Application of SHIP1 agonist AQX-1125 in preparation of medicine for treating focal segmental glomerulosclerosis

Abstract

The invention discloses application of an SHIP1 agonist AQX-1125 in preparation of a medicine for treating focal segmental glomerulosclerosis, and belongs to the technical field of medicines.The endogenous regulatory factor SHIP1 agonist is used for conducting precise speed limiting on a PI3K / Akt pathway, so that a new effective means is provided for FSGS treatment, and the application form of the SHIP1 agonist AQX-1125 can be an oral preparation and the like; tests prove that the AQX-1125 can remarkably repair renal functions, reduce pathological proteinuria, improve pathological damage of kidney tissues, maintain phenotypic homeostasis and ultrastructural integrity of podocyte and accurately regulate and control pathogenic signal pathways and metabolic homeostasis, so that the AQX-1125 shows remarkable technical progress and clinical application potential in the aspect of FSGS treatment.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more particularly to the use of the SHIP1 agonist AQX-1125 in the preparation of a medicament for treating focal segmental glomerulosclerosis. Background Technology

[0002] Focal segmental glomerulosclerosis (FSGS) is a progressive kidney disease characterized by podocyte damage, clinically manifesting as severe proteinuria and ultimately leading to renal failure. Currently, first-line clinical treatment for FSGS relies on hormones and immunosuppressants, but these treatments have drawbacks such as significant side effects, high relapse rates, and limited ability to repair podocyte structure. Abnormal activation of the PI3K / Akt pathway is a core element mediating podocyte injury, but direct use of kinase inhibitors can easily produce severe systemic toxicity.

[0003] Specifically, despite some exploration of existing technologies in the field of nephrology, the following significant technical bottlenecks remain in targeted therapy for focal segmental glomerulosclerosis (FSGS):

[0004] (1) Existing drugs cannot effectively restore the physical structural homeostasis of podocytes: The initiating factor of FSGS lies in the dissociation of the podocyte slit septum (SD) structure. Existing hormones and immunosuppressants are mostly systemic anti-inflammatory and cannot directly act on the spatial localization of the podocyte cytoskeleton and its key proteins (such as Nephrin), making it difficult to control proteinuria leakage from the root cause;

[0005] (2) Lack of endogenous negative feedback regulation of the PI3K / Akt pathway: Although it is known in the existing technology that overactivation of the PI3K / Akt pathway can lead to podocyte damage, kinase inhibitors are often used to "block" it. These drugs have large side effects and poor specificity.

[0006] (3) Imbalance in podocyte metabolic homeostasis and poor intervention effect: Severe autophagy flow blockage is accompanied by FSGS process. Current treatment methods often cannot achieve the dual tasks of "inhibiting apoptosis" and "unblocking autophagy", resulting in the continuous accumulation of damaged organelles and metabolic substrates (such as P62) in podocytes, making it difficult to restore the physiological balance of cells.

[0007] (4) Lack of highly tissue-specific protective strategies: Many existing drugs, while improving renal pathology, are prone to causing systemic metabolic disorders. There is an urgent clinical need for a precise treatment plan that protects the local filtration barrier of the kidney while minimizing overall metabolic disturbance. Existing drugs are unable to directly repair the structure of the podocyte slit septum (SD) and the spatial localization of its key proteins;

[0008] Therefore, there is an urgent need in the field for a new drug that can solve the above problems and be used to treat FSGS. Summary of the Invention

[0009] The purpose of this invention is to provide the application of the SHIP1 agonist AQX-1125 in the preparation of a medicament for treating focal segmental glomerulosclerosis, in order to solve the above-mentioned problems.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] Application of the SHIP1 agonist AQX-1125 in the preparation of drugs for the treatment of focal segmental glomerulosclerosis.

[0012] Through extensive experimental research, the inventors of this application have developed a new and effective approach for the treatment of FSGS by using an endogenous regulator SHIP1 agonist to "precisely limit the rate" of the PI3K / Akt pathway.

[0013] As a preferred technical means, the dosage form of the drug is an oral formulation of AQX-1125.

[0014] Compared with existing technologies, the advantages of this invention are as follows: This invention demonstrates significant technological advancements and clinical application potential in the treatment of FSGS by pharmacologically activating SHIP1, a key negative regulatory target. Specifically:

[0015] (1) Significantly repairs kidney function and reduces pathological proteinuria: Experimental results showed that after AQX-1125 intervention, the urine albumin / creatinine ratio (UACR) of the FSGS mouse model was significantly lower than that of the model group; the improvement of this physiological indicator directly proves that the present invention can effectively repair the glomerular filtration barrier and curb the core proteinuria leakage problem in the FSGS process.

[0016] (2) Improvement of renal tissue pathological damage: PAS staining and HE staining confirmed that AQX-1125 significantly reduced glomerular segmental sclerosis and mesangial matrix proliferation, and the sclerosis index decreased significantly. Observation revealed that the formation of protein casts in the renal tubules was significantly reduced, effectively delaying the renal tubulointerstitial damage caused by protein reabsorption;

[0017] (3) Maintaining the integrity of podocyte ultrastructure: This invention is the first to demonstrate that AQX-1125 can restore the spatial colocalization of SHIP1 and Nephrin. This stabilizing effect on the physical association of the slit diaphragm complex is a deep structural protective effect that existing immunosuppressants do not possess.

[0018] (4) Precise regulation of pathogenic signaling pathways and metabolic homeostasis: By activating SHIP1, this invention specifically suppresses pathological PI3K / Akt pathway signal overload, avoiding the widespread systemic toxicity caused by direct kinase inhibitors. It significantly corrects the autophagic flux blockade in podocytes, manifested as a reduction in the accumulation of the autophagy substrate P62, accompanied by the downregulation of apoptosis-related proteins Bax / Caspase-3, synergistically achieving comprehensive protection for podocyte survival. Attached Figure Description

[0019] Figure 1 Western blot was used to detect changes in the expression and phosphorylation levels of p-PI3K and total PI3K in podocytes of each group;

[0020] Figure 2 To quantitatively analyze the changes in the expression and phosphorylation levels of p-PI3K and total PI3K in podocytes of each group, data are expressed as mean ± SEM (n=3).

[0021] Figure 3 Western blot was used to detect changes in the expression and phosphorylation levels of p-PI3K and total PI3K in podocytes of each group;

[0022] Figure 4 To quantitatively analyze the changes in the expression and phosphorylation levels of p-PI3K and total PI3K in podocytes of each group, data are expressed as mean ± SEM (n=3).

[0023] Figure 5 To detect the apoptosis rate of podocytes in each group by flow cytometry;

[0024] Figure 6 To detect the apoptosis rate of podocytes in each group by flow cytometry, the data are expressed as mean ± SEM (n=3).

[0025] Figure 7 Western blot was used to detect the expression levels of caspase3, Bax, Bcl-2, and other proteins in podocytes of each group.

[0026] Figure 8 , Figure 9 , Figure 10 The expression levels of caspase3, Bax, and Bcl-2 proteins in podocytes of each group were quantitatively analyzed. Data are expressed as mean ± SEM (n=3).

[0027] Figure 11 The expression levels of proteins such as P62 and LC3B in podocytes of each group were detected by Western blot.

[0028] Figure 12 , Figure 13 The expression levels of proteins such as P62 and LC3B in podocytes of each group were quantitatively analyzed. The data are expressed as mean ± SEM (n=3).

[0029] Figure 14 To observe the urinary albumin / creatinine ratio (UACR) of mice in groups 0, 2, 4, and 6, the data are expressed as Mean±SEM (n=12).

[0030] Figure 15 Pathological morphological observation images of kidney tissue from each group of mice;

[0031] Figure 16 This is an immunofluorescence double staining spatial colocalization map of SHIP1 and Nephrin in the glomeruli of mice in each group. Detailed Implementation

[0032] To explain the technical content, objectives, and effects of the present invention in detail, the following specific embodiments further illustrate the content of the present invention. However, the content of the present invention is far more than the following embodiments.

[0033] Unless otherwise specified, all materials and reagents used in the following examples were commercially available. The immortalized mouse glomerular podocyte cell line (MPC-5) was purchased from Guangzhou Yuanjing Biotechnology, and its STR and Amelogenin loci genotyping results were satisfactory. AQX-1125 was purchased from Taoshu Biotechnology.

[0034] DMEM basal culture medium, fetal bovine serum, and trypsin were purchased from Gibco, USA.

[0035] Doxorubicin and P62 rabbit monoclonal antibody (YA2136) were purchased from Med Chem Express, USA.

[0036] Annexin V-APC / 7-AAD apoptosis detection kit was purchased from Wuhan Elite Biotechnology Co., Ltd.

[0037] RIPA lysis buffer, protease inhibitor, PMSF, BCA protein concentration assay kit, 5×SDS-PAGE protein loading buffer, and 1×SDS-PAGE protein loading buffer were purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0038] The 10% SDS-PAGE gel preparation kit and the 12.5% ​​SDS-PAGE gel preparation kit were purchased from Shanghai Yamei Biomedical Technology Co., Ltd.

[0039] The molecular weight standards for the prestained proteins were purchased from Thermo Fisher Scientific, USA.

[0040] The PVDF membrane was purchased from Millipore, USA.

[0041] Tween-20 was purchased from Shanghai White Shark Biotechnology Co., Ltd.

[0042] Electrophoresis buffer, rapid transfer buffer, and TBS were purchased from Wuhan Saiweier Biotechnology Co., Ltd.

[0043] SHIP1 rabbit monoclonal antibody (ab45142) and Caspase-3 rabbit monoclonal antibody (ab184787) were purchased from Abcam, Inc., USA.

[0044] Bax rabbit monoclonal antibody (ET1603-34) was purchased from Hangzhou Huaan Biotechnology Co., Ltd.

[0045] LC3B rabbit monoclonal antibody (T55992F), Phospho-Akt rabbit monoclonal antibody (T40067), Akt rabbit monoclonal antibody (T55561), Phospho-PI3-kinase rabbit monoclonal antibody (T40116), and PI3Kinase rabbit monoclonal antibody (T40115) were purchased from Abdominal Pharma Technology (Shanghai) Co., Ltd.

[0046] Bcl-2 rabbit monoclonal antibody (80313-1-RR) and Nephrin rabbit monoclonal antibody (22912-1-Ap) were purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0047] β-actin mouse monoclonal antibody (YM3028), horseradish peroxidase-labeled goat anti-rabbit IgG, and horseradish peroxidase-labeled goat anti-mouse IgG were purchased from Immunoway, Inc., USA.

[0048] The ECL chemiluminescence reagent kit was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0049] Skim milk powder and 4% paraformaldehyde were purchased from Wuhan Saiweier Biotechnology Co., Ltd.

[0050] 20×citric acid antigen retrieval solution (pH 6.0), 20×Tris-EDTA antigen retrieval solution (pH 9.0), 20×Tris-EDTA antigen retrieval solution (pH 8.0), PBS buffer, general tissue fixative (neutral), bovine serum albumin (BSA), normal rabbit serum (concentrated), hematoxylin staining solution, hematoxylin differentiation solution, hematoxylin blueing solution, ultra-clean fast-drying mounting adhesive, histochemistry kit DAB chromogenic agent, tissue autofluorescence quencher, DAPI staining reagent, and anti-fluorescence quenching mounting medium were purchased from Wuhan Saiweier Biotechnology Co., Ltd.

[0051] Urine protein test kits and creatinine test kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0052] The 3% hydrogen peroxide disinfectant was purchased from Shandong Anjie Gaoke Disinfection Technology Co., Ltd.

[0053] The SpectraMaxi 3X multi-functional microplate reader was purchased from Thermo Fisher Scientific, USA; the CytoFLEX flow cytometer was purchased from Beckman Coulter, Ltd.; and the chemiluminescence imaging analysis system was purchased from Bio-Rad, USA.

[0054] Unless otherwise specified, the technical processes involved are all methods known in the field, such as lentivirus-mediated SHIP1 knockdown (KD) technology, Western blotting technology, flow cytometry, etc.

[0055] Example 1

[0056] In vitro validation (rescue experiment) of AQX-1125 alleviating podocyte injury through the SHIP1 target.

[0057] 1.1 Experimental Design

[0058] To verify the protective mechanism of the SHIP1 agonist AQX-1125 on podocytes at the molecular and cellular levels, this study used MPC-5 podocytes. A stable low-expression SHIP1 strain (KD) was established in mouse kidney podocytes. The negative control used a non-targeted shRNA (shNC) constructed using the same vector. The lentivirus was constructed and packaged by Fuzhou Zaiji Biotechnology Co., Ltd. The following six experimental groups were set up:

[0059] (1) shNC+PBS negative control: Add an equal volume of PBS buffer to doxorubicin (ADR); this group is used to establish a baseline for the normal physiological state of podocytes;

[0060] (2) shNC+AQX-1125: Add SHIP1 agonist AQX-1125 (concentration of 500 nmol / L). This group is used to observe whether the drug alone has toxicity or side effects on normal podocytes under no-damage conditions.

[0061] (3) shNC+ADR: Add 0.2 μg / mL doxorubicin (ADR) to induce damage; this group is used to simulate the podocyte damage under the pathological state of FSGS;

[0062] (4) shNC+ADR+AQX-1125: While adding 0.2 μg / mL doxorubicin to induce damage, AQX-1125 at a concentration of 500 nmol / L was added for intervention; this group was used to verify the therapeutic effect of AQX-1125 on podocyte injury under the premise of normal SHIP1 expression.

[0063] (5) KD+ADR: This group represents the addition of 0.2 μg / mL doxorubicin in the SHIP1 knockdown state. This group aims to observe whether podocyte injury will be further aggravated in the absence of endogenous negative regulator SHIP1.

[0064] (6) KD+ADR+AQX-1125: Under the background of SHIP1 knockdown, repeat the ADR injury and AQX-1125 intervention process; by comparing the effect difference between this group and the "core efficacy group", clarify whether the renal protective effect of AQX-1125 is indeed achieved by activating the SHIP1 target. If the efficacy of this group is significantly weaker than that of group 4, it proves that the target dependence is established.

[0065] 1.2 Detection Methods and Procedures

[0066] 1.2.1 Western blot

[0067] To quantitatively analyze the expression levels of target proteins in podocytes of each experimental group, the present invention employs the following specific experimental steps:

[0068] ① Total cell protein extraction: Collect MPC-5 podocytes from each group after treatment and wash twice with pre-chilled PBS. Add RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors and lyse thoroughly on ice for 30 minutes. Centrifuge at 12,000 rpm for 15 minutes at 4°C and carefully collect the supernatant, which is the whole-cell protein extract.

[0069] ② Protein quantification (BCA method): The concentration of extracted protein was determined using a BCA kit. Based on the measurement results, the protein concentrations of each group were adjusted to be consistent using 5×SDS protein loading buffer, and then denatured at 100℃ for 10 minutes.

[0070] ③ Electrophoresis (SDS-PAGE): Prepare an SDS-PAGE gel with a concentration of 10 wt.% to 12.5 wt.% according to the molecular weight of the target protein. Add an equal amount of protein (20 μg) to each well and perform electrophoretic separation under constant pressure.

[0071] ④ Transfer and Blocking: The protein on the gel was transferred to the PVDF membrane using a wet transfer method. After the transfer was completed, the membrane was blocked in 5% skim milk on a shaker at room temperature for 1 hour to reduce non-specific binding.

[0072] ⑤ Antibody incubation: Primary antibody incubation: Add diluted primary antibody (including SHIP1, p-PI3K, PI3K, p-Akt, Akt, LC3B, P62, Bcl-2, Caspase-3, Bax and internal control β-actin, dilution ratio refers to the instructions, most are 1:1000), and incubate overnight at 4°C.

[0073] ⑥ Secondary antibody incubation: Wash the membrane three times with TBST for 10 minutes each time. Then add the corresponding species secondary antibody labeled with HRP and incubate at room temperature for 1 hour.

[0074] ⑦ Development and Band Analysis: The membrane was washed three times with TBST, then placed in a developer and developed with ECL chemiluminescent substrate. The grayscale values ​​of the target bands were measured using ImageJ image analysis software, and the relative ratio of the target protein to the internal control β-actin was calculated to evaluate the protein expression level.

[0075] Western blotting: Detects the expression of p-PI3K, PI3K, p-AKT, AKT, etc.

[0076] Autophagy flow monitoring (Western Blot): Detects the accumulation of P62 protein and the LC3BII / I ratio conversion.

[0077] Apoptosis molecular detection (Western Blot): Detection of the executive protein Caspase3, the pro-apoptotic protein Bax, and the anti-apoptotic protein Bcl-2.

[0078] 1.2.2 Flow cytometry

[0079] The flow cytometry apoptosis assay was performed according to the Annexin V-APC / 7-AAD apoptosis detection kit instructions. The specific steps are as follows:

[0080] (1) The day before the experiment, the cells were seeded in 6-well plates. The cells were expected to reach 80%-90% confluence under a microscope the next day, so that the apoptosis experiment could be carried out. The cells were induced to undergo apoptosis according to the experimental protocol.

[0081] (2) Transfer the old cell culture medium to a centrifuge tube, wash the cells once with PBS, and add 1 ml of 0.25% (w / v) trypsin (without EDTA) to digest the cells. Incubate at room temperature until the adherent cells can be gently pipetted off, then add the freshly collected cell culture medium to stop the digestion, transfer to a centrifuge tube, centrifuge at 800 rpm for 5 min, discard the supernatant, collect the cells, gently resuspend the cells in PBS, and count them.

[0082] (3) Take 1×10 5Each resuspended cell was centrifuged at 800 rpm for 5 min, the supernatant was discarded, and 500 μl of diluted 1× Annexin BVinding Buffer was added to resuspend the cells.

[0083] (4) Add 5 μl of Annexin V-APC staining solution and 5 μl of 7-AAD staining solution to the cell suspension and mix gently.

[0084] (5) After gently vortexing to mix, incubate at room temperature in the dark for 15-20 minutes. Test immediately. If testing cannot be performed in time, place on ice in the dark and complete the test within 1 hour.

[0085] (6) In addition, blank tubes and single-stain tubes need to be prepared for voltage adjustment and compensation. CytExpertforSpectral software is used for analysis to calculate the percentage of apoptotic cells.

[0086] 1.2.3 Experimental Results and Analysis

[0087] All experimental data are expressed as mean ± standard error (Mean ± SME). Statistical analysis was performed using GraphPadPrism 9.5 software. One-way ANOVA was used for comparisons among multiple groups, with P < 0.05 considered statistically significant.

[0088] The results are as follows Figures 1 to 4 As shown, Figures 1 to 4 The results show the effect of AQX-1125 on the activity of the PI3K / Akt pathway in podocytes. Among them, Figure 1 , 3 The images show Western blot electrophoresis results of the expression levels of p-PI3K, p-Akt, and their corresponding total proteins in podocytes of each group. Figure 2 , Figure 4 The figures show the quantitative statistical analysis of the corresponding protein phosphorylation levels. ADR induction (shNC+ADR group) led to an abnormally high phosphorylation level of PI3K and Akt in podocytes. In SHIP1-normal cells (shNC+ADR+AQX-1125 group), AQX-1125 significantly reduced pathway activity. In the SHIP1 knockdown background (KD+ADR+AQX-1125 group), although AQX-1125 still showed a certain inhibitory trend, its ability to suppress downstream signal overload was significantly limited compared to the normal group. This demonstrates that the "precise rate limiting" of the PI3K / Akt pathway by AQX-1125 is highly dependent on the mediation of the endogenous regulator SHIP1, and the absence of this target significantly reduces the quality of cellular response to drug-regulated signals.

[0089] Flow cytometry quantitative analysis results as follows Figure 5 and Figure 6As shown. Among them, Figure 5 Scatter plot for flow cytometry analysis of podocyte apoptosis distribution in each group; Figure 6 A bar chart showing the quantitative statistical analysis of the total apoptosis rate in each group. Figure 6 In this context, the apoptosis rate (%) refers to the percentage of apoptotic cells detected by flow cytometry out of the total cell count. From... Figure 5 and Figure 6 The results show that the total apoptosis rate in the shNC+ADR group was significantly higher than that in the control group. The shNC+ADR+AQX-1125 group exhibited the strongest rescue effect, with a significantly reduced total apoptosis rate (P<0.0001). Crucially, after SHIP1 knockdown (KD+ADR+AQX-1125 group), the ability of AQX-1125 to reduce apoptosis was significantly weakened, and its apoptosis rate remained at a high level, showing a statistically significant difference compared to the shNC+ADR+AQX-1125 group. This "significant failure of protective effect upon target loss" confirms that AQX-1125-mediated cell repair is clearly SHIP1-dependent.

[0090] Molecular expression confirms such as Figures 7 to 10 The image shows the detection results of apoptosis-related proteins in podocytes in each group. Among them, Figure 7 Western blot electrophoresis images of Caspase-3, Bax, Bcl-2 and internal control β-actin (raw bands); Figure 8 , Figure 9 , Figure 10 The following are bar charts showing the expression levels of the above proteins after normalization. Figures 7 to 10 The results show that under shNC conditions, AQX-1125 effectively reversed ADR-induced Caspase-3 and Bax activation and upregulated the Bcl-2 ratio. However, under SHIP1 knockdown (KD) conditions, although AQX-1125 maintained a protein reversion trend similar to the normal group, its binding... Figure 5 and Figure 6 Flow cytometry data on apoptosis showed that at this point, changes at the molecular level could no longer effectively translate into cellular survival gains. Specifically, the recovery of the anti-apoptotic protein Bcl-2 was significantly upregulated in the KD+ADR+AQX-1125 group, even exceeding that in the normal group. This abnormal compensatory overexpression failed to improve the final cell survival status (the apoptosis rate remained significantly higher than in the normal group), further confirming the irreplaceable role of SHIP1 as a signal regulation "waypoint": without precise regulation by SHIP1, the pathological compensation of a single anti-apoptotic molecule is insufficient to resist the comprehensive apoptosis cascade in the course of FSGS.

[0091] In the pathological progression of FSGS, podocytes are often accompanied by impaired clearance of metabolic waste. This study evaluated the effects of drugs on cellular metabolism by detecting the autophagy marker LC3B and the autophagy substrate p62. Figures 11 to 13 The results shown are the detection results of autophagy markers in podocytes for each group. Among them, Figure 11 Here are Western blot electrophoresis images of P62, LC3B, and the internal control β-actin; Figure Figure 12 , Figure 13 This is a statistical bar chart showing the corresponding protein expression levels.

[0092] from Figures 11 to 13 The results show that: In the model groups (shNC+ADR and KD+ADR groups), significant pathological accumulation of P62 protein was observed in podocytes after ADR treatment, indicating impaired autophagic degradation and inability to properly clear metabolic substrates. In the treatment groups (shNC+ADR+AQX-1125 group), significant pathological accumulation of P62 protein was also observed, indicating impaired autophagic degradation and inability to properly clear metabolic substrates. After AQX-1125 intervention (shNC+ADR+AQX-1125 and KD+ADR+AQX-1125 groups), P62 levels decreased compared to the corresponding model groups, accompanied by a conversion of LC3B-I to LC3B-II. This result indicates that AQX-1125 has a significant effect on facilitating autophagic flux and promoting the clearance of podocyte metabolic substrates. Although the SHIP1 knockdown group and the normal group showed similar improvement trends in the degradation of single metabolic indicators such as P62, the combined effect was significant. Figure 5 and Figure 6 Flow cytometry data clearly show that only when the SHIP1 target function is intact can this improvement in autophagic flux effectively translate into significant anti-apoptotic benefits. This suggests that AQX-1125 has multidimensional regulatory properties for the restoration of podocyte metabolic homeostasis, and SHIP1 is the core key factor determining whether it can ultimately achieve cell survival and repair.

[0093] Example 2

[0094] The restorative effect of AQX-1125 on renal function in a mouse FSGS model

[0095] 2.1 Experimental Modeling

[0096] Six-week-old BALB / c mice, weighing 20-22g, were purchased from the Shanghai Slack Laboratory Animal Center. Mice were housed in standard cages (6 mice per cage), with free access to food and water. The laboratory environment was maintained at 25±2℃ and humidity at 50-70%, with a 12-hour light / 12-hour dark setting. After one week of acclimatization, mice were randomly divided into three groups: a saline control group (Saline), an doxorubicin nephropathy group (ADR), and an AQX-1125 treatment group (ADR+AQX-1125), with 12 mice in each group. Mice in the doxorubicin nephropathy model were given a single intravenous injection of ADR1 (0.5 mg / kg). The treatment group received a SHIP1 agonist (AQX-1125) at a dose of 10 mg / kg via gavage, starting two weeks after modeling, administered every other day for four weeks. Mice in the Saline group received a single intravenous injection or gavage of an equal volume of saline. Six weeks after doxorubicin injection, the mice were sacrificed, and their urine, serum, and kidney tissue were collected and preserved for future use.

[0097] 2.2 Experimental Observation Indicators and Methods

[0098] At weeks 0, 2, 4, and 6, mice were placed in metabolic cages and 24-hour urine samples were collected. The inhibitory effect of AQX-1125 on massive proteinuria was observed according to the instructions of the urine protein assay kit and creatinine assay kit.

[0099] 2.2.1 Specific steps for detecting urinary protein concentration (CBB method): This experiment used the Coomassie Brilliant Blue (CBB) method to quantitatively detect urinary protein concentration in each group of mice. The specific operating procedure is as follows:

[0100] ① Reagent preparation and instrument configuration

[0101] Reagent preparation: Before use, mix CBB reagent with distilled water at a volume ratio of 1:4 to prepare CBB application solution. Prepare fresh solution before use.

[0102] Preparation of standard: Use protein standard solution with a concentration of 524 mg / L.

[0103] Instrument preparation: Use a microplate reader with a wavelength of 595nm.

[0104] ② Measurement Operation Procedure

[0105] Add the corresponding reagents to the test tubes or 96-well plates according to Table 1 below:

[0106] Table 1. Reagents Added

[0107]

[0108] Mixing and color development: After adding the reagents, mix thoroughly using a vortex mixer and let stand at room temperature for 5 minutes. Pipette 200 μL from each of the blank tube, standard tube, and test tube into a 96-well plate and read the results using a microplate reader (wavelength 595 nm).

[0109] ③ Result Calculation and Sample Processing

[0110] Calculation formula: Urine protein concentration (mg / L) = (standard tube OD − blank tube OD) / test tube (OD − blank tube OD) × 524 mg / L × sample dilution factor.

[0111] 2.2.2 Specific Procedures for Detecting Urinary Creatinine Concentration (Crocrine Oxidase Method) This experiment uses the creatine oxidase method (microplate method) to quantitatively determine the creatinine content in mouse urine. The specific procedure is as follows:

[0112] ① Reagent Preparation and Sample Pretreatment: Reagent composition: Reagent 1 (enzyme solution A), Reagent 2 (enzyme solution B), Reagent 3 (442 μmol / L standard). Sample Dilution: Before testing, dilute the urine sample 2-10 times with physiological saline (depending on the conditions of the preliminary experiment). The final result needs to be multiplied by the corresponding dilution factor. Instrument Preparation: Microplate reader (wavelength set to 546 nm), 37℃ incubator.

[0113] Using a 96-well plate, add reagents in the order shown in Table 2 below:

[0114] Table 2. Reagents Added

[0115]

[0116] Note: Before measurement, read the empty plate OD value at 546nm. After the experiment, when calculating, the values ​​of A1 and A2 must be subtracted from the corresponding empty plate OD values ​​before they can be substituted into the calculation formula; K is the dilution factor, and the value is: K = (sample volume + enzyme solution A volume) / (sample volume + enzyme solution A volume + enzyme solution B volume) = 155 / 205.

[0117] ③ Calculate the absorbance change for each well:

[0118] Creatinine content (umol / L) = (∆determined - ∆blank) / (∆standard - ∆blank) * C 标准 ;

[0119] Finally, the urine protein / creatinine ratio (UACR) was calculated, in mg / mg.

[0120] 2.2.3 Renal function assessment

[0121] Urinary protein repair: Figure 14This is a bar chart showing the results of the urinary albumin / creatinine ratio (UACR) in each group of mice. Data are presented as mean ± standard error (Mean ± SME), n=12. Figure 14 As shown, compared with the saline control group, ADR mice showed increased urinary protein excretion and significantly elevated urinary albumin / creatinine ratio (UACR) levels after modeling (P < 0.0001). After treatment with AQX-1125, at week 4 (i.e. week 6) after treatment, the UACR of the ADR+AQX-1125 group mice showed a significant downward trend compared with the ADR model group (P = 0.0001).

[0122] Example 3

[0123] AQX-1125's protective effect on renal histopathology and podocyte ultrastructure

[0124] 3.1 The experimental modeling and grouping are the same as in Example 2.

[0125] 3.2 Experimental Procedure

[0126] 3.2.1 HE staining

[0127] ① Dewaxing paraffin sections to water: Immerse the sections sequentially in environmentally friendly dewaxing solution I for 20 min, environmentally friendly dewaxing solution II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, then rinse with tap water. Frozen sections are then thawed and fixed: Remove the frozen sections from the -20℃ freezer and allow them to reach room temperature. Fix with tissue fixative for 15 min, then rinse with running water.

[0128] ②Pretreatment: The sections were immersed in high-resolution constant staining pretreatment solution for 1 min.

[0129] ③ Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, blue with blueing solution, and rinse with running water.

[0130] ④ Eosin staining: Dehydrate the sections in 95% alcohol for 1 minute, then stain them in eosin staining solution for 15 seconds.

[0131] ⑤ Dehydration and mounting: The sections are sequentially immersed in anhydrous ethanol I for 2 min, anhydrous ethanol II for 2 min, anhydrous ethanol III for 2 min, n-butanol I for 2 min, n-butanol II for 2 min, xylene I for 2 min, and xylene II for 2 min, and then cleared and mounted with neutral resin.

[0132] 3.2.2 PAS staining

[0133] ① Dewaxing paraffin sections to water: Immerse the sections in environmentally friendly dewaxing solution I for 20 minutes, environmentally friendly dewaxing solution II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and 75% alcohol for 5 minutes, then rinse with tap water.

[0134] ② Stain the sections in PAS staining solution B for 10-15 minutes, then wash with tap water and distilled water twice.

[0135] ③Immerse the sections in PAS staining solution A for 25-30 minutes, protect from light, and rinse with running water for 5 minutes;

[0136] ④ Stain the sections with PAS staining solution C for 30 seconds, wash with tap water, differentiate with hydrochloric acid solution, wash with tap water, turn blue with ammonia solution, and rinse with running water.

[0137] ⑤ Dehydration and mounting: The sections are sequentially immersed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene for 5 min, and xylene II for 5 min, and then cleared and mounted with neutral resin.

[0138] 3.2.3 Immunofluorescence staining

[0139] ① Dewaxing paraffin sections to water: Immerse the sections in environmentally friendly dewaxing solution I for 10 min, environmentally friendly dewaxing solution II for 10 min, environmentally friendly dewaxing solution III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and then wash with distilled water.

[0140] ② Antigen retrieval: Place the slide in EDTA 8.0 for heat retrieval for 30 minutes. After cooling the retrieval slide, place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time.

[0141] ③ Circle blocking with serum: After slightly drying the tissue section, draw a circle around the tissue with a histochemical pen, add BSA, and block for 30 minutes.

[0142] ④ Add the mixed reagent of the first and second primary antibodies: Mix the two primary antibodies from different sources, add the prepared primary antibody dropwise, and incubate the slices in a humidified chamber at 4°C overnight.

[0143] ⑤ Add secondary antibody: Place the slide in PBS (pH 7.4) and wash three times on a decolorizing shaker for 5 minutes each time. Add the corresponding secondary antibody and incubate at room temperature in the dark for 50 minutes.

[0144] ⑥ Place the slide in PBS (pH 7.4) and wash three times on a decolorizing shaker for 5 minutes each time. Add DAPI staining solution and incubate at room temperature in the dark for 10 minutes.

[0145] ⑦ Quenching tissue autofluorescence: Place the slide in PBS (pH 7.4) and wash three times on a decolorizing shaker, 5 minutes each time. Add autofluorescence quencher solution B for 5 minutes, then rinse with running water for 10 minutes.

[0146] ⑧ Mounting: Mount the slide with anti-fluorescence quenching mounting medium.

[0147] ⑨ Image acquisition: DAPI excitation wavelength 330~380nm, emission wavelength 420nm; 488 excitation wavelength 465~495nm, emission wavelength 515~555nm; CY excitation wavelength 510~560nm, emission wavelength 590nm; CY5 excitation wavelength 608~648nm, emission wavelength 672~712nm.

[0148] 3.3 Improvement in pathological morphology

[0149] Figure 15 Photomicrographs of HE-stained and PAS-stained kidney tissues from each group of mice. Pathological morphological analysis revealed ( Figure 15 The saline control group (Saline) showed clear renal cortical structure, regular glomerular morphology, no mesangial matrix proliferation, and no abnormal exudate in the renal tubules. The doxorubicin group (ADR) exhibited typical and severe FSGS-like pathological damage. Glomeruli showed significant segmental sclerosis and capillary loop collapse; PAS staining revealed significant thickening of the mesangial area and accumulation of basement membrane-like material. Particularly noteworthy was the presence of numerous homogeneous, pinkish protein casts filling the renal tubules due to severe damage to the filtration barrier. The ADR+AQX-1125 group showed significant improvement in the aforementioned lesions. Glomerular sclerosis was significantly reduced, capillary loop compression was alleviated, and the number of protein casts in the renal tubules was greatly reduced.

[0150] 3.4 Physical Correlation of Stabilized Crack Diaphragm (SD)

[0151] Figure 16 This image shows the spatial colocalization of SHIP1 and Nephrin in the glomeruli of mice in each group, stained with immunofluorescence. Immunofluorescence double staining is the first demonstration that AQX-1125 can restore the spatial colocalization of SHIP1 and Nephrin within the glomerulus. This effect directly stabilizes the physical association of the podocyte cleft septum complex, overcoming the bottleneck of existing hormone therapies in repairing the physical structure of podocytes.

[0152] 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 SHIP1 agonist AQX-1125 in the preparation of drugs for the treatment of focal segmental glomerulosclerosis.

2. The application according to claim 1, characterized in that, The drug is in the form of an oral formulation, AQX-1125.

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