A method for constructing a rat passive Heymann nephritis model
By injecting Sheep Anti-Rat Fx1A Serum into the tail vein twice, 24 hours apart, a rat PHN model with a long course of disease and difficulty in remission was established. This solves the problem of short course of disease and easy remission in existing technologies, and achieves long-term observation and research needs similar to the pathological changes of human membranous nephropathy.
Patent Information
- Application Number
- CN202311238626.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-25
- Publication Date
- 2025-10-17
- Estimated Expiration
- 2043-09-25
AI Technical Summary
In the existing technology, the Heimann nephritis model has a short course and easy remission, which cannot meet the needs of long-term observation, and the model is not similar enough to the pathological changes of human membranous nephropathy.
A passive Heimann nephritis model was established in rats by injecting Sheep Anti-Rat Fx1A Serum into the tail vein twice 24 hours apart. The injection volume was 3-8 mL/kg, preferably 5 mL/kg. During the injection, the rats were placed in a prone position. The tail was wiped with alcohol and the needle was inserted distally to ensure that the needle entered the blood vessel. The injection was completed within 1 minute.
A rat PHN model with a long course of disease and difficult-to-relieve condition was successfully constructed, showing nephrotic syndrome with massive proteinuria, which is suitable for long-term observation. The pathological changes are similar to those of human membranous nephropathy, providing assistance for dynamic observation and drug treatment research.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological medicine, and particularly relates to a method for constructing a passive Heymann nephritis model of rats. BACKGROUND
[0002] Primary membranous nephropathy is the main pathological type of nephrotic syndrome in adults in clinic, but the mechanism of tissue damage and progression thereof is still unclear. The Heymann nephritis model is a classic animal model for studying human nephropathy, and can be used for the research of primary membranous nephropathy, and is widely used.
[0003] The Heymann nephritis model belongs to heteroimmune anti-glomerular basement membrane nephritis, and the modeling mechanism is to immunize a second animal with a kidney cortex homogenate of a first animal, so that the second animal produces anti-kidney serum (anti-kidney antibody) against the first animal, and then the anti-kidney serum is injected into a healthy first animal to induce the production of nephritis. Since this nephritis model is formed by passively injecting antibodies, it is also called passive Heymann nephritis (PHN).
[0004] PHN is an ideal animal model for studying proteinuria-induced kidney damage, because the kidney is the only target organ of the model animal damage, the antibody used is very small, and the pathological changes appear quickly, and the pathological changes are similar to human membranous nephropathy. According to previous research reports, a one-time intravenous injection of anti-beta 2 microglobulin antiserum is usually used to establish a PHN model, although the model develops rapidly, but due to its short course and easy remission, it cannot meet the needs of long-term observation. Therefore, a PHN model with a longer course is needed to meet the needs of long-term observation. SUMMARY
[0005] The purpose of the present application is to provide a method for constructing a passive Heymann nephritis model of rats, to solve the problems in the background art.
[0006] In order to achieve the above-mentioned purpose, the present application designs a method for constructing a passive Heymann nephritis model of rats, which comprises the following steps: injecting Sheep Anti-Rat Fx1A Serum into the tail vein of the rat multiple times at intervals of 24 hours.
[0007] Further, the rat is male, the species and strain are Lewis, the level is SPF, and the weight is 200-220g.
[0008] Further, the rat is injected with the Sheep Anti-Rat Fx1A Serum into the tail vein twice at intervals of 24 hours, wherein the first needle is injected at 0-1 min, and the second needle is injected at 0-1 min after 24 hours. Further, the rat is injected with the Sheep Anti-Rat Fx1A Serum into the tail vein twice at intervals of 24 hours, wherein the first needle is injected at 0-1 min, and the second needle is injected at 0-1 min after 24 hours.
[0009] Further, the injection volume of the tail vein injection is 3-8 mL / kg, preferably 5 mL / kg.
[0010] Further, when the tail vein injection is performed, the rat is in a prone position, the rat tail is wiped with alcohol to make the blood vessel clear, the needle is inserted at the far end 1 / 3, and the blood return is observed to confirm that the needle enters the blood vessel, and the injection is completed within 1 min.
[0011] Compared with the prior art, the method for constructing the rat passive Heymann nephritis model has the following advantages:
[0012] In the present application, by twice tail vein injection of Sheep Anti-Rat Fx1A Serum to rats, a rat PHN model is successfully constructed, and the model rats show nephrotic syndrome characterized by a large amount of proteinuria, long disease course, and difficult to relieve, which can meet the needs of long-term observation. The modeling method of the present application is simple and easy to implement, and can provide research help for dynamic observation of kidney damage of model animals, exploration of the pathogenesis of PHN, and research and development of drug treatment of human model kidney disease. BRIEF DESCRIPTION OF DRAWINGS
[0013] Figure 1 : G1, G2 and G3 group rats' body weight change curve.
[0014] Among them, Two-way ANOVA: *P<0.05, ***P<0.001 vs. Control serum (G1); ###P<0.001 vs. Fx1A Serum (5 mL / kg) (G2).
[0015] Figure 2 : G1, G2 and G3 group rats' 24h urine albumin content change curve.
[0016] Among them, Two-way ANOVA: *P<0.01, ***P<0.001 vs. Control serum (G1); ###P<0.001 vs. Fx1A Serum (5 mL / kg) (G2).
[0017] Figure 3 : G1, G2 and G3 group rats' 24h urine total protein content change curve.
[0018] Among them, Two-way ANOVA: *P<0.01, ***P<0.001 vs. Control serum (G1); ###P<0.001 vs. Fx1A Serum (5 mL / kg) (G2).
[0019] Figure 4: Graph of the change in the 24h urinary albumin creatinine ratio content of the G1, G2, and G3 group rats.
[0020] Two-way ANOVA: ***P < 0.001 vs. Control serum (G1); ###P < 0.001 vs. Fx1A Serum (5 mL / kg) (G2).
[0021] Figure 5 : Illustration of the kidney PAS staining of the G1, G2, and G3 group rats.
[0022] Figure 6 : Bar graph of the proportion of the positive area of the glomerular PAS staining of the G1, G2, and G3 group rats.
[0023] One-way ANOVA: *p < 0.05 vs. Control serum (G1).
[0024] Figure 7 : Illustration of the glomerular C3d immunohistochemical staining of the G1, G2, and G3 group rats.
[0025] Figure 8 : Bar graph of the intensity score of the glomerular C3d immunohistochemical staining of the G1, G2, and G3 group rats.
[0026] One-way ANOVA: ***P < 0.001 vs. Control serum (G1); ##P < 0.01 vs. Fx1A Serum (5 mL / kg) (G2). DETAILED DESCRIPTION
[0027] The embodiments of the present application will be described in detail below with specific examples, which are only used to illustrate the present application and should not be considered as limiting the scope of the present application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer.
[0028] I. Purchase and feeding of rats
[0029] The information of the purchased rats is shown in Table 1.
[0030] Table 1 Information of rats
[0031]
[0032]
[0033] II. Feeding of rats
[0034] After the rats were purchased, the veterinarian performed a physical examination on them and then quarantined and acclimated them for the initial period of the experiment. The initial period of the experiment lasted for at least 3 days, from the day the rats arrived to the day the model was induced.
[0035] Rats were housed in a barrier system, with environmental conditions maintained at 20-26°C and humidity at 40-70%. Animal care staff recorded daily morning and afternoon air changes. The air in the animal room was changed 10-20 times per hour, and lighting followed a 12 / 12-hour light / dark cycle, except for necessary interruptions during experimental activities, which were recorded. Following enrollment, rats were housed together, with 3-4 rats per cage, in polysulfone plastic cages with corncob bedding. Cages contained toys for the rats to chew or hide in, providing an enriched living environment. Cage cards were provided with the trial number, group, sex, animal name, and number of rats, and rats were arranged in order according to group.
[0036] Rats were fed a normal diet supplied by Beijing Keao Xieli Feed Co., Ltd., with free access. Any addition of additional food items required the approval of the project leader and was recorded. Water for the rats was provided by an automatic drinking water dispenser with secondary reverse osmosis water.
[0037] 3. Main instruments and reagents
[0038] The instruments and reagents used in the experiment are shown in Tables 2 and 3.
[0039] Table 2 Main instruments
[0040]
[0041]
[0042] Table 3 Main reagents
[0043] Reagent name Catalogue number Manufacturer Creatinine assay kit CRE-1070 Shanghai Prolifune Pharmaceutical Science and Technology Urea assay kit URE-2070 Shanghai Prolifune Pharmaceutical Science and Technology Albumin assay kit ALB-6070 Shanghai Prolifune Pharmaceutical Science and Technology Total protein assay kit TP-6070 Shanghai Prolifune Pharmaceutical Science and Technology Control serum PTX-000S biosharp Sheep Anti-Rat Fx1A Serum PTX-002S Probetex
[0044] Example 1 Rat grouping and model establishment
[0045] After the quarantine period, the rats were divided into three groups. Before grouping, urine biochemical indicators were tested, albumin / creatinine values were calculated, and the rats were weighed. The statistical t-test for albumin / creatinine values and body weight between any two groups was performed to ensure that p > 0.05, i.e., the differences in body weight and albumin / creatinine values between the groups were minimal.
[0046] They are:
[0047] Group G1: recorded as control serum, n = 3;
[0048] G2 group: Fx1A Serum (5 mL / kg), n = 7, the PHN model of the rats in the G2 group was established by injecting Sheep Anti-Rat Fx1A Serum through the tail vein, and the injection day was taken as Day 0, the rats in the G2 group were injected with antiserum on Day 0; when injecting, the rats were in prone position, the tail was wiped with alcohol to make the blood vessels clear, the needle was inserted at 1 / 3 of the distal end, and the needle was observed to enter the blood vessels, and the injection was completed within 1 min.
[0049] G3 group: Fx1A Serum (5 mL / kg*2), n = 7, the PHN model of the rats in the G3 group was established by injecting Sheep Anti-Rat Fx1A Serum through the tail vein, and the injection day was taken as Day 0, the rats in the G3 group were injected with antiserum on Day 0 and Day 1. When injecting, the rats were in prone position, the tail was wiped with alcohol to make the blood vessels clear, the needle was inserted at 1 / 3 of the distal end, and the needle was observed to enter the blood vessels, and the injection was completed within 1 min; the first needle was injected at 0-1 min, and the second needle was injected at 0-1 min after 24 h. The interval of 24 h was because after injecting the first needle of Sheep Anti-Rat Fx1A Serum through the tail vein, the animals showed symptoms of listlessness and slow movement, which recovered after about 1 h of injection, in order to reduce the adverse reactions of injecting Sheep Anti-Rat Fx1A Serum on the rats and affect the test results, the second injection was performed after 24 h.
[0050] Model index detection of Example 2
[0051] 2.1 Body weight
[0052] For the rats in the G1 group, the body weight was measured twice a week from the test start day.
[0053] For the rats in the G2 group and the G3 group, the body weight was measured twice a week from the day when the first needle of Sheep Anti-Rat Fx1A Serum was injected.
[0054] 2.2 Urine biochemical index detection
[0055] For the rats in the G1 group, the G2 group and the G3 group, urine biochemical index detection was performed on Day 2, Day 7, Day 14 and Day 28, respectively, taking the injection day as Day 0.
[0056] The detection indexes included albumin, total protein and albumin creatinine ratio.
[0057] 2.3 Rat kidney pathological tissue detection
[0058] After deep anesthesia with isoflurane, the experimental animals were euthanized and perfused with normal saline in vivo; the kidneys were fixed in 10% formalin, embedded in paraffin, and cut into 3-μm-thick slices. PAS (Periodic Acid-Schiff) staining or immunohistochemical staining using C3d antibody were performed, and the stained slices were scanned whole using a Hamamatsu NanozoomerS210 slice scanner.
[0059] Example 3 Test Results
[0060] like Figure 1 As shown in the figure, the weight change curves of rats in each group are given. It can be seen that the weight gain of rats in group G2 that were injected with Fx1A Serum once and rats in group G1 that were injected with Control serum were similar, while the weight gain of rats in group G3 that were injected with Fx1A Serum twice was slower, and their weight was significantly lower than that of groups G1 and G2 after Day 13.
[0061] like Figure 2 、 3 Figures 4 and 5 show the change curves of urine albumin, urine total protein, and urine albumin creatinine in each group of rats. It can be seen that the three test indicators all have the same change trend: the three test values of the G2 group rats increased slightly on Day 7, peaked on Day 14, and slightly declined on Day 28; while the three test values of the G3 group rats were always higher than those of the G2 group rats. During the test period, the three test values continued to increase without a significant decline, and the high values were maintained for a longer period of time, indicating that the model of the G3 group rats can be maintained for a longer period of time. At the same time, the three test values of the G1 group rats remained at a lower level.
[0062] like Figure 5 Figure 2 shows an example of PAS staining of the kidneys of rats in each group. The more shaded areas, the more pronounced the glomerular mesangial thickening. As can be seen, the glomerular mesangial thickening and more pronounced nephritis symptoms were observed in the G3 group of rats, which received two tail vein injections.
[0063] like Figure 6 As shown in the figure, the changes in the proportion of PAS-positive staining areas of the glomeruli of rats in each group are given. It can be seen that the positive staining area of the glomeruli of rats in group G2 is higher than that in group G1, but there is no statistical difference, while the positive staining area of the glomeruli of rats in group G3 is significantly higher than that in group G2.
[0064] like Figure 7As shown in the figure, the glomerular C3d immunohistochemical staining of rats in each group is given, and the more coloring and the darker color, the more C3d expression, it can be seen that the glomerular C3d immunohistochemical staining of rats in the G3 group is the most significant.
[0065] As shown in the figure, the glomerular C3d immunohistochemical staining of rats in each group is given, and the more coloring and the darker color, the more C3d expression, it can be seen that the glomerular C3d immunohistochemical staining of rats in the G3 group is the most significant. Figure 8 As shown in the figure, the glomerular C3d immunohistochemical staining intensity score of rats in each group is given, it can be seen that the glomerular C3d immunohistochemical staining intensity score of rats in the G2 group is higher than that of rats in the G1 group, but there is no statistical difference, and the glomerular C3d immunohistochemical staining intensity score of rats in the G3 group is significantly higher than that of rats in the G1 group and the G2 group.
[0066] The present application successfully establishes a rat PHN model on the basis of one-time tail vein injection of antiserum, and the model animals show nephrotic syndrome characterized by a large amount of proteinuria, and the pathological performance is diffuse thickening of glomerular basement membrane, deposition of immune complex outside the glomerular basement membrane and fusion of foot process.
[0067] The above is only the preferred embodiment of the present application, and is not used to limit the application, any modification, equivalent replacement, improvement, etc. within the design concept of the present application should be included in the protection scope of the present application.
Claims
1. A method for constructing a rat passive Heymann nephritis model, characterized by: Rats were injected with Sheep Anti-Rat Fx1ASerum into the tail vein multiple times at 24-h intervals; Rats were injected twice into the tail vein 24 hours apart, with the first injection at 0-1 min and the second injection at 0-1 min after 24 hours.
2. The method for constructing a rat passive Heymann nephritis model according to claim 1, wherein: The rats were male, of Lewis species and strain, SPF grade, and weighed 200-220 g.
3. The method for constructing a rat passive Heymann nephritis model according to claim 1, wherein: The injection volume of the tail vein injection is 3-8 mL / kg.
4. The method for constructing a rat passive Heymann nephritis model according to claim 1, wherein: When performing tail vein injection, the rat is in prone position, the tail is wiped with alcohol to make the blood vessels clear, the needle is inserted at the distal 1 / 3, and the return of blood is observed to confirm that the needle has entered the blood vessel. The injection is completed within 1 minute.