Construction and application of podocyte-specific S1PR1 gene knockout mouse model

Abstract

This invention belongs to the field of biomedical technology and discloses a method for constructing and applying a podocyte-specific S1PR1 gene knockout mouse model. Podocyte-specific S1PR1 knockout mice were constructed using the Cre-LoxP system, and then an FSGS model was established via doxorubicin injection. The model was validated using urine, serum, histopathological, and molecular mechanism detection methods. The study found that decreased S1PR1 expression induces podocyte senescence and FSGS progression by downregulating Xab2, leading to abnormal POLR2A splicing and reduced expression. This invention is the first to construct such a model, revealing the regulatory role of the S1PR1-Xab2-POLR2A pathway in podocyte senescence, providing a reliable tool for studying the pathogenesis of FSGS, clarifying the potential of S1PR1 as a therapeutic target, and enabling drug screening for FSGS treatment. It has significant clinical translational value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for constructing a research model of focal segmental glomerulosclerosis (FSGS) based on S1PR1 (sphingosine 1-phosphate receptor 1) regulating podocyte senescence, and the application of S1PR1 in the treatment of FSGS. Background Technology

[0002] Focal segmental glomerulosclerosis (FSGS) is a glomerular disease characterized primarily by damage to the visceral epithelial cells (podocytes) of the glomeruli. Clinically, it mainly manifests as nonselective proteinuria, microscopic hematuria, and hypertension. It is often resistant to hormone therapy, and its continued progression can lead to renal function decline, severely impacting the growth, development, and quality of life of patients, especially children. Statistics show that FSGS accounts for 10%-15% of childhood nephrotic syndromes, with a male-to-female ratio of approximately 3:2, and a 15-year mortality rate as high as 50%. Currently, there is no specific treatment, and there is an urgent need to explore new pathogenesis and therapeutic targets.

[0003] Sphingosine 1-phosphate receptor 1 (S1PR1) is a seven-transmembrane G protein-coupled receptor widely expressed in glomerular mesangial cells, vascular endothelial cells, and podocytes. It inhibits apoptosis by regulating the PI3K / Akt pathway and promotes cytoskeleton rearrangement by activating Rac, playing a crucial role in maintaining the blood-brain barrier and the localization of tight junction proteins. However, the role of S1PR1 in the pathogenesis of FSGS has not been reported, and its association with podocyte senescence and FSGS progression lacks systematic research.

[0004] Existing FSGS research models suffer from problems such as long modeling cycles and low simulation of pathological features, making it difficult to accurately reflect the relationship between podocyte damage and disease progression. Therefore, constructing an FSGS research model based on S1PR1 regulation and clarifying the mechanism of action of S1PR1 in podocyte senescence is of great significance for the development of targeted therapies for FSGS. Summary of the Invention

[0005] In view of this, one of the objectives of this invention is to provide a method for constructing a podocyte-specific S1PR1 gene knockout mouse model and a method for constructing an FSGS research model based on S1PR1 regulating podocyte senescence, to reveal the mechanism by which S1PR1 affects podocyte senescence by regulating downstream molecules Xab2 and POLR2A, and to provide the application of S1PR1 in FSGS treatment.

[0006] The present invention solves the above-mentioned technical problems through the following technical means: 1. Construction of the S1PR1 gene knockout mouse model: Podocyte-specific S1PR1 knockout mice (S1PR1-CKO) were constructed using the Cre-LoxP system: S1PR1-flox mice were crossed with NPHS2-Cre mice, and genotypes were identified by PCR to screen out homozygous S1PR1 knockout mice.

[0007] Genotyping method: DNA was extracted from mouse tails, amplified by PCR using specific primers, and detected by 2% agarose gel electrophoresis. Wild-type (WT) showed a 299bp band, heterozygotes (Heterozygous) showed two bands, 299bp and 353bp, and homozygotes showed a 353bp band.

[0008] 2. Establishment of the FSGS model: Modeling Groups: Mice were divided into four groups: 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: Mice in the FLOX+ADR group and S1PR1-CKO+ADR group were intraperitoneally injected with anti-glomerular basement membrane antibody serum (NTS) at a dose of 16 μL / g, as a single injection, to establish the FSGS model; the normal control group was injected with an equal volume of physiological saline.

[0009] 3. Model validation metrics: Urine testing: Regularly test 24-hour urine protein quantification and urine protein / creatinine ratio (UACR) to assess glomerular filtration function impairment.

[0010] Serum tests: Detect serum albumin and blood urea nitrogen levels to assess kidney function.

[0011] Histopathological examination: Kidney index calculation: bilateral kidney weight / body weight (KI) to assess the degree of kidney enlargement.

[0012] Pathological staining: HE staining was used to observe glomerular structure, PAS staining was used to observe glomerular mesangial matrix proliferation, and immunohistochemistry was used to detect the expression of podocyte-related proteins (Nephrin, ZO-1, and Podocin).

[0013] Electron microscopy: Observe the fusion of podocyte foot processes and the damage to the glomerular basement membrane.

[0014] Molecular mechanism detection: The expression levels of S1PR1, Xab2, POLR2A and cell senescence-related markers (such as p16 and p21) were detected by Western blot and immunofluorescence.

[0015] 4. Application of S1PR1 in the treatment of FSGS Based on the above model validation, decreased S1PR1 expression can induce podocyte senescence and FSGS progression by downregulating Xab2, leading to abnormal POLR2A splicing and reduced expression. Therefore, S1PR1 agonists or Xab2 activators can be used to prepare drugs for the treatment of FSGS.

[0016] The beneficial effects of this invention are: This invention is the first to construct a podocyte-specific S1PR1 knockout FSGS model, which accurately simulates the pathological process of podocyte damage in FSGS, providing a reliable tool for studying the pathogenesis of FSGS.

[0017] This study reveals for the first time the regulatory role of the S1PR1-Xab2-POLR2A pathway in podocyte senescence and clarifies the potential of S1PR1 as a therapeutic target for FSGS.

[0018] This model can be used to screen FSGS treatments targeting S1PR1 or Xab2, and has significant clinical translational value. Attached Figure Description

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Figure 1 Electrophoresis diagram of S1PR1-CKO mouse genotype identification; Figure 2 Trends in 24-hour urinary protein quantification in each group of mice; Figure 3 Comparison of kidney index among different groups of mice (2 weeks and 4 weeks after modeling and normal control). Figure 4 HE staining shows the glomerular structure of mice in each group (×400). Figure 5 PAS staining shows the glomerular structure of mice in each group (×400). Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings: I. Experimental Materials Laboratory animals: SPF-grade C57BL / 6 mice, S1PR1-flox mice, and NPHS2-Cre transgenic mice were all purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0022] Reagents: Rabbit anti-S1PR1 antibody (Abcam), mouse anti-Xab2 antibody (CST), Nephrin, ZO-1, and Podocin antibodies (Santa Cruz), PCR primers (Shanghai Sangon Biotech Co., Ltd.).

[0023] Instruments: PCR instrument (Bio-Rad), electrophoresis instrument (Bio-Rad), fluorescence microscope (Zeiss), transmission electron microscope (Hitachi).

[0024] II. Experimental Procedure Construction and Identification of S1PR1-CKO Mice S1PR1-flox mice and NPHS2-Cre mice were mated at a 1:1 ratio. Tail tissue was collected from the offspring mice 7 days after birth, and genomic DNA was extracted using the phenol-chloroform method.

[0025] PCR reaction system (20 μL): DNA template 2 μL, forward and reverse primers 0.5 μL each, 2×TaqMix 10 μL, ddH2O 7 μL.

[0026] Reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ final extension for 10 min.

[0027] Electrophoretic identification: 2% agarose gel electrophoresis was performed, and the bands were observed under UV light to screen for homozygous S1PR1-CKO mice. (See attached instruction manual) Figure 1 The image shows an electrophoresis diagram of S1PR1-CKO mice for genotyping identification, from which the bands corresponding to wild type, heterozygote and homozygote can be clearly seen.

[0028] Establishment of the FSGS model Eight-week-old male S1PR1-CKO mice and their littermate FLOX mice were randomly divided into five groups (n=6-8): normal control group, FLOX group, S1PR1-CKO group, FLOX+ADR group, and S1PR1-CKO+ADR group.

[0029] Mice in the FLOX+ADR and S1PR1-CKO+ADR groups were injected intraperitoneally with serum NTS (anti-glomerular basement membrane antibody) at a dose of 16 μL / g in a single injection to establish the FSGS model; the normal control group was injected with an equal volume of physiological saline.

[0030] Indicator Testing Urine protein detection: 24-hour urine samples were collected from mice at 0, 1, 2, 3, and 4 weeks after modeling. Urine protein concentration was measured using the Coomassie brilliant blue method, and the 24-hour urinary protein quantification was calculated. Simultaneously, urinary creatinine levels were measured, and the UACR was calculated. (Refer to the appendix in the instruction manual.) Figure 2 The figure shows the trend of 24-hour urinary protein quantification in each group of mice, and can intuitively show the differences in urinary protein quantification in different groups at different time points.

[0031] Serum marker detection: Blood was collected by enucleation of the eyeball 4 weeks after modeling, and the serum was separated by centrifugation. The serum albumin and urea nitrogen levels were detected by a fully automated biochemical analyzer.

[0032] Kidney Index Calculation: After euthanizing the mouse, weigh it, remove both kidneys, weigh them, and calculate the KI (kidney mass / body weight × 100%). Refer to the instruction manual appendix. Figure 3 The study showed a comparison of the kidney index in each group of mice (2 weeks and 4 weeks after modeling and normal control), which allowed for a comparison of the degree of kidney enlargement in different groups.

[0033] Pathological examination: Kidney tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (4 μm), and stained with hematoxylin and eosin (HE) and eosin (PAS). Glomerular morphology was observed under a light microscope. (See attached instruction manual.) Figure 4 and Figure 5 The glomerular structures of mice in each group after HE staining and PAS staining are shown (×400), and the differences in glomerular structure between different groups can be clearly seen.

[0034] Immunohistochemistry: Sections were dewaxed to water, and after antigen retrieval, they were incubated with primary antibodies (Nephrin 1:200, ZO-11:200), and DAB staining was performed to observe protein expression localization.

[0035] Electron microscopy: Renal cortical tissue was taken, fixed with 2.5% glutaraldehyde, ultrathinly sectioned, stained with uranium acetate, and the foot process structure of podocytes was observed under a transmission electron microscope.

[0036] Molecular detection: Western blot: Total protein was extracted from the renal cortex, and the expression of S1PR1, Xab2, POLR2A, and p16 proteins was detected, with GAPDH as an internal control.

[0037] 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.

[0038] III. Experimental Results 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.

[0039] 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.

[0040] 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).

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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 anti-glomerular 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.

Images