A human-derived renal tubular epithelial cell organoid-based diabetic nephropathy pathological model and a construction method and application thereof

Abstract

The application discloses a diabetic nephropathy pathological model based on human renal tubular epithelial cell organoids and a construction method and application thereof, and belongs to the field of biomedical technology. The construction method comprises the following steps: jointly inducing the renal tubular epithelial cell organoids in a culture medium containing glucose, advanced glycation end products and tumor necrosis factor alpha to prepare the diabetic nephropathy pathological model. The diabetic nephropathy pathological model is constructed by joint induction, the model shows the core pathological phenotypes of diabetic nephropathy, including down-regulation of renal tubular epithelial cell marker expression, up-regulation of fibrosis marker expression, thickening of the basement membrane and abnormal albumin uptake function, the construction method is simple and standardized, the construction period is short, the model has good repeatability and stability, the model has wide application value in high-throughput screening of traditional Chinese medicine monomers or compound medicines with kidney protection, and provides a new platform for mechanism research and treatment strategy development of diabetic nephropathy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids, its construction method, and its application. Background Technology

[0002] Diabetic nephropathy, one of the most serious and common microvascular complications of diabetes, is a leading cause of end-stage renal disease, placing a heavy burden on global public health systems. Its pathological features are complex, including glomerular sclerosis, tubulointerstitial fibrosis, inflammatory cell infiltration, and progressive loss of renal function. Although controlling blood glucose and blood pressure can slow disease progression to some extent in clinical practice, effective treatments to reverse or cure diabetic nephropathy remain extremely limited.

[0003] Currently, research on diabetic nephropathy heavily relies on animal models, such as rodent models induced by streptozotocin combined with a high-fat diet. However, these models have significant limitations: firstly, the modeling process takes months to a year, resulting in high time and economic costs; secondly, due to significant species differences between humans and rodents in kidney anatomy, gene expression profiles, and immune systems, animal experimental results often cannot directly and accurately predict the efficacy and safety of drugs in humans, leading to low clinical translation success rates. Traditional two-dimensional cell culture models, such as immortalized human renal tubular epithelial cell lines, while simple to operate, cannot simulate the complex three-dimensional structure, intercellular interactions, and complete physiological functions of in vivo kidney tissue, greatly limiting their application value in in-depth research on pathological mechanisms and accurate assessment of drug efficacy and toxicity.

[0004] In recent years, organoid technology, as a revolutionary biotechnology, has brought new hope to disease modeling. Organoids are three-dimensional micro-organs formed in vitro using stem cells, capable of highly mimicking the cell types, spatial structures, and some physiological functions of the source organ. However, how to generate highly mature and stable renal tubular epithelial organoids from human renal tubular tissue, and on this basis, accurately reproduce the key pathological features of diabetic nephropathy (such as renal tubular epithelial cell damage, inflammation, fibrosis, and functional disorders) through controlled pathological stimulation, thereby constructing a standardized pathological model that can reliably be used for drug screening, remains a critical technical challenge in this field. Especially when applying such models to the research of traditional Chinese medicines with complex components and diverse mechanisms of action, a reliable platform that reflects the complexity of the disease is needed to verify their multi-target effects. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids, along with its construction method and application, in order to solve the problems of the lack of existing organoid models for diabetic nephropathy and the difficulty in accurately reproducing the key pathological features of diabetic nephropathy.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the present invention provides a method for constructing a pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids, comprising the following steps: A pathological model of diabetic nephropathy was obtained by co-inducing renal tubular epithelial cell organoids in a culture medium containing glucose, advanced glycation end products, and tumor necrosis factor-α.

[0007] The beneficial effects of this invention are as follows: This invention constructs a pathological model of diabetic nephropathy through combined induction, enabling the model to exhibit the core pathological phenotypes of diabetic nephropathy, including downregulation of renal tubular epithelial cell markers, upregulation of fibrosis markers, basement membrane thickening, and abnormal albumin uptake function. The construction method is simple and standardized, with a short construction cycle, good reproducibility and stability. A highly biomimetic in vitro three-dimensional model of human diabetic nephropathy has been established, and its application value in high-throughput screening of traditional Chinese medicine monomers or compound drugs with renal protective effects has been clarified, providing a new platform for the study of the mechanism of diabetic nephropathy and the development of treatment strategies.

[0008] Furthermore, renal tubular epithelial organoids were prepared by the following method: S1. Pre-treat the renal tubular tissue to obtain renal tubular cortical tissue; S2. First, the renal tubular cortical tissue obtained in S1 is cut, digested, then filtered and the filtrate is collected. Finally, it is centrifuged to obtain renal tubular epithelial organoids.

[0009] The beneficial effects of adopting the above-mentioned further technical solutions are as follows: The present invention provides a method for establishing a renal tubular epithelial cell organoid model. The method is simple to construct, has low cost, and produces organoids with clear boundaries and uniform morphology with a high yield. It is the basis for the construction of the diabetic nephropathy model in this application.

[0010] Furthermore, the pretreatment in S1 includes removing the capsule and renal pedicle tissue from the renal tubular tissue, followed by rinsing with physiological saline and separating the renal tubular cortex tissue.

[0011] Furthermore, the size of the renal tubular cortex tissue cut in S2 is 1-2 mm. 3 .

[0012] Furthermore, the digestion time in S2 is 5-30 min; the centrifugation conditions are: 1-10℃, 200-300 ×g, centrifugation for 1-5 min, repeated 2-5 times.

[0013] Furthermore, the digestion process in S2 includes: adding 5-10 times the volume of tissue digestion solution, placing it in a constant temperature shaker at 37°C for digestion, and setting the rotation speed to 50 rpm.

[0014] Furthermore, the digestion process in S2 also includes: centrifugation at 1-10℃, 200-300 ×g for 1-5 min to remove the digestion fluid; washing the precipitate with Advanced DMEM / F12 and then passing it through a 70 μm cell sieve to remove undigested tissue.

[0015] Furthermore, in a culture medium containing glucose, advanced glycation end products (AGEs), and tumor necrosis factor-α, the concentration of glucose was 25-30 mmol / L, the concentration of AGEs was 100-200 μg / mL, and the concentration of tumor necrosis factor-α was 10-20 mg / mL; the combined induction time was 14-28 days.

[0016] Furthermore, renal tubular epithelial cell organoids were cultured for 5-10 days in three-dimensional matrix gel and renal tubular epithelial organoid culture medium before combined induction.

[0017] Furthermore, the three-dimensional matrix adhesive is Matrigel or a recombinant basement membrane analog, with a mass concentration of 50%-70%.

[0018] Furthermore, the culture conditions are as follows: culture at 35-10℃ and 4%-6% CO2, with the medium changed every 2 days.

[0019] Preferably, the culture conditions are: 37℃, 5% CO2, with the medium changed every 2 days.

[0020] In a second aspect, the present invention provides a pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids, which is prepared by the above-described construction method.

[0021] A third aspect of the present invention provides the application of the above-described pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids in screening or evaluating candidate drugs for the prevention and / or treatment of diabetic nephropathy.

[0022] The beneficial effects of this invention are as follows: The pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids obtained by this invention can be used to evaluate the effects of drugs on fibrosis, inflammation and renal function markers, providing a tool for the study of multi-target drug mechanisms and accelerating the development of new drugs for diabetic nephropathy; at the same time, it also provides a reliable in vitro evaluation system for traditional Chinese medicine with complex components and diverse mechanisms of action, which helps to verify its therapeutic potential and promote the application and transformation of traditional Chinese medicine in the treatment of diabetic nephropathy.

[0023] A fourth aspect of the present invention provides a method for screening or evaluating candidate drugs for the prevention and / or treatment of diabetic nephropathy, comprising the following steps: (1) Apply the candidate drug to the above-mentioned diabetic nephropathy pathological model based on human renal tubular epithelial cell organoids; (2) Detect pathological indicators of the diabetic nephropathy pathological model based on human renal tubular epithelial cell organoids before and after drug administration. The pathological indicators include at least one of the following: expression level of fibrosis markers, secretion of inflammatory factors, and expression level of renal tubular epithelial cell markers. (3) Compare the changes in pathological indicators obtained in step (2) with the control group that did not receive the drug. If the candidate drug can significantly reverse the case indicators, the candidate drug is determined to have the potential to prevent and / or treat diabetic nephropathy.

[0024] This invention provides a method for screening or evaluating candidate drugs for the prevention and / or treatment of diabetic nephropathy. Based on the pathological model of diabetic nephropathy obtained in this invention, this method provides a reliable in vitro evaluation system for traditional Chinese medicines with complex components and diverse mechanisms of action, which helps to verify their therapeutic potential, promotes the application and transformation of traditional Chinese medicines in the treatment of diabetic nephropathy, and can evaluate the effects of drugs on fibrosis, inflammation and renal function markers through high-throughput screening technology, providing tools for the study of multi-target drug mechanisms and accelerating the development of new drugs for diabetic nephropathy.

[0025] Furthermore, in step (1), the candidate drug is administered by incubating it in a suspension of a diabetic nephropathy pathological model based on human renal tubular epithelial cell organoids.

[0026] Furthermore, the pathological model suspension of diabetic nephropathy based on human renal tubular epithelial cell organoids was prepared by the following method: first, the original culture medium was discarded, and organoid digestion solution was added for digestion; then, the cell was centrifuged, the supernatant was discarded, and the cell was washed twice with pre-cooled Advance DMEM / F12, and filtered through a 70 μm cell filter; next, the cell was centrifuged, the supernatant was discarded, and the cell was resuspended in human renal tubular epithelial cell organoid culture medium containing 50%-70% Matrigel; finally, the renal tubular epithelial cell organoids were seeded into plates and cultured in a 37°C, 5% CO2 incubator.

[0027] Furthermore, the incubation time is 48-72 hours.

[0028] Furthermore, the candidate drugs are single Chinese medicine monomers, extracts of compound Chinese medicines, or natural product derivatives.

[0029] Preferably, the candidate drug is at least one of astragaloside A, rhein, ligustrazine, total saponins of Panax notoginseng, and puerarin.

[0030] In a fifth aspect, the present invention provides a tool or kit for drug screening of diabetic nephropathy, comprising the above-described pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids.

[0031] The present invention has the following beneficial effects: 1. This invention, based on human renal tubular epithelial cell organoids, successfully simulates key pathological features of human diabetic nephropathy, including extracellular matrix deposition, epithelial-mesenchymal transition (EMT), inflammatory responses, and functional impairment, through the combined induction of high glucose, advanced glycation end products (AGEs), and inflammatory factors (TNF-α). The model is highly similar to clinical diabetic nephropathy in pathological morphology and molecular expression (such as upregulation of fibrosis markers and downregulation of renal tubular markers), providing an in vitro research platform that more closely approximates the physiological state of the human body.

[0032] 2. Compared to animal models (such as rodents), the method of this invention avoids the problems of long cycles (modeling requires several months to a year), large species differences, and low clinical conversion rates, thus reducing time and economic costs. Compared to two-dimensional cell models, this invention utilizes three-dimensional organoid culture to simulate the complex microenvironment, cell polarity, and intercellular interactions of kidney tissue, more accurately reflecting disease mechanisms and drug responses.

[0033] 3. The model construction method of this invention is standardized, the induction conditions are clear, and it has good repeatability and stability, making it suitable for large-scale experiments.

[0034] 4. The model constructed in this invention is particularly suitable for high-throughput screening of single or compound Chinese medicines. It can evaluate the effects of drugs on fibrosis, inflammation and renal function markers, provide tools for multi-target drug mechanism research, and accelerate the development of new drugs for diabetic nephropathy.

[0035] 5. The model constructed in this invention provides a reliable in vitro evaluation system for traditional Chinese medicines with complex components and diverse mechanisms of action, which helps to verify their therapeutic potential and promote the application and transformation of traditional Chinese medicines in the treatment of diabetic nephropathy. Attached Figure Description

[0036] Figure 1 Images of the renal tubular epithelial cell organoids P2 generation obtained in Example 1 on day 3; where A is 4×; B is 10×; C is 20×; Figure 2 The images show fluorescence images of CFTR and Pax8, the P2 generation markers of renal tubular epithelial cell organoids obtained in Example 1; where A is the CFTR fluorescence image and B is the Pax8 fluorescence image. Figure 3 Images of the human diabetic nephropathy renal tubular epithelial organoid model obtained in Example 1 on day 14 of induction, where A is 4×; B is 10×; C is 20×; Figure 4 The graphs show the results of cytokine levels after treatment with different concentrations of astragaloside A in Example 2. In the graphs, A represents the statistical results of α-SMA level, B represents the statistical results of Fibronectin level, C represents the statistical results of AQP1 level, D represents the statistical results of E-Cadherin level, E represents the statistical results of IL-1β level, and F represents the statistical results of TNF-α level. Figure 5 The graphs show the results of cytokine levels after treatment with different concentrations of rhein in Example 2. In the graphs, A is the statistical result of α-SMA level, B is the statistical result of Fibronectin level, C is the statistical result of AQP1 level, D is the statistical result of E-Cadherin level, E is the statistical result of IL-1β level, and F is the statistical result of TNF-α level. Figure 6 The graphs show the results of cytokine level detection after treatment with different concentrations of tetramethylpyrazine in Example 2. In the graphs, A is the statistical result of α-SMA level, B is the statistical result of Fibronectin level, C is the statistical result of AQP1 level, D is the statistical result of E-Cadherin level, E is the statistical result of IL-1β level, and F is the statistical result of TNF-α level. Figure 7 The graphs show the results of cytokine levels after treatment with different concentrations of Panax notoginseng total saponins in Example 2. In the graphs, A is the statistical result of α-SMA level, B is the statistical result of Fibronectin level, C is the statistical result of AQP1 level, D is the statistical result of E-Cadherin level, E is the statistical result of IL-1β level, and F is the statistical result of TNF-α level. Figure 8The graphs show the results of cytokine level detection after treatment with different concentrations of puerarin in Example 2. In the graphs, A is the statistical result of α-SMA level, B is the statistical result of Fibronectin level, C is the statistical result of AQP1 level, D is the statistical result of E-Cadherin level, E is the statistical result of IL-1β level, and F is the statistical result of TNF-α level. Detailed Implementation The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0037] Example 1: Construction of a pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids A method for constructing a pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids includes the following steps: S1. Preparation of renal tubular cortical tissue Fresh surgical tissue was obtained and immediately placed in pre-cooled 4°C epithelial organoid basal culture medium (manufacturer: Bozhen Biotechnology Co., Ltd., catalog number: B213151) for repeated rinsing until the supernatant was clear and free of impurities. The renal tubular tissue specimen was transferred to a 7 cm cell culture dish, the capsule and renal pedicle tissue were removed, and the tissue was rinsed with physiological saline to obtain renal tubular cortical tissue.

[0038] S2, Preparation of renal tubular epithelial organoids First, the renal tubular cortical tissue obtained from S1 is cut into pieces of 1-2 mm. 3The tissue fragments were transferred to 50 mL centrifuge tubes using a pipette. After sedimentation, the supernatant was aspirated, and pre-chilled (4°C) epithelial organoid basal culture medium was added. The tubes were resuspended 3-5 times until the supernatant was clear and free of impurities. The supernatant was then removed, and 10 mL of tissue digestion solution was added. The tubes were then placed in a 37°C shaker at 50 rpm for 20 min. The digestion solution was removed by centrifugation at 4°C, 250 × g, for 3 min. The precipitate was washed with Advanced DMEM / F12 and then passed through a 70 μm sieve to remove undigested tissue. Finally, the filtrate was collected in a clean 50 mL centrifuge tube, centrifuged at 4°C, 250 × g for 3 min, and 1 mL of pre-chilled (4°C) epithelial organoid basal culture medium was added. Lyse the red blood cells in mL of erythrocyte lysis buffer at room temperature for 3 min, centrifuge again, resuspend the organoid pellet in epithelial organoid basal medium, repeat centrifugation and washing twice to completely remove digestion residues, and obtain a renal tubular epithelial cell organoid suspension; add an appropriate volume of epithelial organoid basal medium according to the amount of organoids to resuspend the pellet, aspirate 20 μL onto a hemocytometer, and count the number of organoids in the 20 μL sample until the organoid density is resuspended to 20-30 intact organoids / μL.

[0039] S3, Pre-culture of renal tubular epithelial organoids First, 200 μL of renal tubular epithelial cell organoid suspension was taken and mixed with an equal volume of undiluted Matrigel. The mixture was then thoroughly mixed by pipetting. Next, 50 μL of the organoid suspension (Mattrigel mixture) was added to the center of the bottom of each well in a 96-well plate. The plate was incubated upside down in 37°C, 5% CO2 medium for 20 min to form solid droplets. Finally, 200 μL of room-temperature prepared organoid culture medium was added to each well, and sterile PBS was added to the uninoculated wells to maintain humidity during culture. The plate was then capped, and the culture medium was replaced with fresh medium every two days. The plate was cultured at 37°C and 5% CO2 for 8 days to complete the pre-culture. The image of the third day of the P2 generation of renal tubular epithelial organoids is shown below. Figure 1 As shown, the fluorescence spectra of the P2 generation markers CFTR and Pax8 are as follows: Figure 2 As shown in the figure, the results indicate that the prepared organoids have clear boundaries and uniform morphology.

[0040] S4. Construction of a human diabetic nephropathy renal tubular epithelial organoid model First, glucose, advanced glycation end products (AGEs), and tumor necrosis factor-α (TNF-α) were vortexed and added to the complete organoid culture medium. The concentration of glucose in the medium was 30 mmol / L, the concentration of AGEs was 200 μg / mL, and the concentration of TNF-α was 10 ng / mL. Then, the pre-culture medium was discarded, and induction medium containing glucose, AGEs, and TNF-α was added. The culture was carried out at 37℃ and 5% CO2 for 15 consecutive days. During this period, the induction medium containing glucose, AGEs, and TNF-α was replaced every 2 days to obtain a human diabetic nephropathy renal tubular epithelial organoid model. An image of the human diabetic nephropathy renal tubular epithelial organoid model on day 14 of induction is shown below. Figure 3 As shown, the obtained model has clear boundaries and a uniform shape.

[0041] Example 2: Application of human renal tubular epithelial cell organoid models in drug screening for the treatment of diabetic nephropathy I. Experimental Methods (1) Experimental grouping and treatment Using the human diabetic nephropathy renal tubular epithelial organoid model obtained in Example 1, experiments were set up for five drugs: astragaloside A, rhein, ligustrazine, total saponins of Panax notoginseng, and puerarin. Each drug was divided into 5 groups: model control group (HG group), 5 μmol / L treatment group, 25 μmol / L treatment group, 50 μmol / L treatment group, and 100 μmol / L treatment group, with 3 replicates for each group.

[0042] Organoid culture medium containing the corresponding concentration of drug (final DMSO concentration ≤ 0.1%) was added to each concentration treatment group, while organoid culture medium containing an equal amount of DMSO was added to the model control group. The samples were cultured at 37℃ in a 5% CO2 incubator for 48 h.

[0043] (2) Administration method The drug was administered by incubating the drug in a suspension of a diabetic nephropathy pathological model based on human renal tubular epithelial cell organoids for 60 hours.

[0044] The pathological model suspension of diabetic nephropathy based on human renal tubular epithelial cell organoids was prepared by the following method: First, the original culture medium was discarded, and organoid digestion solution was added for digestion; then, the cell was centrifuged, the supernatant was discarded, and the cell was washed twice with pre-cooled Advance DMEM / F12 and filtered through a 70 μm cell filter; then, the cell was centrifuged again, the supernatant was discarded, and the cell was resuspended in human renal tubular epithelial cell organoid culture medium containing 60% Matrigel; finally, the renal tubular epithelial cell organoids were seeded into plates and cultured in a 37°C, 5% CO2 incubator.

[0045] (3) Indicator detection Supernatants from tissue cultures and organoids were collected. The levels of inflammatory factors were detected using ELISA kits (TNF-α, manufacturer: abcam, catalog number: ab181421; IL-1β, manufacturer: abcam, catalog number: ab260072). Simultaneously, the expression levels of α-SMA, Fibronectin, AQP1, and E-Cadherin in organoids were detected by qPCR. All procedures were performed according to the corresponding instructions.

[0046] II. Experimental Results Experimental results are as follows Figures 4-8 The figures show the detection results of inflammatory factor levels and RNA expression levels after treatment with different concentrations of astragaloside A, rhein, ligustrazine, total saponins of Panax notoginseng, and puerarin.

[0047] The results showed that, compared with the model control group (HG group), all concentrations of the five drugs significantly reduced the levels of α-SMA, Fibronectin, AQP1, E-Cadherin, and inflammatory factors (TNF-α, IL-1β) in the organoids (P<0.05); and as the drug concentration increased from 5 μmol / L to 100 μmol / L, the reduction in these indicators gradually increased, showing a concentration-dependent relationship (P<0.05). These results indicate that the diabetic nephropathy organoid model constructed in this invention has good sensitivity to different concentrations of drugs for treating diabetic nephropathy and can be used for dose-dependent screening of small molecule traditional Chinese medicine drugs for the treatment of diabetic nephropathy.

[0048] In summary, this invention provides a method for constructing a renal tubular epithelial cell organoid model. This method is simple, low-cost, and produces organoids with clear boundaries, uniform morphology, and a high yield. The renal tubular epithelial cell organoid model constructed using this method can also be used to construct diabetic nephropathy models. The constructed diabetic nephropathy models are highly responsive to drugs for treating diabetic nephropathy, enabling high-throughput screening of these drugs and providing a basis for the clinical screening of novel diabetic nephropathy drugs.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids, characterized in that, Includes the following steps: A pathological model of diabetic nephropathy was obtained by co-inducing renal tubular epithelial cell organoids in a culture medium containing glucose, advanced glycation end products, and tumor necrosis factor-α.

2. The method for constructing a pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids according to claim 1, characterized in that, The renal tubular epithelial organoids were prepared by the following method: S1. Pre-treat the renal tubular tissue to obtain renal tubular cortical tissue; S2. First, the renal tubular cortical tissue obtained in S1 is cut, digested, then filtered and the filtrate is collected. Finally, it is centrifuged to obtain renal tubular epithelial organoids.

3. The method for constructing a pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids according to claim 2, characterized in that, The pretreatment in S1 includes removing the capsule and renal pedicle tissue from the renal tubular tissue, and separating the renal tubular cortex tissue after rinsing with physiological saline.

4. The method for constructing a pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids according to claim 2, characterized in that, The digestion time in S2 is 5-30 min; the centrifugation conditions are: 1-10℃, 200-300 ×g, centrifugation for 1-5 min, repeated 2-5 times.

5. The method for constructing a pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids according to claim 1, characterized in that, The culture medium containing glucose, advanced glycation end products (AGEs), and tumor necrosis factor-α has a glucose concentration of 25-30 mmol / L, an AGE concentration of 100-200 μg / mL, and a tumor necrosis factor-α concentration of 10-20 mg / mL; the combined induction time is 14-28 days.

6. The method for constructing a pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids according to claim 1, characterized in that, Renal tubular epithelial cell organoids were cultured in three-dimensional matrix gel and renal tubular epithelial organoid culture medium for 5-10 days before combined induction.

7. A pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids, characterized in that, It is prepared by the construction method according to any one of claims 1-6.

8. The use of the pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids as described in claim 7 in screening or evaluating candidate drugs for the prevention and / or treatment of diabetic nephropathy.

9. A method for screening or evaluating candidate drugs for the prevention and / or treatment of diabetic nephropathy, characterized in that, Includes the following steps: (1) Applying the candidate drug to the diabetic nephropathy pathological model based on human renal tubular epithelial cell organoids as described in claim 7; (2) Detect pathological indicators of the diabetic nephropathy pathological model based on human renal tubular epithelial cell organoids before and after drug administration. The pathological indicators include at least one of the following: expression level of fibrosis markers, secretion of inflammatory factors, and expression level of renal tubular epithelial cell markers. (3) Compare the changes in pathological indicators obtained in step (2) with the control group that did not receive the drug. If the candidate drug can significantly reverse the case indicators, the candidate drug is determined to have the potential to prevent and / or treat diabetic nephropathy.

10. A tool or kit for drug screening in diabetic nephropathy, characterized in that, This includes the pathological model of diabetic nephropathy based on human renal tubular epithelial cell organoids as described in claim 7.

Images