Construction method and application of renal fibrosis model

Abstract

The invention discloses a construction method and application of a renal fibrosis model. The invention provides a fibrosis promoting effect of a TRPM3 agonist CIM0216, and human pluripotent stem cells are induced to be differentiated into kidney organs through active ingredients such as CHIR-99021, FGF9 and heparin; the process relates to accurate regulation and control of cell fate determinants so as to simulate a natural path of kidney development; further, a fibrosis promoting drug CIM0216 is introduced to induce fibrosis reaction in kidney organs, and a reliable renal fibrosis model is created; the method can be used for evaluating and screening potential anti-fibrosis treatment drugs. The model not only can simulate the disease progress, but also can accelerate the research and development process of new drugs, and provides a more effective strategy for clinical treatment.

Description

Technical Field

[0001] The present invention relates to a method for constructing a disease model, and in particular to a method for constructing a renal fibrosis model and an application thereof. Background Art

[0002] Renal fibrosis refers to the abnormal proliferation and deposition of collagen fibers in renal tissue, leading to the gradual loss of renal structure and function. This process is usually caused by damage to renal tubular epithelial cells and inflammatory response. Inflammatory factors stimulate fibroblast activation, excessive production of collagen and other matrix components, and thus cause proliferation of renal interstitial and peritubular connective tissue. Renal fibrosis may be caused by a variety of factors, including trauma, infection, inflammation, blood circulation disorders, and immune response and other pathogenic factors, leading to damage to intrinsic cells. In the later stages, a large amount of collagen deposition and accumulation occurs, causing the renal parenchyma to gradually harden and form scars until the kidney completely loses its organ function.

[0003] Establishing an appropriate renal fibrosis model is the primary task in developing treatment options. Currently, the main methods for renal fibrosis modeling are:

[0004] ①Unilateral Ureteral Obstruction (UUO) model is to block the flow of urine by ligating one side of the rat's ureter, thereby causing tubulointerstitial damage and fibrosis. This model can quickly produce renal interstitial fibrosis and is suitable for studying the pathogenesis and treatment of renal fibrosis.

[0005] ② Cyclosporine A-induced model: Cyclosporine A (CsA) is an immunosuppressant, and long-term use can lead to nephrotoxicity and renal interstitial fibrosis. Subcutaneous injection of CsA can induce a similar renal fibrosis process in animals. This model is mainly used to study the mechanism of renal fibrosis caused by CsA and to find new drugs to prevent CsA nephrotoxicity.

[0006] ③Gentamicin-induced model: Gentamicin is an antibiotic that can induce tubulointerstitial fibrosis by injection. This model can simulate drug-induced renal damage and is used to study the mechanism of drug-induced nephropathy.

[0007] ④Unilateral renal vein ligation model: By ligating one side of the rat's renal vein, the blood supply to the kidney can be blocked, leading to ischemia and hypoxia of the tubular interstitium, and then inducing fibrosis. This model is suitable for studying the pathogenesis of ischemic nephropathy.

[0008] Each of these models has its own advantages and disadvantages, and the choice of model depends on the purpose and specific requirements of the study. For example, although the UUO model is simple and easy to perform, it is an acute mechanical injury and may not be suitable for all types of tubular and interstitial injuries and fibrosis. Although the cyclosporine A-induced model can simulate the real nephrotoxic process, it is expensive and has liver toxicity, which limits its scope of application.

[0009] Moreover, experimental animal models play an important role in medical research, but they also have some limitations, including:

[0010] ① Species differences: Although animal models play an indispensable role in the study of human diseases and drugs, due to species differences, the responses of animal models may not be exactly the same as the clinical manifestations of human diseases and syndromes.

[0011] ②Reliability of the model: Animal models may not be able to fully simulate the complexity of human diseases, such as the genetic factors, pathophysiological mechanisms, and connections between diseases.

[0012] ③ Ethical and moral issues: When using animal models for experimental research, animal welfare and the avoidance of unnecessary suffering must be considered. This means that when conducting animal experiments, ethical guidelines and animal protection regulations must be followed to ensure that the experiments are conducted within the scope of ethics and law.

[0013] ④ Extrapolation of experimental results: The results obtained through animal models may not be reliable when extrapolated to other species, especially in the safety testing of drugs and chemicals. In addition, it may also be unreliable to confirm certain theories in biomedicine through animal experiments.

[0014] ⑤ Complexity and cost of the experiment: The construction process of some animal models is complicated and requires a large number of animals and resources. For example, the chronic social frustration model requires the use of a large number of animals, resulting in the consumption of experimental facilities and human resources.

[0015] ⑥ Misleading experimental results: Some abnormal phenomena observed in animal experiments may be normal biological characteristics of the test animal species, or may be changes caused by unnatural means, or due to stress reactions in the laboratory environment. Such abnormalities may have nothing to do with human pathological changes.

[0016] ⑦ Repeatability and consistency of experiments: The experimental results of animal models may be affected by multiple factors such as individual differences, experimental conditions, and feeding management, making it difficult to ensure the repeatability and consistency of the experimental results.

[0017] ⑧ Experimental alternatives: Although animal models are indispensable in some studies, alternative methods should be sought when possible, such as the use of in vitro cell models, computer simulations, etc., to reduce dependence on animals.

[0018] Kidney organoids are three-dimensional constructs composed of multiple cell types that simulate real human kidneys in structure and function. These models can not only be used for experiments in an in vitro environment, avoiding the ethical issues of animal models, but can also be used quickly and easily to conduct experiments, accelerating the research process. In recent years, research on kidney organoids has made significant progress. Researchers have been able to culture organoids with certain kidney functions in vitro. These organs contain nephron-like structures such as glomeruli, renal tubules, and collecting ducts. These organoids not only show potential in constructing kidney disease models, but can also be used for drug screening and evaluating drug nephrotoxicity. Summary of the invention

[0019] The purpose of the first aspect of the present invention is to provide the use of a TRPM3 agonist in constructing a fibrosis model or preparing a product for constructing a fibrosis model.

[0020] The second aspect of the present invention aims to provide the use of a TRPM3 agonist in constructing a renal tubular injury model or in preparing a product for constructing a renal tubular injury model.

[0021] The third aspect of the present invention aims to provide a method for constructing a renal fibrosis and / or renal tubular injury model.

[0022] The fourth aspect of the present invention is directed to a model of renal fibrosis and / or tubular injury.

[0023] The purpose of the fifth aspect of the present invention is to provide an application of the renal fibrosis and / or tubular injury model of the fourth aspect of the present invention.

[0024] The sixth aspect of the present invention aims to provide a product for constructing a renal fibrosis and / or tubular injury model.

[0025] The seventh aspect of the present invention aims to provide a method for constructing kidney organoids.

[0026] The technical solution adopted by the present invention is:

[0027] The first aspect of the present invention provides the use of a TRPM3 agonist in constructing a fibrosis model or preparing a product for constructing a fibrosis model.

[0028] In some embodiments of the invention, the fibrosis is renal fibrosis.

[0029] In some embodiments of the present invention, the TRPM3 agonist includes CIM0216 or a pharmaceutically acceptable salt thereof.

[0030] The second aspect of the present invention provides the use of a TRPM3 agonist in constructing a renal tubular injury model or preparing a product for constructing a renal tubular injury model.

[0031] In some embodiments of the present invention, the renal tubular injury is renal organoid tubular injury

[0032] In some embodiments of the present invention, the TRPM3 agonist includes CIM0216 or a pharmaceutically acceptable salt thereof.

[0033] The third aspect of the present invention provides a method for constructing a renal fibrosis and / or tubular injury model, comprising the following steps: using pro-fibrotic drugs to induce renal organoids to form renal organoid fibrosis and / or tubular injury.

[0034] In some embodiments of the present invention, the profibrotic drug is a TRPM3 agonist; the TRPM3 agonist includes CIM0216 or a pharmaceutically acceptable salt thereof.

[0035] In some embodiments of the present invention, the working concentration of the TRPM3 agonist is 50-400 μM.

[0036] In some embodiments of the present invention, the induction time is 24 to 72 hours.

[0037] In some embodiments of the present invention, the method for constructing the kidney-like organ comprises the following steps:

[0038] S1: culturing human pluripotent stem cells in a first culture medium for 3 to 6 days;

[0039] S2: replacing the first culture medium with the second culture medium and culturing for 1 to 3 days;

[0040] S3: replacing the second culture medium with the first culture medium and culturing for 0.5 to 2 hours;

[0041] S4: replacing the first culture medium with the second culture medium and culturing for 4 to 6 days;

[0042] S5: Replace the third culture medium with the basal culture medium and culture in suspension for 2 to 8 days to obtain kidney organoids.

[0043] In some embodiments of the present invention, the first culture medium comprises a GSK-3 inhibitor; and the second culture medium comprises heparin and fibroblast growth factor.

[0044] In some embodiments of the present invention, the working concentration of the GSK-3 inhibitor is 2 to 16 μM.

[0045] In some embodiments of the present invention, the GSK-3 inhibitor comprises at least one of B216763, TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, and CHIR-99021.

[0046] In some embodiments of the present invention, the working concentration of heparin is 0.5-10 μg / mL.

[0047] In some embodiments of the present invention, the working concentration of the fibroblast growth factor is 100-300 ng / mL.

[0048] In some embodiments of the present invention, the fibroblast growth factor is fibroblast growth factor-9.

[0049] In the present invention, the method for constructing the kidney organoid is not particularly limited, and the kidney organoids obtained by conventional methods in the art all fall within the protection scope of the present invention.

[0050] The fourth aspect of the present invention provides a renal fibrosis and / or tubular injury model constructed by the method of the third aspect of the present invention.

[0051] The fifth aspect of the present invention provides the use of the renal fibrosis and / or tubular injury model described in the third aspect of the present invention in the development and screening of drugs for treating renal fibrosis and / or tubular diseases.

[0052] In a sixth aspect, the present invention provides a product for constructing a renal fibrosis and / or tubular injury model, the product comprising a renal organoid culture medium and a profibrotic drug; the profibrotic drug is a TRPM3 agonist.

[0053] In some embodiments of the present invention, the kidney organoid culture medium comprises a first culture medium and a second culture medium, wherein the first culture medium comprises a GSK-3 inhibitor, and the second culture medium comprises heparin and fibroblast growth factor.

[0054] In some embodiments of the present invention, the working concentration of the GSK-3 inhibitor is 2 to 16 μM.

[0055] In some embodiments of the present invention, the GSK-3 inhibitor comprises at least one of B216763, TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, and CHIR-99021.

[0056] In some embodiments of the present invention, the working concentration of heparin is 0.5-10 μg / mL.

[0057] In some embodiments of the present invention, the working concentration of the fibroblast growth factor is 100-300 ng / mL.

[0058] In some embodiments of the present invention, the fibroblast growth factor is fibroblast growth factor-9.

[0059] A seventh aspect of the present invention provides a method for constructing a kidney organoid, comprising the following steps:

[0060] S1: Human pluripotent stem cells are cultured in suspension into a cluster;

[0061] S2: Replace the medium containing GSK-3 inhibitor and culture the cell clusters in suspension for 3 to 6 days;

[0062] S3: Replace the culture medium containing fibroblast growth factor and heparin, and continue to suspend and culture the cell clusters for 1 to 3 days;

[0063] S4: Replace the medium containing GSK-3 inhibitor and continue to suspend and culture the cell clusters for 0.5 to 2 hours;

[0064] S5: Replace the culture medium containing fibroblast growth factor and heparin, and continue to culture the cell clusters in suspension for 3 to 7 days;

[0065] S6: Continue to suspend and culture the cell clusters in basal medium for at least 2 days to obtain kidney organoids.

[0066] In some embodiments of the present invention, the working concentration of the GSK-3 inhibitor is 2 to 16 μM;

[0067] In some embodiments of the present invention, the GSK-3 inhibitor comprises at least one of B216763, TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, and CHIR-99021.

[0068] In some embodiments of the present invention, the working concentration of heparin is 0.5-10 μg / mL.

[0069] In some embodiments of the present invention, the working concentration of the fibroblast growth factor is 100-300 ng / mL.

[0070] In some embodiments of the present invention, the fibroblast growth factor is fibroblast growth factor-9.

[0071] In some embodiments of the present invention, the suspension culture is performed by placing a culture vessel carrying the cell clusters in a shaker for suspension culture.

[0072] Kidney organoid modeling has many advantages in kidney disease research and drug development, including:

[0073] 1) Simulating the human kidney environment: Kidney organoids can simulate the structure and partial functions of the kidney in an in vitro environment, which helps researchers better understand the pathogenesis and treatment response of kidney diseases.

[0074] 2) Personalized disease models: By using the patient’s own cells to establish kidney organoids, personalized disease models can be created, which is particularly important for studying the disease characteristics and treatment effects in specific patient populations.

[0075] 3) Reduce animal experiments: The kidney organoid model provides an alternative to traditional animal models, reducing dependence on animals and reducing ethical controversy.

[0076] 4) Drug screening and toxicity testing: Kidney organoids can be used to screen drugs for the treatment of kidney diseases and detect drug nephrotoxicity, which is crucial for drug development and safety assessment.

[0077] 5) High-throughput screening: The culture and analysis of kidney organoids can be achieved with high throughput, which is very useful for rapidly screening a large number of potential therapeutic drugs or testing the nephrotoxicity of drugs.

[0078] 6) Long-term stability: Some studies have shown that kidney organoids can be expanded and cultured stably in vitro for a long time, which provides the possibility for long-term research and drug testing.

[0079] 7) Potential in regenerative medicine: Although not yet clinically used, kidney organoids have potential applications in the field of regenerative medicine, such as as a source of kidneys for transplantation.

[0080] 8) Diversity of disease models: Kidney organoids can be used to construct models of a variety of kidney diseases, including genetic diseases, infectious diseases, and cancer, which helps to fully understand the complexity of these diseases.

[0081] Kidney organoid modeling shows significant advantages and potential in kidney disease research, drug development, toxicology evaluation, and future regenerative medicine.

[0082] The beneficial effects of the present invention are:

[0083] The present invention provides the profibrotic effect of TRPM3 agonist CIM0216, and also provides a renal fibrosis model constructed based on TRPM3 agonist CIM0216, specifically inducing human pluripotent stem cells (hPSCs) to differentiate into renal organoids through active ingredients such as CHIR-99021, FGF9, heparin, etc.; this process involves precise regulation of cell fate determining factors to simulate the natural path of renal development; then, by introducing profibrotic drugs to induce fibrotic reactions in renal organoids, a reliable renal fibrosis model is created; it can be used to evaluate and screen potential anti-fibrotic therapeutic drugs.

[0084] In the process of preparing kidney organoids, the whole process is suspension culture, which only requires the use of conventional culture dishes / culture bottles, etc. It is simple to operate, lower cost, and has a large output. There is no need to use transwell cell culture plates, low-adsorption 96-well plates, etc.

[0085] This model can not only simulate disease progression, but also accelerate the development of new drugs and provide more effective strategies for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 This is the differentiated mature kidney organoid in Example 1; scale bar: 500 μm.

[0087] Figure 2 This is the differentiated mature kidney organoid of Example 2; scale bar: 500 μm.

[0088] Figure 3 This is the expression of fibrosis-related genes in kidney organoids after administration of CIM0216 in Example 1.

[0089] Figure 4 The immunofluorescence results of kidney organoids after administration of CIM0216 in Example 2. The blue color is DAPI staining, representing the cell nucleus, the green color LTL represents the renal tubule, and the red color FN1 represents fibronectin. The scale bar is 100 μm.

[0090] Figure 5 The results of hematoxylin-eosin (HE) staining of kidney organoids in Example 3. The scale bar is 500 μm. DETAILED DESCRIPTION

[0091] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0092] Example 1 Construction of kidney organoids

[0093] Human pluripotent stem cells were cultured in suspension into cell clusters on an orbital shaker at 40-80 rpm in a constant temperature and humidity cell culture incubator. The culture medium was refreshed daily with PSC culture medium (Thermo Fisher Scientific, Inc., TM ).

[0094] After the cells are clustered, use a mesh to screen out cell clusters with a diameter of less than 50μm. The cell clusters are transferred to APEL2 medium containing CHIR99021 (working concentration is 2μM) and suspended for 6 days, with the medium changed once a day. Then replace the APEL2 medium containing FGF9 (100ng / mL) and heparin (1μg / mL) to continue to suspend and culture the cell clusters for 2 days. Then replace the APEL2 medium containing CHIR99021 (2μM) and culture the cell clusters for 1.5h. Then replace the APEL2 containing FGF9 (100ng / mL) and heparin (0.5μg / mL) and continue to suspend and culture for 5 days. Then replace the APEL2 medium and continue to suspend and culture the cell clusters for 5 days to obtain kidney organoids ( Figure 1 ).

[0095] Example 2 Construction of kidney organoids

[0096] Human pluripotent stem cells were cultured in suspension into cell clusters on an orbital shaker at 40-80 rpm in a constant temperature and humidity cell culture incubator. The culture medium was refreshed daily with PSC culture medium (Thermo Fisher Scientific, Inc., TM ).

[0097] After the cells are clustered, use a mesh to screen out cell clusters with a diameter of less than 50μm. Transfer the cell clusters to APEL2 medium containing CHIR99021 (working concentration is 16μM) and culture them in suspension for 3 days, changing the medium once a day. Then replace the APEL2 medium containing FGF9 (300ng / mL) and heparin (10μg / mL) to continue to suspend and culture the cell clusters for 2 days. Then replace the APEL2 medium containing CHIR99021 (12μM) and culture the cell clusters for 1h. Then replace the APEL2 containing FGF9 (300ng / mL) and heparin (5μg / mL) and continue to suspend and culture for 6 days. Then replace the APEL2 medium and continue to suspend and culture the cell clusters for 2 days to obtain kidney organoids ( Figure 2 ).

[0098] Example 3 Renal organoid fibrosis modeling

[0099] The kidney organoids obtained in Example 1 were transferred to an ultra-low adsorption 96-well plate (U bottom), with one kidney organoid per well. 200 μL APEL2 medium was added to each well of the control group, and the same volume of fresh medium containing 50 μM CIM0216 (CAS No.: 1031496-06-6) (MedChemEepress) was added to the modeling group, and cultured for 72 hours. After 72 hours, kidney organoid fibrosis samples were collected.

[0100] Example 4 Renal Organoid Fibrosis Modeling

[0101] The kidney organoids obtained in Example 2 were transferred to an ultra-low attachment 96-well plate (U bottom), with one kidney organoid per well. 200 μL APEL2 medium was added to each well of the control group, and the same volume of APEL2 medium containing 400 μM CIM0216 (CAS No.: 1031496-06-6) was added to the modeling group for 24 h. After 24 h, kidney organoid fibrosis samples were collected.

[0102] Example 5 Detection of kidney organoid fibrosis model

[0103] 1. RT-qPCR

[0104] Collect the kidney organoids (control group and modeling group) of Example 3 with EP tubes and wash once with DPBS. Add 1 mL trizol (ThermoFisher) to dissolve the cells. Add 200 μL chloroform, shake for 15 seconds, and let stand for 2 min. Centrifuge at 4°C, 12000 rpm×15 min, and take the supernatant. Add the same volume of isopropanol to the supernatant and mix it upside down. Place at -20°C for 10 min. Centrifuge at 12000 rpm 4°C for 10 min and discard the supernatant. Add 500 μL of pre-cooled anhydrous ethanol to the precipitate, blow off the precipitate to wash RNA, wash twice, and centrifuge at 12000 rpm 4°C and discard the supernatant. Dry the precipitate at room temperature for about 5 min. Add 20 μL DPEC water to the centrifuge tube to dissolve the RNA. Synthesize cDNA with RevertAid FirstStrand cDNA Synthesis Kit according to the instructions, and cDNA is used for RT-qPCR detection.

[0105] The results showed that after administration of CIM0216, the fibrosis-related gene FN1 in the organoids was significantly increased, and the kidney injury-related molecule KIM-1 was also significantly increased ( Figure 3 ).

[0106] 2. Immunofluorescence

[0107] The kidney organoids (control group and modeling group) of Example 4 were outsourced to make paraffin sections, and then the following steps were performed:

[0108] ① Place the slices in a 60℃ oven for 1.5h.

[0109] ② Place the slices into xylene I, II, and III in turn, soaking for 10 minutes each.

[0110] ③ Place the slices in 100% alcohol for 10 minutes, then soak them in 95%, 80%, 70% alcohol and pure water for 5 minutes.

[0111] ④ Dilute the antigen repair solution in proportion, put the slices into the repair solution, put them into the microwave, and heat them on medium heat for 8 minutes, stop the heat for 8 minutes, and then on medium-low heat for 7 minutes.

[0112] ⑤ After the antigen repair solution used to soak the slices cools down to room temperature, take out the slices, rinse them in PBS, wipe them dry, and circle the tissue slices with an immunohistochemistry pen.

[0113] ⑥ Add 10% donkey serum + 0.1% Triton X-100 blocking solution into the circle and incubate at room temperature for 30 minutes.

[0114] ⑦Discard the blocking solution and add the primary antibody diluted in proportion (anti-FN1 antibody, Boster, BA1772, 1:200; LTL lectin, Vector lab, B1325-2, 1:200) and incubate at 4°C overnight.

[0115] ⑧The next day, soak the slices in PBS and wash them on a shaker for 15 minutes.

[0116] ⑨Take out the slices, add the secondary antibody diluted in proportion, and incubate at room temperature in the dark for 1 hour. (Thermo Fisher, A11012, 1:1000; Vector lab, SA-5488-1, 1:200)

[0117] ⑩Discard the secondary antibody, add 1 μg / mL DAPI (Sigma Aldrich), and stain the nucleus for 5 minutes.

[0118] Discard DAPI, soak the slices in PBS, and wash on a shaker for 15 minutes.

[0119] Take out the slices, wipe the area around the tissue dry, add a drop of sealing agent, and seal the slices with a coverslip.

[0120] Microscopic examination.

[0121] The results showed that after administration of CIM0216, the expression of FN1 in the renal organoids of the modeling group was significantly increased, while the LTL staining signal characterizing the renal tubules decreased and the arrangement of renal tubular epithelial cells disappeared ( Figure 4 ).

[0122] 3. HE staining

[0123] The kidney organoids (control group and modeling group) of Example 3 were outsourced to make paraffin sections, and then stained according to the operating instructions of H&EStaining Kit (Hematoxylin and Eosin).

[0124] Results: In the CIM0216-administered modeling group, renal tubular epithelial cells were lost and some renal tubular structures collapsed ( Figure 5 ).

[0125] Combined with immunofluorescence, qPCR and HE staining structures, CIM0216 can cause fibrosis in kidney organoids and cause certain damage to the renal tubules.

[0126] The above specific implementations have been described in detail for the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of a TRPM3 agonist in constructing a fibrosis model or preparing a product for constructing a fibrosis model; preferably, the fibrosis is renal fibrosis.

2. The use of TRPM3 agonists in constructing a renal tubular injury model or preparing products for constructing a renal tubular injury model; Preferably, the renal tubular injury is renal tubular injury in a kidney organoid.

3. The use according to claim 1 or 2, characterized in that: The TRPM3 agonist includes CIM0216 or a pharmaceutically acceptable salt thereof.

4. A method for constructing a renal fibrosis and / or renal tubular injury model, comprising the following steps: Renal organoid formation using profibrotic drugs to induce fibrosis and / or tubular injury in renal organoids.

5. The construction method according to claim 4, characterized in that: The profibrotic drug is a TRPM3 agonist; the TRPM3 agonist includes CIM0216 or a pharmaceutically acceptable salt thereof; Preferably, the working concentration of the TRPM3 agonist is 50-400 μM.

6. A renal fibrosis and / or renal tubular injury model, wherein the renal fibrosis and / or renal tubular injury model is constructed by the construction method described in claim 4 or 5.

7. Use of the renal fibrosis and / or tubular injury model according to claim 6 in developing and screening drugs for treating renal fibrosis and / or tubular diseases.

8. A product comprising a kidney organoid culture medium and a profibrotic drug; the profibrotic drug is a TRPM3 agonist.

9. The product according to claim 8, characterized in that The TRPM3 agonist includes CIM0216 or a pharmaceutically acceptable salt thereof; preferably, the working concentration of the TRPM3 agonist is 50-400 μM.

10. A method for constructing a kidney organoid, comprising the following steps: S1: Human pluripotent stem cells are cultured in suspension into a cluster; S2: Replace the medium containing GSK-3 inhibitor and culture the cell clusters in suspension for 3 to 6 days; S3: Replace the culture medium containing fibroblast growth factor and heparin, and continue to suspend and culture the cell clusters for 1 to 3 days; S4: Replace the medium containing GSK-3 inhibitor and continue to suspend and culture the cell clusters for 0.5 to 2 hours; S5: Replace the culture medium containing fibroblast growth factor and heparin, and continue to culture the cell clusters in suspension for 3 to 7 days; S6: Continue to suspend and culture the cell clusters in basal medium for at least 2 days to obtain kidney organoids.

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