A high-throughput construction method and application of human kidney organoids simulating pathological characteristics of lupus nephritis

Abstract

This invention relates to the fields of regenerative medicine, disease models, and drug screening, and particularly to a high-throughput method and application for constructing human kidney organoids that simulate the pathological characteristics of lupus nephritis. The kidney organoids are mixed and cultured with plasma from lupus nephritis patients in low-adsorption multi-well plates. This invention utilizes a standardized process to prepare human kidney organoids in high throughput, and then stimulates them with plasma from lupus nephritis patients, thereby rapidly and in large quantities constructing disease models in vitro that simulate the severity of individual patients' diseases and reproduce key pathological features (such as podocyte damage and inflammatory fibrosis). This platform provides a closed-loop research tool for lupus nephritis research, from "clinical samples" to "in vitro models" and then to "clinical validation."

Description

Technical Field

[0001] This invention relates to the fields of regenerative medicine, disease models and drug screening technologies, and in particular to a high-throughput method and application for constructing human kidney organoids that simulate the pathological characteristics of lupus nephritis. Background Technology

[0002] Lupus nephritis is one of the most serious complications of systemic lupus erythematosus (SLE). Its pathogenesis is complex and varies greatly from person to person. There is a lack of humanized, scalable disease models that can accurately simulate the process of "systemic autoimmune attack - kidney-specific damage". Existing animal models have species differences, and traditional 2D cell culture cannot reproduce the three-dimensional structure and cell interactions of the kidney.

[0003] In recent years, the technology of kidney organoids derived from human induced pluripotent stem cells has matured, providing new tools for modeling human kidney diseases. Existing research has successfully established a high-throughput protocol for mass production of kidney organoids in low-adsorption 96-well plates, achieving a leap in yield from single digits to thousands. However, current technologies primarily produce "healthy" or genetically modified organoids. There is still no mature method to effectively introduce complex systemic pathogenic factors (present in plasma) from lupus patients into organoids to construct pathological models that can differentiate disease severity. This technological gap severely restricts the discovery of targets and the development of precision treatments for lupus nephritis. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides a high-throughput method and application for constructing human kidney organoids that mimic the pathological characteristics of lupus nephritis. This invention utilizes plasma from lupus nephritis patients to induce disease-specific pathological phenotypes in human induced pluripotent stem cell-derived kidney organoids on a 96-well high-throughput platform. This platform can be used for mechanistic studies of lupus nephritis, biomarker discovery, high-throughput drug screening, and personalized medicine.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a high-throughput method for constructing human kidney organoids that simulate the pathological characteristics of lupus nephritis, comprising the following steps: mixing and culturing kidney organoids with plasma from patients with lupus nephritis in a low-adsorption multi-well plate.

[0007] Preferably, the plasma from the lupus nephritis patient is mixed in the form of a culture medium, wherein the volume percentage of plasma in the culture medium is 10%.

[0008] Preferably, the culture time is 72 hours.

[0009] Preferably, the low-adsorption porous plate includes a 96-well V-shaped base plate.

[0010] Preferably, the kidney organoid is obtained by induction and differentiation of human induced pluripotent stem cells.

[0011] Preferably, the induced pluripotent stem cells are derived from human urine.

[0012] Preferably, the lupus nephritis patients include patients with mild lupus nephritis and / or patients with severe lupus nephritis.

[0013] The present invention also provides the application of the lupus nephritis pathological model obtained by the construction method described above in the preparation of reagents for screening drugs for the treatment of lupus nephritis.

[0014] The beneficial effects of this invention are:

[0015] This invention utilizes a standardized process to prepare human kidney organoids in high throughput, and then stimulates them with plasma from lupus nephritis patients. This allows for the rapid and large-scale in vitro construction of disease models that simulate the severity of individual patients' diseases and reproduce key pathological features (such as podocyte damage and inflammatory fibrosis). This platform provides a closed-loop research tool for lupus nephritis research, from "clinical samples" to "in vitro models" and then to "clinical validation."

[0016] (1) Pioneering pathological model: For the first time, patient plasma was successfully used to induce a damage phenotype in human kidney organoids that closely matches the clinical pathology of lupus nephritis, achieving a leap from "healthy organoids" to "disease organoids".

[0017] (2) High throughput and standardization: Based on the integrated scheme of 96-well plates, the batch parallel preparation and analysis of disease models are realized. The efficiency is far superior to traditional methods such as Transwell, and the cost is significantly reduced, making it suitable for large-scale drug screening.

[0018] (3) Clear application prospects: This platform can be directly used for: ① disease mechanism research; ② high-throughput drug efficacy and toxicity assessment; ③ discovery and verification of new diagnostic biomarkers and therapeutic targets; ④ guidance of clinical individualized treatment strategies.

[0019] Plasma stimulation of kidney organoids from different lupus nephritis patients induced structural and molecular phenotypic changes highly consistent with the pathological characteristics of lupus nephritis kidneys in these patients. These changes included glomerular and tubular structural disorder, loss of function-related molecules, and upregulation of damage-related markers. Furthermore, these changes correlated with the severity of the disease, enabling organoid models to reproduce the pathological changes of lupus nephritis of varying severities in vitro. Transforming the complex pathological environment in vivo into high-throughput disease organoids in vitro provides a new technological pathway for disease severity assessment, classification, and drug screening. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0021] Figure 1 The flowchart of the construction and experimental analysis of the "high-throughput platform for human lupus nephritis kidney organoids" provided by the present invention shows the complete technical process of processing kidney organoids using healthy donor plasma, plasma from patients with mild or severe lupus nephritis, and performing multi-dimensional characterization.

[0022] Figure 2 The image shows a characterization of immunofluorescence (IF) staining, illustrating the difference in expression between proximal tubular cell marker LTL (green) and podocyte marker SYNPO (red) in kidney organoids treated with healthy donor plasma (CTR), plasma from patients with mild lupus nephritis (LN-M), and plasma from patients with severe lupus nephritis (LN-S). Scale bar: 50 µm.

[0023] Figure 3 The immunofluorescence staining comparison of kidney injury and fibrosis markers shows the expression of the kidney injury marker KIM-1 and the fibrosis marker protein fibronectin 1 (FN-1) in three groups of kidney organoids, revealing the injury and fibrosis process related to the severity of the disease.

[0024] Figure 4 The images are ultrastructural analysis images obtained by transmission electron microscopy (TEM), showing the structural damage (such as fusion and disappearance) of podocyte foot processes in kidney organoids treated with patient plasma. This visually demonstrates the model's ability to simulate pathological changes at the organelle level. Scale bar: 5 µm (×1,900); 1 µm (×6,800).

[0025] Figure 5 A heatmap of differentially expressed genes was created, showing the normalized expression patterns of differentially expressed genes in three groups of kidney organoids: CTR, LN-M, and LN-S. Significantly enriched biological pathways were annotated with side notes, revealing the molecular characteristics of disease progression at the transcriptomic level.

[0026] Figure 6 The correlation heatmap systematically demonstrates the statistical association between the expression levels of key biomarkers (such as functional biomarkers and damage biomarkers) in kidney organoids and corresponding patient clinical parameters (such as eGFR, SLEDAI score, etc.).

[0027] Figure 7As a heatmap for multi-omics integrated correlation analysis, A and B respectively show the sets of genes that were significantly downregulated and upregulated in kidney organoids of the lupus nephritis model, and the correlation network between them and the peripheral blood circulating plasma cytokine levels of the corresponding LN patients, revealing the intrinsic link between systemic immune factors and local molecular damage to the kidney. Detailed Implementation

[0028] The present invention also provides a high-throughput method for constructing human kidney organoids that simulate the pathological characteristics of lupus nephritis, comprising the following steps: mixing and culturing kidney organoids with plasma from patients with lupus nephritis in a low-adsorption multi-well plate.

[0029] In this invention, the plasma from the lupus nephritis patient is preferably mixed in the form of a culture medium, wherein the volume percentage of plasma in the culture medium is preferably 10%. In this invention, the culture time is preferably 72 hours. In this invention, the low-adsorption porous plate preferably comprises a 96-well V-shaped bottom plate, model MS-9096VZ.

[0030] In this invention, the kidney organoid is preferably obtained by induced differentiation of human induced pluripotent stem cells. This invention does not impose any particular limitations on the conditions for the induced differentiation; those skilled in the art can use conventional methods. In this invention, the induced pluripotent stem cells are preferably derived from human urine. In this invention, the lupus nephritis patients preferably include patients with mild lupus nephritis and / or patients with severe lupus nephritis.

[0031] The present invention also provides the application of the lupus nephritis pathological model obtained by the construction method described above in the preparation of reagents for screening drugs for the treatment of lupus nephritis.

[0032] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

[0034] High-throughput preparation of kidney organoids

[0035] First, human induced pluripotent stem cells (iPSCs) derived from human urine were cultured at a rate of 6.5 × 10⁻⁶. 4Cells were seeded into six-well plates and mTeSR PLUS medium containing the ROCK inhibitor Y27632 (final concentration 10 µmol / L) was added. From days 1 to 4, 2 ml of STEMdiff APEL2 medium (with CHIR99021 added, final concentration 8 µmol / L) was added to the cells to induce differentiation. From days 5 to 7, the medium was replaced with 2 ml of STEMdiff APEL2 medium supplemented with CHIR99021 (final concentration 8 µmol / L), FGF-9 (final concentration 200 ng / ml), and heparin (final concentration 1 µg / ml) to further induce differentiation. On day 7, the previously cultured cells were digested into a single-cell suspension and cultured at 5.0 × 10⁶ cells per well. 4 Cells were introduced into 96-well V-type cell culture plates and cultured in 3D suspension for 24 h to allow them to aggregate into spheroids. On day 8, STEMdiff APEL2 medium containing CHIR99021 (final concentration 5 µmol / L) was added to stimulate differentiation for 60 min; finally, STEMdiff APEL2 medium containing FGF-9 and heparin was added to continue differentiation. From day 9 to 14, organoids gradually differentiated, and under a microscope, cells at the edge of the spheroids were observed to differentiate gradually, forming radial tentacles, and later tubular structures appeared. From day 15 to 30, numerous small tubular structures were observed under a microscope, radiating outwards (from the center to the edge of the spheroid). At this point, kidney organoids had begun to form, kidney-specific genes began to be expressed, and renal tubules formed. The STEMdiff APEL2 medium was then replaced, and the kidney organoids further matured. Results: Hundreds of mature kidney organoids with uniform structure and containing renal tubules and podocytes were successfully obtained from a single 96-well plate.

[0036] Example 2

[0037] Construction and validation of a lupus nephritis disease model

[0038] Grouping: The mature kidney organoids obtained in Example 1 were each well stimulated for 72 hours with a culture medium containing 180 µL of plasma from healthy individuals (10% by volume), 180 µL of plasma from patients with mild LN (10% by volume), and 180 µL of plasma from patients with severe LN (10% by volume) in a CO2 incubator at 37°C with 5% CO2. The culture medium was StemDIF medium from StemCell.

[0039] Phenotypic analysis: Immunofluorescence showed that SYNPO and LTL signaling in LN plasma organoids was weakened in a severity-dependent manner. Figure 2 ); signal enhancement of damage marker KIM-1 and fibrosis marker FN-1 ( Figure 3 Transmission electron microscopy confirmed significant foot process fusion in the severe LN group. Figure 4 ).

[0040] Mechanism validation: RNA-seq analysis showed that in organoids of severe LN patients, characteristic pathways of lupus, such as interferon response and interleukin-17 signaling, were significantly enriched. Figure 5 ).

[0041] Clinical correlation: Linear regression analysis showed that the expression level of HAVCR1 in organoids was significantly negatively correlated with the estimated glomerular filtration rate (eGFR) of donor patients (R² < 0.8, p < 0.005). Figure 6 ).

[0042] 1. Immunofluorescence staining procedure: Organoids are fixed in 4% paraformaldehyde and then embedded and sectioned. After dewaxing, hydration, and antigen retrieval, the sections are permeabilized according to the protein target. Non-specific binding sites are then blocked with 10% styrene-glucosamine, and a primary antibody targeting the target protein is added. After incubation under suitable conditions, the sections are washed. A fluorescently labeled secondary antibody matching the primary antibody is then added, followed by incubation and washing. Finally, counterstaining is performed using nuclear dyes. After anti-quenching mounting, images are acquired using a fluorescence microscope or confocal microscope. Image analysis can be performed using uniform parameters for background correction and threshold segmentation, and quantitative indicators such as fluorescence intensity, positive area ratio, or positive cell number can be quantified. Multiple random fields of view can be selected for each sample, and the mean values ​​are calculated for comparative analysis.

[0043] 2. Transmission electron microscopy (TEM) experimental procedures: Tissue or cell samples are cut into small pieces and chemically fixed, followed by post-fixation. After gradient dehydration, the samples are replaced and embedded for solidification, and ultrathin sections are prepared using an ultramicrotome. The sections are placed on a grid for contrast staining. Images are observed and acquired using a transmission electron microscope under appropriate accelerating voltage conditions. The ultrastructural characteristics (mainly observing the morphology and structure of tubular cells and podocytes, such as foot processes) are described and analyzed according to the experimental objectives.

[0044] 3. Clinical association analysis: The expression level of HAVCR1 in organoids was paired with the estimated glomerular filtration rate (eGFR) of the corresponding donor patients. Linear regression analysis was performed using GraphPad Prism 9 software. Scatter plots were generated and regression models were established to assess the correlation between HAVCR1 expression and renal function indicators.

[0045] 4. Mechanism Validation: Total RNA was extracted from samples using RNA-seq. After passing quality control, sequencing libraries were constructed, and transcriptome sequencing was performed using a high-throughput sequencing platform. The raw sequencing data underwent quality control and filtering to remove low-quality and adapter sequences. The cleaned, valid sequences were aligned to a reference genome for quantitative analysis to obtain gene expression abundance information. Differences in gene expression among different samples were compared and analyzed. The expression matrix data obtained from sequencing was imported into the R language software environment for data processing and statistical analysis, including data preparation, standardization, gene expression calculation, differential expression analysis, and visualization. This data was used to screen differentially expressed genes and functional pathways related to the studied phenotype.

[0046] Example 3

[0047] Platform-based biomarker discovery

[0048] An integrated analysis was performed on the organoid transcriptome data from Example 2 and the corresponding patient plasma 46-factor detection data (commissioned to Shanghai Unimicron Biotechnology Co., Ltd.). It was found that the levels of factors such as CCL19 and IL-8 in plasma were strongly correlated with organoid injury scores. Figure 7 ).

[0049] Example 4

[0050] Examples of drug screening applications

[0051] (1) Inducing lupus nephritis-like lesion phenotype by contacting conventional organoids or organoids containing a reporter system with plasma derived from patients with lupus nephritis for 24–96 hours; (2) Adding the candidate drug to the culture system during or after induction; (3) Adding the candidate drug to the culture system during or after induction; (4) Adding the candidate drug to the culture system during or after induction.

[0052] (5) Compare the drug-treated group with the untreated control group, and determine the potential therapeutic effect of the candidate drug on lupus nephritis based on the status of the reporting system or the expression of damage-related markers, the integrity of organoid structure and the expression of function-related genes.

[0053] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A high-throughput method for constructing human kidney organoids that simulate the pathological characteristics of lupus nephritis, characterized in that, Includes the following steps: Kidney organoids were mixed with plasma from patients with lupus nephritis and cultured in low-adsorption multi-well plates.

2. The high-throughput construction method for human kidney organoids according to claim 1, characterized in that, The plasma from the lupus nephritis patient was mixed in the form of a culture medium, wherein the plasma volume percentage in the culture medium was 10%.

3. The high-throughput construction method for human kidney organoids according to claim 1, characterized in that, The incubation period was 72 hours.

4. The high-throughput construction method for human kidney organoids according to claim 1, characterized in that, The low-adsorption porous plate includes a 96-well V-shaped base plate.

5. The high-throughput construction method for human kidney organoids according to claim 1, characterized in that, The kidney organoids were obtained by induction and differentiation of human induced pluripotent stem cells.

6. The high-throughput construction method for human kidney organoids according to claim 1, characterized in that, The induced pluripotent stem cells are derived from human urine.

7. The high-throughput construction method for human kidney organoids according to claim 1, characterized in that, The lupus nephritis patients include those with mild lupus nephritis and / or those with severe lupus nephritis.

8. The use of the lupus nephritis pathological model obtained by the construction method according to any one of claims 1 to 7 in the preparation of reagents for screening drugs for the treatment of lupus nephritis.

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