Construction method and application of podocyte specific S1PR1 gene knockout mouse model
By constructing a podocyte-specific S1PR1 gene knockout mouse model, the shortcomings of existing FSGS research models were addressed, the podocyte injury process was accurately simulated, the mechanism of action of S1PR1 in FSGS was revealed, and a new direction was provided for targeted therapy of FSGS.
Patent Information
- Application Number
- CN202511138450.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-14
- Publication Date
- 2025-11-21
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Existing FSGS research models suffer from long modeling cycles, low simulation of pathological features, difficulty in accurately reflecting the relationship between podocyte damage and disease progression, lack of systematic research on the role of S1PR1 in the pathogenesis of FSGS, and no effective treatment methods.
A podocyte-specific S1PR1 gene knockout mouse model was constructed. Homozygous S1PR1 knockout mice were selected by hybridization using the Cre-LoxP system. Combined with anti-glomerular basement membrane antibody modeling, a precise FSGS model was established. Urinary protein, renal function and histopathological indicators were detected to reveal the regulatory role of the S1PR1-Xab2-POLR2A pathway in podocyte senescence.
This study provides a precise FSGS research tool, reveals the mechanism of action of S1PR1 in podocyte senescence, clarifies its potential as a therapeutic target for FSGS, and supports the development of therapeutic drugs targeting S1PR1 or Xab2.
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Figure CN120982471A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological medicine, and particularly relates to a construction method of a focal segmental glomerulosclerosis (FSGS) research model based on S1PR1 (1-phospho-sphingosine receptor 1) regulation of podocyte aging, and application of S1PR1 in FSGS treatment. BACKGROUND
[0002] Focal segmental glomerulosclerosis (FSGS) is a glomerular disease mainly characterized by damage to glomerular visceral epithelial cells (podocytes), and is mainly manifested clinically as non-selective proteinuria, microscopic hematuria and hypertension, and is often resistant to hormone therapy, and continuously progresses to lead to reduced renal function, seriously affecting the growth and development and life quality of patients (especially children). According to statistics, FSGS accounts for 10%-15% of nephrotic syndrome in children, the male to female ratio is about 3:2, and the 15-year mortality rate is as high as 50%, and there is currently no specific treatment method, and new pathogenesis and treatment targets need to be explored.
[0003] 1-phospho-sphingosine receptor 1 (S1PR1) is a seven-transmembrane G protein-coupled receptor, which is widely expressed in glomerular mesangial cells, vascular endothelial cells and podocytes, can inhibit cell apoptosis by regulating the PI3K / Akt pathway, promote cytoskeleton rearrangement by activating Rac, and plays an important role in maintaining the blood-brain barrier and tight junction protein localization. However, the role of S1PR1 in the pathogenesis of FSGS has not been reported, and its correlation with podocyte aging and FSGS progression lacks systematic research.
[0004] The existing FSGS research model has problems such as long modeling cycle and low simulation degree of pathological characteristics, and it is difficult to accurately reflect the relationship between podocyte damage and disease progression. Therefore, it is of great significance to construct a FSGS research model based on S1PR1 regulation, and to clarify the mechanism of S1PR1 in podocyte aging, for the development of targeted therapeutic drugs for FSGS. SUMMARY
[0005] Therefore, one of the purposes of the present application is to provide a construction method of a podocyte-specific S1PR1 gene knockout mouse model and a FSGS research model based on S1PR1 regulation of podocyte aging, to reveal the mechanism of S1PR1 affecting podocyte aging by regulating downstream molecules Xab2 and POLR2A, and to provide application of S1PR1 in FSGS treatment.
[0006] The present application solves the above technical problems through the following technical means:
[0007] 1. Construction of S1PR1 gene knockout mouse model:
[0008] Construction of S1PR1-CKO mice by Cre-LoxP system: S1PR1-flox mice were crossed with NPHS2-Cre mice, and genotypes were identified by PCR to screen S1PR1 homozygous knockout mice.
[0009] Genotype identification method: Tail DNA was extracted, and specific primers were used for PCR amplification, and 2% agarose gel electrophoresis was used for detection. Wild type (WT) showed a 299bp band, heterozygous (Heterozygous) showed two bands of 299bp and 353bp, and homozygous showed a 353bp band.
[0010] 2. Establishment of FSGS model:
[0011] Modeling grouping: The mice were divided into 4 groups, namely normal control group, FLOX group (S1PR1-flox mice), S1PR1-CKO group (S1PR1 knockout mice), FLOX+ADR group (S1PR1-flox mice), and S1PR1-CKO+ADR group (S1PR1 knockout mice + anti-glomerular basement membrane antibody serum). Modeling method: The FLOX+ADR group and S1PR1-CKO+ADR group mice were injected intraperitoneally with anti-glomerular basement membrane antibody serum NTS, with a dose of 16 μL / g, and a single injection to construct the FSGS model; the normal control group was injected with an equal amount of normal saline.
[0012] 3. Model verification index:
[0013] Urine detection: 24-hour urine protein quantification and urine protein / creatinine ratio (UACR) were detected regularly to evaluate the damage of glomerular filtration function.
[0014] Serum detection: Serum albumin and urea nitrogen levels were detected to evaluate renal function.
[0015] Histopathological detection:
[0016] Kidney index calculation: Double kidney weight / body weight (KI) to evaluate the degree of kidney enlargement.
[0017] Pathological staining: HE staining to observe glomerular structure, PAS staining to observe mesangial matrix proliferation, and immunohistochemical detection of podocyte-related proteins (Nephrin, ZO-1, Podocin) expression.
[0018] Electron microscopy observation: Observation of podocyte foot process fusion and glomerular basement membrane damage.
[0019] Molecular mechanism detection: The expression levels of S1PR1, Xab2, POLR2A and cell aging related markers (such as p16, p21) are detected by Western blot and immunofluorescence.
[0020] 4. Application of S1PR1 in FSGS treatment
[0021] Based on the above model verification, the decrease of S1PR1 expression can cause the decrease of POLR2A splicing and expression by down-regulating Xab2, and further induce podocyte aging and FSGS progression. Therefore, S1PR1 agonists or Xab2 activators can be used for preparing drugs for treating FSGS.
[0022] Advantages of the present application:
[0023] The present application first constructs a FSGS model with podocyte-specific S1PR1 knockout, accurately simulates the pathological process of podocyte damage in FSGS, and provides a reliable tool for studying the pathogenesis of FSGS.
[0024] The present application first discloses the regulatory role of S1PR1-Xab2-POLR2A pathway in podocyte aging, and determines the potential of S1PR1 as a target for FSGS treatment.
[0025] The model can be used for screening FSGS treatment drugs targeting S1PR1 or Xab2, and has important clinical transformation value. BRIEF DESCRIPTION OF DRAWINGS
[0026] The present application will be further described below in combination with the drawings and examples.
[0027] Figure 1 : S1PR1-CKO mouse genotype identification electrophoresis map;
[0028] Figure 2 : 24-hour urine protein quantitative change trend of mice in each group;
[0029] Figure 3 : Comparison of kidney index of mice in each group (2 weeks, 4 weeks after modeling and normal control);
[0030] Figure 4 : HE staining shows the glomerular structure of mice in each group (400X);
[0031] Figure 5 : PAS staining shows the glomerular structure of mice in each group (400X). DETAILED DESCRIPTION
[0032] The present application will be described in detail below in combination with the drawings:
[0033] I. Experimental materials
[0034] Experimental animals: SPF grade C57BL / 6 mice, S1PR1-flox mice, NPHS2-Cre transgenic mice, all purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.
[0035] Reagents: Rabbit anti-S1PR1 antibody (Abeam), mouse anti-Xab2 antibody (CST), Nephrin, ZO-1, Podocin antibody (Santa Cruz), PCR primers (Shanghai Sangon Biological Engineering Co., Ltd.).
[0036] Instruments: PCR instrument (Bio-Rad), electrophoresis instrument (Bio-Rad), fluorescence microscope (Zeiss), transmission electron microscope (Hitachi).
[0037] II. Experimental steps
[0038] Construction and identification of S1PR1-CKO mice
[0039] S1PR1-flox mice and NPHS2-Cre mice were mated at a ratio of 1:1, and the tail tissues of the offspring mice were taken 7 days after birth. Genomic DNA was extracted by phenol-chloroform method.
[0040] PCR reaction system (20 μL): DNA template 2 μL, upstream and downstream primers 0.5 μL each, 2 × TaqMix 10 μL, ddH2O 7 μL.
[0041] Reaction conditions: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s, a total of 35 cycles; 72°C final extension for 10 min.
[0042] Electrophoresis identification: 2% agarose gel electrophoresis, band observation under ultraviolet lamp, selection of homozygous S1PR1-CKO mice. As shown in the specification Figure 1 The electrophoretogram for identification of S1PR1-CKO mouse genotype is shown, from which the bands corresponding to wild type, heterozygote and homozygote can be clearly seen.
[0043] Establishment of FSGS model
[0044] Eight-week-old male S1PR1-CKO mice and littermates FLOX mice were selected and randomly divided into 5 groups (n=6-8): normal control group, FLOX group, S1PR1-CKO group, FLOX+ADR group, S1PR1-CKO+ADR group.
[0045] The FLOX+ADR group and the S1PR1-CKO+ADR group of mice were injected intraperitoneally with anti-glomerular basement membrane antibody serum NTS at a dose of 16 μL / g, single injection, to construct the FSGS model; the normal control group was injected with an equal amount of normal saline.
[0046] Index detection
[0047] Urine protein detection: collect 24-hour urine of mice at 0 weeks, 1 week, 2 weeks, 3 weeks, and 4 weeks after modeling, detect urine protein concentration by Coomassie brilliant blue method, and calculate 24-hour urine protein quantification; at the same time, detect urine creatinine level, and calculate UACR. Refer to the attached Figure 2 , the figure presents the change trend of 24-hour urine protein quantification of mice in each group, which can directly show the difference in urine protein quantification of different groups at different time points.
[0048] Serum index detection: enucleate and take blood at 4 weeks after modeling, centrifugal separate serum, and detect serum albumin and urea nitrogen level by automatic biochemical analyzer.
[0049] Kidney index calculation: weigh the mice after sacrifice, extract and weigh the double kidneys, and calculate KI (double kidney weight / body weight x 100%). Refer to the attached Figure 3 , which shows the comparison of kidney index of mice in each group (2 weeks and 4 weeks after modeling and normal control), which can compare the degree of kidney enlargement of different groups.
[0050] Pathological detection:
[0051] Fix the kidney tissue in 4% paraformaldehyde, paraffin-embedded, slice (4 μm), and perform HE staining and PAS staining, and observe the glomerular morphology under light microscope. Refer to the attached Figure 4 and Figure 5 , which respectively show the glomerular structure of mice in each group after HE staining and PAS staining (x400), which can clearly see the difference in glomerular structure of different groups.
[0052] Immunohistochemistry: deparaffinize the slice to water, incubate the primary antibody (Nephrin 1:200, ZO-11:200) after antigen repair, develop by DAB, and observe the protein expression location.
[0053] Electron microscopy observation: take the kidney cortex tissue, fix it in 2.5% glutaraldehyde, slice it ultra-thin, stain it with uranyl acetate, and observe the podocyte foot process structure under transmission electron microscope.
[0054] Molecular detection:
[0055] Western blot: extract total protein of kidney cortex, detect S1PR1, Xab2, POLR2A, and p16 protein expression, and take GAPDH as internal reference.
[0056] Immunofluorescence: Frozen sections were incubated with S1PR1 (1:100) and Xab2 (1:100) antibodies, labeled with fluorescent secondary antibodies, and observed for protein colocalization using confocal microscopy.
[0057] III. Experimental Results
[0058] Genotyping: The PCR product of S1PR1-CKO mice showed a single 353bp band, confirming successful knockout, consistent with the instructions. Figure 1 The results are consistent.
[0059] Urinary protein and UACR: The 24-hour urinary protein and UACR in the S1PR1-CKO+ADR group were significantly higher than those in the FLOX+ADR group (P<0.05), and gradually increased with the extension of modeling time, consistent with the instructions. Figure 2 The trend is consistent.
[0060] Serum markers: Serum albumin levels were significantly lower and blood urea nitrogen levels were significantly higher in the S1PR1-CKO+ADR group (P<0.05).
[0061] Kidney index: The KI in the S1PR1-CKO+ADR group was significantly higher than that in the FLOX+ADR group (P<0.05), consistent with the instructions. Figure 3 The results echoed each other.
[0062] Pathological results: The S1PR1-CKO+ADR group showed significant segmental glomerular sclerosis, mesangial matrix proliferation, severe podocyte foot process fusion, and significantly decreased expression of Nephrin and ZO-1, consistent with the product manual. Figure 4 and 5 The observations were consistent.
[0063] Molecular mechanism: The expression of Xab2 and POLR2A was decreased and the expression of p16 was increased in the S1PR1-CKO+ADR group, suggesting enhanced podocyte senescence.
[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.
Claims
1. A method for constructing a podocyte-specific S1PR1 gene knockout mouse model, characterized in that, Includes the following steps: S1) Cross S1PR1-flox mice with NPHS2-Cre mice to obtain offspring mice; S2) Extract DNA from the tails of offspring mice, identify genotypes by PCR, and screen out S1PR1 homozygous knockout mice S1PR1-CKO. In the PCR identification, the wild type showed a 299bp band, and the homozygous showed a 353bp band.
2. A method for constructing a focal segmental glomerulosclerosis (FSGS) model, characterized in that, Includes the following steps: A) Construct S1PR1-CKO mice using the method described in claim 1; B) Mice were divided into a normal control group, a FLOX group, a S1PR1-CKO group, a FLOX+ADR group, and a S1PR1-CKO+ADR group. C) FLOX+ADR group and S1PR1-CKO+ADR group mice were intraperitoneally injected with serum NTS antiglomerular basement membrane antibody at a dose of 16 μL / g, once, to construct the FSGS model.
3. The method according to claim 2, characterized in that, It also includes a model validation step, which includes detecting 24-hour urinary protein quantification, urinary protein / creatinine ratio, serum albumin, blood urea nitrogen, glomerular pathological staining, and podocyte-related protein expression.
4. The application of the FSGS model described in claim 2 with S1PR1 as the target in the preparation of a drug for treating focal segmental glomerulosclerosis (FSGS).
5. The application according to claim 4, characterized in that, The drug is an S1PR1 agonist.
6. A pharmaceutical composition for treating focal segmental glomerulosclerosis (FSGS), characterized in that, It contains an S1PR1 agonist.
7. The pharmaceutical composition according to claim 6, characterized in that, It also includes pharmaceutically acceptable carriers.