Construction method of renal tubular interstitial nephritis-uveitis syndrome mouse model

Abstract

The invention belongs to the technical field of biology, and relates to a construction method of a renal tubular interstitial nephritis-uveitis syndrome mouse model. Comprising the following steps: anesthetizing a mouse; carrying out subcutaneous injection on photoreceptor intercellular vitamin A binding proteins and a complete Freund's adjuvant on the front neck, the thighs on the two sides and the back of the anesthetized mouse, and carrying out active immunization; and performing intraperitoneal injection on the actively immunized mouse by adopting pertussis toxin, and performing final immunization. According to the invention, the renal tubular interstitial nephritis-uveitis syndrome mouse model is successfully constructed, so that a foundation is laid for subsequent pathological mechanism research of the disease, and a support can be provided for further development of medicine research for treating the disease.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for constructing a mouse model of tubulointerstitial nephritis-uveitis syndrome. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Tubulointerstitial nephritis and uveitis (TINU) syndrome is a rare disease characterized primarily by tubulointerstitial inflammation and uveitis, and is mainly autoimmune. The incidence is relatively high in middle-aged women and young men, with a female-to-male ratio of approximately 3:1. However, animal models of this disease are currently lacking. Therefore, limited research on its pathological mechanisms hinders further investigation. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a method for constructing a mouse model of tubulointerstitial nephritis-uveitis syndrome. This invention successfully constructs a mouse model of tubulointerstitial nephritis-uveitis syndrome, thus laying the foundation for subsequent research on the pathological mechanisms of this disease and providing support for further drug research to treat it.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: Firstly, the application of an experimental autoimmune uveoretinitis (EAU) mouse model in constructing a mouse model of tubulointerstitial nephritis-uveitis syndrome.

[0006] Experimental autoimmune uveoretinitis (EAU) is a classic animal model of human uveitis, established through peripheral immunization of susceptible animals. Increased expression of serum creatinine (CREA), urea (UREA), and urinary β2-microglobulin (β2M) are diagnostic indicators of renal dysfunction induced by interstitial nephritis-uveitis syndrome. This study found that EAU mice exhibited significantly increased levels of CREA, UREA in serum and β2M in urine; therefore, the experimental autoimmune uveoretinitis mouse model can serve as a mouse model for tubulointerstitial nephritis-uveitis syndrome.

[0007] On the other hand, a method for constructing a mouse model of tubulointerstitial nephritis-uveitis syndrome includes the following steps: Anesthetize the mice; Active immunization was performed by subcutaneous injection of interphotoreceptor retinoid binding protein (IRBP) and complete Freunds adjuvant (CFA) into the anterior neck, bilateral thighs, and back of anesthetized mice. Mice were injected intraperitoneally with pertussis toxin (PTX) after active immunization.

[0008] Before constructing a mouse model of tubulointerstitial nephritis-uveitis syndrome, the mice need to be anesthetized. The traditional method of anesthetizing mice is injection anesthesia, which is prone to over-anesthesia, which can easily lead to the death of the mice. In order to reduce the mortality rate of mice, the method of anesthetizing mice is inhalation anesthesia.

[0009] To prepare a composite emulsion, IRBP-CFA emulsion, for subcutaneous injection, IRBP-CFA emulsion is used. Traditional laboratory methods for preparing drug emulsions involve injection mixing using medical three-way stopcocks. However, studies have shown that this method is prone to clogging and difficulty in pushing the mixture, resulting in wasted time, effort, and materials. Furthermore, it easily leads to the inactivation of IRBP (especially the IRBP1-20 antigenic peptide), resulting in low susceptibility in mice. To address this issue, this invention employs ultrasonic emulsification to prepare an emulsion of IRBP and CFA. Since IRBP is easily degraded by heat, this invention uses an ice bath for ultrasonic emulsification to prevent IRBP inactivation during the process.

[0010] Furthermore, studies have shown that the injection volumes of IRBP, CFA, and PTX also affect the success rate of model establishment. To further improve the success rate, in this invention, the subcutaneous injection masses of IRBP in the anterior neck, bilateral thighs, and back are 120-130 μg, 120-130 μg, and 120-130 μg, respectively; the subcutaneous injection masses of CFA in the anterior neck, bilateral thighs, and back are 120-130 μg, 120-130 μg, and 120-130 μg, respectively; and the intraperitoneal injection mass of PTX is 0.4-0.6 μg.

[0011] Thirdly, the application of a mouse model of tubulointerstitial nephritis-uveitis syndrome in screening or preparing drugs for treating tubulointerstitial nephritis-uveitis syndrome, wherein the mouse model of tubulointerstitial nephritis-uveitis syndrome is obtained by the above-described construction method.

[0012] The beneficial effects of this invention are as follows: This invention successfully constructed a mouse model of tubulointerstitial nephritis-uveitis syndrome. With this mouse model, researchers can observe the disease's development and progression in vivo more closely and gain a detailed understanding of its pathophysiological changes. Through long-term tracking and monitoring of the model mice, the pathogenesis, progression patterns, and intrinsic relationship between tubulointerstitial inflammation and uveitis can be precisely grasped. This helps in the discovery of new disease biomarkers and potential therapeutic targets, providing a theoretical basis for early diagnosis and personalized treatment.

[0013] Meanwhile, in drug development, this model can be used to screen and evaluate the efficacy and safety of various candidate drugs. Researchers can simulate different treatment regimens on model mice, observing the improvement of disease symptoms and the effects on other aspects of the body. This can not only improve the efficiency of drug development but also reduce development costs and risks, accelerate the market launch of new therapeutic drugs, and bring more treatment options and hope for recovery to patients with tubulointerstitial nephritis-uveitis syndrome. Furthermore, research findings based on this model may also provide reference and guidance for the research and treatment of other related autoimmune diseases, promoting the development of the entire medical field in autoimmune disease research.

[0014] In addition, by optimizing the modeling method, this invention has solved the problems of low success rate and high mortality rate, thus improving the application prospects of this mouse model of tubulointerstitial nephritis-uveitis syndrome. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0016] Figure 1 The images shown are the results of detecting the pathological manifestations of the fundus of mice in each group using a small animal fundus imaging system in this embodiment of the invention. A is the blank group and B is the model group. Figure 2 The image shows the results of observing the pathological manifestations of retinal tissue using hematoxylin and eosin (HE) staining in an embodiment of the present invention. A is the blank group, B is the model group, and the scale bar is 100 μm. Figure 3 The images show the results of fluorescein sodium angiography in various groups of mice in this invention, where A is the blank group and B is the model group. Figure 4 The images show the clinical (A) and pathological (B) scores of mice in each group in this embodiment of the invention. Figure 5The image shows the results of HE staining observation of pathological changes in the kidney tissue of mice in each group in this embodiment of the invention. A is the blank group and B is the model group. The scale bar is 50 μm. Figure 6 The image shows the results of MASSON staining observation of pathological changes in the kidney tissue of mice in each group in this embodiment of the invention. A is the blank group and B is the model group. The scale bar is 50 μm. Figure 7 The image shows the results of pertannic acid Schiff (PAS) staining observation of the pathological changes in the kidney tissue of mice in each group in this embodiment of the invention. A is the blank group and B is the model group. The scale bar is 50 μm. Figure 8 This is a graph showing the results of detecting the concentrations of CREA(B) and UREA(A) in the serum of mice in each group using a fully automated biochemical analyzer in an embodiment of the present invention. Figure 9 This is a graph showing the results of detecting the concentration of β2M in the urine of mice in each group using the ELISA method in an embodiment of the present invention. Detailed Implementation

[0017] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0018] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0019] Given the current lack of animal models of tubulointerstitial nephritis-uveitis syndrome, which makes it difficult to study its pathological mechanisms, this invention proposes a method for constructing a mouse model of tubulointerstitial nephritis-uveitis syndrome.

[0020] One typical embodiment of the present invention provides the application of an experimental autoimmune uveoretinitis mouse model in constructing a mouse model of tubulointerstitial nephritis-uveitis syndrome.

[0021] Another embodiment of the present invention provides a method for constructing a mouse model of tubulointerstitial nephritis-uveitis syndrome, comprising the following steps: Anesthetize the mice; Active immunization was performed by subcutaneous injection of vitamin A-binding protein between photoreceptor cells and complete Freund's adjuvant into the anterior neck, bilateral thighs, and back of anesthetized mice. Pertussis toxin was administered intraperitoneally to mice after active immunization.

[0022] In some embodiments, the mouse is an SPF-grade wild-type C57BL / 6 female mouse.

[0023] In some embodiments, the method of anesthetizing mice is inhalation anesthesia. This method can avoid over-anesthesia and reduce the mortality rate of mice during the modeling process.

[0024] In some embodiments, the intercellular vitamin A-binding protein is the IRBP1-20 peptide.

[0025] In some embodiments, an emulsion is prepared by combining intercellular vitamin A-binding protein (ICP-A) with complete Freund's adjuvant, and then the emulsion is administered subcutaneously. Specifically, the ICP-A is prepared by ultrasonic emulsification. This method avoids clogging and prevents IRBP inactivation caused by clogging impurities. More specifically, the ultrasonic emulsification is performed in an ice bath. This avoids temperature rise due to mechanical vibration during the ultrasonic process, thereby preventing thermal degradation of the IRBP.

[0026] In some embodiments, the mass of vitamin A-binding protein between photoreceptor cells injected subcutaneously into the anterior neck, bilateral thighs, and back is 120-130 μg, 120-130 μg, and 120-130 μg, respectively.

[0027] In some embodiments, the mass of complete Freund's adjuvant injected subcutaneously into the anterior neck, bilateral thighs, and back is 120-130 μg, 120-130 μg, and 120-130 μg, respectively.

[0028] In some embodiments, the mass of pertussis toxin injected intraperitoneally is 0.4~0.6 μg.

[0029] A third embodiment of the present invention provides the application of a mouse model of tubulointerstitial nephritis-uveitis syndrome in screening or preparing drugs for treating tubulointerstitial nephritis-uveitis syndrome, wherein the mouse model of tubulointerstitial nephritis-uveitis syndrome is obtained by the above-described construction method.

[0030] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0031] Example Eight to ten-week-old SPF-grade wild-type C57BL / 6 female mice were selected, and three reagents were used: 5 mg / ml IRBP1-20 peptide, 5 mg / ml Complete Freunds Adjuvant (CFA) containing Mycobacterium tuberculosis, and 5 μg / ml Pertussis toxin (PTX).

[0032] Drug preparation method: Weigh 10 mg of human IRBP 1-20 antigenic peptide powder into a 1.5 ml centrifuge tube using a microbalance. Add 2 ml of sterile PBS containing 2% DMSO to the centrifuge tube containing the IRBP powder and shake on a shaker to accelerate dissolution. Then, centrifuge at 3000 rpm for 30 s to remove air bubbles and prepare an IRBP solution with a concentration of 5 mg / ml. Subsequently, add 2 ml of CFA containing 5 mg / ml Mycobacterium tuberculosis at a volume ratio of 1:1, place on ice, and use an ultrasonic homogenizer to prepare an IRBP-CFA emulsion. This method of emulsion preparation is convenient, quick, and yields good results with minimal waste. Dissolve 50 μg of PTX in 10 ml of sterile PBS to prepare a PTX solution with a concentration of 5 μg / ml and store at -20 ℃ for later use.

[0033] Mouse modeling method: Mice were anesthetized by inhalation anesthesia, and experiments were conducted when the mice were in a well-controlled anesthetized state. 50 μl of IRBP-CFA emulsion was injected subcutaneously (im) into the anterior neck, bilateral thighs, and back of each mouse. After active immunization, 100 μl of PTX was injected intraperitoneally as an adjuvant to enhance the immune effect. On day 25, the model mice reached peak inflammation. Fundus retinal angiography was used to detect fundus lesions. Subsequently, serum, urine, and kidney tissue were collected to observe specific indicators of interstitial nephritis-uveitis syndrome.

[0034] The results are as follows Figures 1-4As shown, in the control group mice, the optic disc and blood vessel margins were clear, without swelling, inflammatory infiltration, or other pathological manifestations. The retinal layers were clearly defined, uniform in thickness, and no other obvious abnormalities were observed. Compared to the control group, the model group mice exhibited optic disc swelling with diffuse and localized inflammatory infiltration. Thickened and tortuous veins were visible in the retinal vessels. The retinal vascular segments were extensively infiltrated by white cuffs, with the cuffs being larger than the vessel diameter. Furthermore, isolated lesions were observed in the retinal tissue. The retina showed thickening, wrinkling, and vascular dilation, with uneven thickness of the inner and outer granular layers, irregular cell arrangement within the visual field, and abundant inflammatory cell infiltration in the retinal tissue. These results indicate that the EAU mouse model was successfully established.

[0035] Next, based on the EAU model, specific indicators consistent with renal interstitial nephritis-uveitis syndrome were detected in mouse serum and kidney tissue. The results showed that the kidney tissue of the control group mice showed no significant pathological changes. Compared with the control group, EAU mice showed extensive inflammatory cell infiltration and renal interstitial fibrosis in the renal interstitium. Simultaneously, the pathological scores and degree of renal interstitial fibrosis in EAU mice were significantly higher than those in the control group. Figures 5-7 As shown in the figure. Increased expression of CREA, UREA, and β2M are diagnostic indicators of renal dysfunction induced by interstitial nephritis-uveitis syndrome. The study showed that, compared with the control group, EAU mice had elevated serum levels of CREA and UREA, as well as elevated urinary levels of β2M (P<0.05). Figures 8-9 This demonstrates that the mouse model of tubulointerstitial nephritis-uveitis syndrome was successfully constructed in this embodiment.

[0036] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of an experimental autoimmune uveoretinitis mouse model in the construction of a mouse model of tubulointerstitial nephritis-uveitis syndrome.

2. A method for constructing a mouse model of tubulointerstitial nephritis-uveitis syndrome, characterized in that, Includes the following steps: Anesthetize the mice; Active immunization was performed by subcutaneous injection of vitamin A-binding protein between photoreceptor cells and complete Freund's adjuvant into the anterior neck, bilateral thighs, and back of anesthetized mice. Pertussis toxin was administered intraperitoneally to mice after active immunization.

3. The construction method as described in claim 2, characterized in that, The mice were SPF-grade wild-type C57BL / 6 female mice.

4. The construction method as described in claim 2, characterized in that, The mice were anesthetized by inhalation anesthesia.

5. The construction method as described in claim 2, characterized in that, The vitamin A-binding protein between photoreceptor cells is the IRBP1-20 peptide.

6. The construction method as described in claim 2, characterized in that, The intercellular vitamin A-binding protein of photoreceptor cells was prepared into an emulsion with complete Freund's adjuvant, and then the emulsion was used for subcutaneous injection.

7. The construction method as described in claim 6, characterized in that, Ultrasonic emulsification was used to prepare an emulsion by combining intercellular vitamin A-binding proteins with complete Freund's adjuvant.

8. The construction method as described in claim 7, characterized in that, The ultrasonic emulsification was performed in an ice bath.

9. The construction method as described in claim 2, characterized in that, The mass of vitamin A-binding protein between photoreceptor cells injected subcutaneously into the anterior neck, bilateral thighs, and back was 120-130 μg, 120-130 μg, and 120-130 μg, respectively. Alternatively, the total mass of Freund's adjuvant injected subcutaneously into the anterior neck, bilateral thighs, and back is 120-130 μg, 120-130 μg, and 120-130 μg, respectively. Alternatively, the mass of pertussis toxin administered via intraperitoneal injection is 0.4–0.6 μg.

10. The use of a mouse model of tubulointerstitial nephritis-uveitis syndrome in screening or preparing drugs for treating tubulointerstitial nephritis-uveitis syndrome, wherein the mouse model of tubulointerstitial nephritis-uveitis syndrome is obtained by the construction method according to any one of claims 2 to 8.

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