 4th weeks of modeling, there was no significant change in the serum uric acid levels of rats in the MSU group compared with the control group. Compared with the control group and the MSU group, the serum uric acid levels of rats in the UA group and the UA+MSU group increased significantly. At the 4th week, the serum uric acid levels of rats in the UA+MSU group increased further and were significantly higher than those in the UA group. This indicates that the pathological damage caused by this modeling method is partly caused by the increase in serum uric acid levels. And the presence of MSU crystals in the kidneys can further increase the serum uric acid level.

[0113] Therefore, the present invention prepares a rat model of renal injury and renal fibrosis. The above pathological changes are caused by the increase of blood uric acid concentration and the focal lesions caused by urate crystals injected into the kidneys.

[0114] 3. Serum creatinine test

[0115] After the rat HN model was prepared in Example 1, blood was collected through the inferior vena artery, and the serum creatinine of the rat was detected by ELISA method. The specific steps were as follows:

[0116] (1) The serum collection procedure is the same as in Example 2.

[0117] (2) Rat serum was subjected to ELISA test to detect creatinine (ELISA kit was purchased from Nanjing Jiancheng Biotechnology Co., Ltd., C011-2-1). The ELISA operation was performed according to the requirements of the kit.

[0118] GraphPad Prism 6.0 was used to analyze the serum creatinine data and statistically plot them. Figure 7 As shown. The inter-group comparison of quantitative data that obeyed the normal distribution was performed using the t-test or analysis of variance (ANOVA), and the non-parametric rank sum test was used when it did not obey the normal distribution. P<0.05 was considered statistically significant. Compared with the control group, the serum creatinine value of rats in the modeling group increased significantly, indicating that the animal model can damage the renal function of rats. Compared with the control group, there was no significant change in the serum urine creatinine of rats in the modeling group, indicating that the animal model did not affect the renal function of rats.

[0119] In the fourth week, the serum creatinine value of the model group (UA+MSU group) of the present invention was significantly increased compared with that of the control group, indicating that this modeling method can damage the renal function of rats in the fourth week.

[0120] The results showed that the rat model of renal injury and renal fibrosis prepared by the scheme of the present invention showed the manifestation of renal function impairment in rats.

[0121] In summary, the animal model of hyperuricemia renal injury prepared by the method of the present invention can simulate the formation of urate crystals in the kidney when uric acid is elevated. When the blood uric acid concentration does not decrease, the kidney damage caused by the coexistence of urate crystals and soluble uric acid in the kidney. The modeling method of the method of the present invention can make it clear that urate crystals and soluble uric acid coexist in the kidney and cause kidney disease, providing an intuitive animal model for studying HN that is consistent with urate crystals and soluble uric acid causing kidney disease, and this model also provides a good tool for drug experiments.

[0122] 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 an animal model of renal injury induced by uric acid crystals and soluble uric acid, characterized in that: The following steps are involved: Urate crystal suspension and a mixture of potassium oxonate and uric acid are administered to experimental animals, wherein the dosage of the urate crystal suspension is 0.5-100 mg / kg; the daily dosage of potassium oxonate in the mixture of potassium oxonate and uric acid is 750 mg / kg, and the daily dosage of uric acid is 300 mg / kg, both based on the body weight of the experimental animals, and the mixture of potassium oxonate and uric acid is administered for 3-4 weeks.

2. The method for constructing an animal model of renal injury induced by uric acid crystals and soluble uric acid according to claim 1, characterized in that: The experimental animals were administered urate crystal suspension by injection and a mixture of potassium oxonate and uric acid by oral gavage.

3. The method for constructing an animal model of renal injury induced by uric acid crystals and soluble uric acid according to claim 2, characterized in that: The experimental animals were administered a suspension of urate crystals by intrarenal injection.

4. The method for constructing an animal model of renal injury induced by uric acid crystals and soluble uric acid according to claim 3, characterized in that: The experimental animals were administered a suspension of urate crystals by intrarenal injection into the renal cortex.

5. The method for constructing an animal model of renal injury induced by uric acid crystals and soluble uric acid according to claim 4, characterized in that: The injection depth is 1 / 3 to 2 / 3 of the total width of the kidney of the experimental animal.

6. The method for constructing an animal model of renal injury induced by uric acid crystals and soluble uric acid according to claim 1, characterized in that: The dosage of the urate crystal suspension is 50-80 mg / kg, based on the body weight of the experimental animal.

7. The method for constructing an animal model of renal injury induced by uric acid crystals and soluble uric acid according to claim 1, characterized in that: The urate crystal suspension is a sodium urate crystal suspension.

8. The method for constructing an animal model of renal injury induced by uric acid crystals and soluble uric acid according to claim 1, characterized in that: The experimental animals are one or more of mice, dogs, monkeys, pigs and cattle.

9. The method for constructing an animal model of renal injury induced by uric acid crystals and soluble uric acid according to claim 1, characterized in that: The construction method also includes verifying the pathological morphology, serum uric acid value and serum creatinine value of the experimental animal after the experimental animal is injected with a urate crystal suspension and gavaged with a mixture of potassium oxonic acid and uric acid.

10. An animal model of renal injury caused by uric acid crystals and soluble uric acid, characterized in that: The method according to any one of claims 1 to 9 is used to construct the obtained product.

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