Diabetic nephropathy kidney organoid model and construction method and application thereof

Abstract

The invention discloses a diabetic nephropathy kidney organoid model which is obtained by inducing a kidney organoid through a composition, and the composition comprises glucose, palmitic acid and linoleic acid. According to the invention, linoleic acid is introduced into the construction of the diabetic nephropathy kidney organ model as an induction factor for the first time, and it is determined that the combined treatment of glucose, palmitic acid and linoleic acid can stably induce kidney organs to generate pathological characteristics conforming to diabetic nephropathy. The invention further discloses a construction method and application of the diabetic nephropathy kidney organoid model.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a diabetic nephropathy kidney organoid model and a construction method and application thereof. BACKGROUND

[0002] Diabetic nephropathy (DN) is one of the common complications of diabetes and seriously threatens human health. The current widely used diabetic nephropathy research models include a streptozotocin-induced mouse model, a high-glucose-treated renal tubular epithelial cell model, etc., but these models can simulate the metabolic disorder, hyperglycemia, proteinuria and glomerular sclerosis and other kidney pathological characteristics under the diabetic state to a certain extent. However, these models have certain limitations, the principle of animal modeling is inconsistent with the pathogenesis of humans, and there are significant differences between the pathogenesis of DN mice and humans, the kidney structure of mice is different from that of humans, and there is no obvious differentiation of renal cortex and medulla; the development of proteinuria in many models is atypical, or cannot progress to end-stage renal disease, and in terms of mechanism research, animal experiments are often difficult to accurately intervene in specific signal pathways or molecular events. And the single cell line model is difficult to fully reflect the three-dimensional structure and complex cell-cell interaction of diabetic nephropathy, and there are significant deficiencies in reproducing the histopathology and microenvironment interaction of diabetic nephropathy.

[0003] The clinical pathological phenotypes of diabetic nephropathy mainly include the following aspects: (1) glomerular basement membrane (GBM) thickening: early manifestation of GBM homogeneous thickening (normal adult about 300-400 nm, DN patients can reach 600-1000 nm), electron microscopy can see basement membrane laminin and collagen IV deposition; (2) excessive deposition of extracellular matrix (ECM) in the mesangial area, mesangial cell proliferation, leading to glomerular sclerosis; (3) podocyte damage and shedding, manifested as foot process fusion and reduced number of podocytes (NPHS1). Urinary podocyte markers (such as podocalyxin, nephrin) can reflect the degree of damage.

[0004] Kidney organoids are a rapidly developing three-dimensional culture technology in recent years, which is derived from pluripotent stem cells and can self-organize into a miniature kidney-like tissue with multiple nephron structures (such as glomerulus, proximal tubule, distal tubule, etc.) by simulating the differentiation trajectory of kidney development. In recent years, kidney organoids have become a research hotspot because they can simulate the three-dimensional spatial structure of kidney development and disease. At present, there are still many challenges in constructing a kidney organoid model of diabetic nephropathy. First, the existing kidney organoid culture technology still has obvious limitations in structural development. The number of podocytes in the formed organoid is small, and the glomerular structure is not perfect, which makes it difficult to truly reproduce the typical pathological features of diabetic nephropathy, such as glomerular basement membrane thickening, podocyte damage, and ECM accumulation. Second, kidney organoids are highly sensitive to changes in the microenvironment of the culture medium. Even slight changes can lead to abnormal morphology or development arrest of the organoid, greatly limiting the simulation of high glucose, high fat and other diabetic pathological factors. Therefore, a reliable, stable and typical diabetic nephropathy phenotype kidney organoid model needs to be constructed. SUMMARY

[0005] The purpose of the present application is to provide a diabetic nephropathy kidney organoid model and its construction method and application, in order to solve the technical problems of the prior art diabetic nephropathy kidney organoid model development structure being imperfect and the model being unstable.

[0006] According to a first aspect of the present application, a diabetic nephropathy kidney organoid model is provided, which is obtained by inducing a kidney organoid with a composition. The composition comprises glucose, palmitic acid and linoleic acid.

[0007] The present application first uses linoleic acid as an inducing factor to introduce a diabetic nephropathy kidney organoid model into the model construction, and determines that the combination of glucose, palmitic acid and linoleic acid can stably induce kidney organoids to produce pathological features consistent with diabetic nephropathy, such as podocyte damage and basement membrane thickening. The technical problem of the prior art that the commonly used inducing factors (such as glucose, palmitic acid, AGEs) cannot induce typical diabetic nephropathy pathological phenotypes when introduced into the kidney organoid system, or cause excessive damage to the organoid, affecting its survival and structural integrity, is solved.

[0008] In some embodiments, the concentration of glucose in the composition is 25-35 mM; the concentration of palmitic acid is 0.15-0.25 mM; and the concentration of linoleic acid is 0.15-0.25 mM. Preferably, the concentration of glucose is 30 mM; the concentration of palmitic acid is 0.2 mM; and the concentration of linoleic acid is 0.2 mM.

[0009] In some embodiments, the time for inducing kidney organoids with the composition is 72-96 hours. Preferably, the time used is 72 hours.

[0010] According to a second aspect of the present application, there is provided a method for constructing a diabetic nephropathy kidney organoid model, comprising the following steps: (1) resuspending human embryonic stem cells in a first culture medium containing a ROCK inhibitor, CHIR 99021 and an antioxidant, culturing the cell suspension, replacing the first culture medium containing the ROCK inhibitor, CHIR 99021 and the antioxidant with the first culture medium containing CHIR 99021 on the second day, culturing until embryoid bodies are formed, transferring the embryoid bodies to a second culture medium for further differentiation, and obtaining kidney organoids; (2) replacing the second culture medium with a third culture medium containing a composition to induce the kidney organoids, and obtaining the same.

[0011] In some embodiments, the components of the first culture medium include IMDM, Ham’s F-12 Nutrient Mix, Protein-Free Hybridoma Medium (PFHM-II), polyvinyl alcohol, GlutaMAX, lipids, ITS-X, 1-thioglycerol, AA2P and antibiotic Plasmocin.

[0012] In some embodiments, the components of the second culture medium include VEGF-A, BMP7, retinoic acid, DMEM high glucose medium, KOSR, polyvinyl alcohol, non-essential amino acids NEAA, GlutaMAX, HEPES and antibiotic Plasmocin; and the components of the third culture medium include VEGF-A, BMP7, retinoic acid, DMEM sugar-free medium, KOSR, polyvinyl alcohol, non-essential amino acids NEAA, GlutaMAX, HEPES, the composition and antibiotic Plasmocin.

[0013] In some embodiments, the components of the first medium comprise IMDM in a ratio of 0.40x~0.45x; Ham’s F-12 Nutrient Mix in a ratio of 0.40x~0.45x; Protein Free Hybridoma Medium in a ratio of 0.03x~0.06x; Polyvinyl alcohol in a volume percentage of 0.20%~0.30%; GlutaMAX in a concentration of 1x; Lipids in a concentration of 0.05x~1x; ITS-X in a concentration of 0.05x~0.1x; 1-Thio-glycerol in a volume percentage of 0.0037%~0.0042%; AA2P in a concentration of 45~55 μg / mL; and antibiotic Plasmocin in a concentration of 2.0~3.0 μg / mL. Preferably, the ratio of IMDM is 0.41x (i.e. 40.13% of the base liquid volume); the ratio of Ham’s F-12 Nutrient Mix is 0.45x (i.e. 45.38% of the base liquid volume); the ratio of Protein Free Hybridoma Medium is 0.05x (i.e. 5% of the base liquid volume); the volume percentage of Polyvinyl alcohol is 0.25%; GlutaMAX is in a concentration of 1x (diluted from 100x stock to a final concentration of 1x); Lipids are in a concentration of 1x (diluted from 100x stock to a final concentration of 1x); ITS-X is in a concentration of 0.1x (diluted 1:1000 from 100x stock); 1-Thio-glycerol is in a volume percentage of 0.0039%; AA2P is in a concentration of 50 μg / mL; and antibiotic Plasmocin is in a concentration of 2.5 μg / mL.

[0014] In some embodiments, the components of the second medium comprise DMEM (11966) in a ratio of 0.79x~1x; KOSR in a volume percentage of 10%~20%; Polyvinyl alcohol in a volume percentage of 0.20%~0.30%; HEPES in a concentration of 8~15 mM; VEGF-A in a concentration of 100~200 ng / mL; BMP-7 in a concentration of 50~100 ng / mL; Retinoic acid in a concentration of 1~2 μM; and antibiotic Plasmocin in a concentration of 2.0~3.0 μg / mL. Preferably, the ratio of DMEM (11966) is 0.79x; the volume percentage of KOSR is 15%; the volume percentage of Polyvinyl alcohol is 0.25%; non-essential amino acids NEAA is in a concentration of 1x (diluted from 100x stock to a final concentration of 1x); GlutaMAX is in a concentration of 1x (diluted from 100x stock to a final concentration of 1x); HEPES is in a concentration of 10 mM; VEGF-A is in a concentration of 200 ng / mL; BMP-7 is in a concentration of 100 ng / mL; Retinoic acid is in a concentration of 2 μM; and antibiotic Plasmocin is in a concentration of 2.5 μg / mL.

[0015] In some embodiments, the components of the third medium include DMEM (11966-025) at 0.79x~1x; KOSR at 10%~20% by volume; polyvinyl alcohol at 0.20%~0.30% by volume; non-essential amino acids (NEAA) at 1x; GlutaMAX at 1x; HEPES at 8~15 mM; and antibiotic Plasmocin at 2.0~3.0 pg / mL. Preferably, the components of the third medium include DMEM (11966-025) at 0.79x; KOSR at 15% by volume; polyvinyl alcohol at 0.25% by volume; non-essential amino acids (NEAA) at 1x (diluted from 100x stock to a final concentration of 1x); GlutaMAX at 1x (diluted from 100x stock to a final concentration of 1x); HEPES at 10 mM; and antibiotic Plasmocin at 2.5 pg / mL.

[0016] In some embodiments, the components of the first medium further include BSA and penicillin-streptomycin.

[0017] In some embodiments, the components of the first medium include BSA at 0.20%~0.30% by volume; and penicillin-streptomycin at 0.5x~2x. Preferably, the components of the first medium include BSA at 0.25% by volume; and penicillin-streptomycin at 1x (including 100 U / mL Penicillin and 100 pg / mL Streptomycin).

[0018] In some embodiments, the components of the second and third media further include penicillin-streptomycin. The components of the second and third media include penicillin-streptomycin at 0.5x~2x; and antibiotic Plasmocin at 2.0~3.0 pg / mL. Preferably, the components of the second and third media include penicillin-streptomycin at 1x (including 100 U / mL Penicillin and 100 pg / mL Streptomycin); and antibiotic Plasmocin at 2.5 pg / mL.

[0019] In some embodiments, the method of constructing a diabetic nephropathy kidney organoid model comprises the following steps: (1) isolating the human embryonic stem cells by dispase, resuspending the human embryonic stem cells in a first medium containing a ROCK inhibitor, CHIR 99021 and an antioxidant, culturing the cell suspension, then replacing half the volume of the first medium containing the ROCK inhibitor, CHIR 99021 and the antioxidant with the first medium containing CHIR 99021 on the second day, culturing until the formation of embryoid bodies, transferring the embryoid bodies to a second medium for continued differentiation, replacing half the volume of the second medium every two days, to obtain kidney organoids; (2) replacing the second medium with a third medium containing a composition to induce the kidney organoids, to obtain a diabetic nephropathy kidney organoid model.

[0020] In some embodiments, in step (1), the first medium containing CHIR 99021 is replaced with the first medium containing a ROCK inhibitor, CHIR 99021 and an antioxidant on the second day, and the embryoid bodies are transferred to the second medium for continued differentiation on the third day.

[0021] In some embodiments, the time used in step (1) is 14 days. It should be noted that the time used is counted from the day of isolating the human embryonic stem cells as day 0.

[0022] In some embodiments, the human embryonic stem cells are commercial human embryonic stem cell H9 cell line; and the dispase is dispase II.

[0023] In some embodiments, the antioxidant is selected from β-mercaptoethanol.

[0024] In some embodiments, the ROCK inhibitor is Y27632.

[0025] In some embodiments, the concentration of the ROCK inhibitor is 3.1-3.5 mM; the concentration of CHIR 99021 is 6-9 μM; and the concentration of the antioxidant is 0.7-1.2 mM. Preferably, the concentration of the ROCK inhibitor is 3.3 μM; the concentration of CHIR 99021 is 8 μM; and the concentration of the antioxidant is 1 mM.

[0026] In some embodiments, the concentration of VEGF-A is 100-200 ng / mL; the concentration of BMP7 is 50-100 ng / mL; and the concentration of retinoic acid is 1-2 μM. Preferably, the concentration of VEGF-A is 150 ng / mL; the concentration of BMP7 is 75 ng / mL; and the concentration of retinoic acid is 1.5 μM.

[0027] By increasing the concentrations of VEGF-A, BMP7 and retinoic acid in the second medium in the second stage of differentiation, kidney organoids with more obvious glomerular structures and higher proportions of basement membranes are successfully obtained, significantly improving the structural maturity of the model.

[0028] According to a third aspect of the present invention, the application of a kidney organoid model for diabetic nephropathy in pathological mechanism research, drug screening, and regenerative medicine research is provided.

[0029] The beneficial effects of this invention are as follows: (1) This invention introduces linoleic acid as an inducing factor into the construction of a kidney organoid model for diabetic nephropathy for the first time, and determines that the combined treatment of glucose with palmitic acid and linoleic acid can stably induce kidney organoids to produce pathological features consistent with diabetic nephropathy, such as podocyte damage and basement membrane thickening. (2) In the second stage of differentiation, this invention optimizes the differentiation culture system by selecting the concentrations of VEGF-A, BMP7 and retinoic acid in the culture medium, enhances the development of podocytes and glomeruli in kidney organoids, and successfully obtains kidney organoids with more obvious glomerular structure and increased basement membrane ratio, which significantly improves the structural maturity of the kidney organoid model of diabetic nephropathy and lays a solid foundation for the structure of DN model. Attached Figure Description

[0030] Figure 1 (A) shows the immunofluorescence staining results of kidney organoids obtained from conventional and optimized culture media. Figure 1 (B) Quantitative statistical data of NPHS1 in kidney organoids obtained from conventional and optimized culture media; Figure 2 The results of qPCR analysis of kidney organoids obtained from conventional and optimized culture media are shown. Figure 3 Immunofluorescence staining results of kidney organoids differentiated from H9 cell lines under optimized culture conditions; Figure 4 (A) shows the immunofluorescence staining results commonly used in cell line induction factors to attempt to construct diabetic organ models. Figure 4 (B) represents the fluorescence intensity of NPHS1 in each group of kidney organoids; Figure 5 (A) shows the immunofluorescence staining results of kidney organoids in each group. Figure 5 (B) represents the fluorescence intensity of NPHS1 in each group of kidney organoids; Figure 6 (A) shows the white light images of each group after Masson staining. Figure 6 (B) is a statistical quantification chart of ECM for each group; Figure 7 Results of qPCR analysis of kidney organoids in each group; Figure 8 The results of Western blot analysis of renal organoid proteins in each group; Figure 9 The transmission electron microscopy characterization results of each group of kidney organoids are compared with the thickness of GBM. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to the accompanying drawings, but the embodiments of the present invention are not limited thereto. The raw materials and reagents involved in the following embodiments are all commercially available.

[0032] I. Materials 1. Preparation of stock solution (1) Glucose stock solution (3000 mM): Weigh 54 g of glucose powder (Glucose, #Sigma G7021), dissolve it in 80 mL of sterile ultrapure water, and bring the volume to 100 mL. Filter sterilize using a 0.1 μM filter. Store at low temperature for later use.

[0033] (2) Palmitic acid stock solution (100 mM): Weigh 128.2 mg of palmitic acid powder (PA, #Sigma-Aldrich P5585) and dissolve it in 5 mL of preheated 0.1 M NaOH (70℃); dissolve 1.92 g of FFA-free BSA (Gibco 15260037) in 40 mL of sterile PBS preheated to 55℃ (final concentration 4.8%, w / v), stir to dissolve, and filter to sterilize to obtain PA-NaOH solution; slowly add PA-NaOH solution dropwise to BSA solution (keep stirring at 60℃), control the molar ratio of PA to BSA to be 3:1~5:1; add PBS to a final volume of 50 mL, continue stirring for 1 hour, the solution should be pale yellow and transparent; filter to sterilize with a 0.1 μM filter, dispense and store at -80℃, avoid repeated freeze-thaw cycles.

[0034] (3) Linoleic acid stock solution (50 mM): Weigh 1.402 g of linoleic acid (LA, Sigma, #L1012) and dissolve it in 100-200 μL of ethanol, using a 37°C water bath to aid dissolution. Dissolve 2.5 g of FAF-BSA (Sigma A8806) in 10 mL of PBS to obtain a BSA solution. Under conditions of 37-45°C, slowly add LA to the BSA solution, continuously stirring gently for 30-60 minutes. Filter through a 0.1 μm filter membrane and collect the sterile LA-BSA composite solution. Aliquot into light-protected EP tubes and store at -20°C, avoiding repeated freeze-thaw cycles.

[0035] (4) Mannitol stock solution (2400 mM): Weigh 43.7 g of mannitol powder (Mannitol, #Sigma M4125), dissolve it in 80 mL of sterile ultrapure water, and bring the volume to 100 mL. Filter sterilize using a 0.1 μM filter. Store at low temperature for later use.

[0036] In the “additives” described below in this invention, “glucose” can be prepared by the above method to form a glucose stock solution and then added to the culture medium; “mannitol” can be prepared by the above method to form a mannitol (Mannitol) stock solution and then added to the culture medium; “linoleic acid” can be prepared by the above method to form a linoleic acid stock solution and then added to the culture medium; and “palmitic acid” can be prepared by the above method to form a palmitic acid stock solution and then added to the culture medium.

[0037] 2. Culture medium formulation (1) First stage culture medium The first stage culture medium (first medium) is the modified bovine serum albumin-polyvinyl alcohol essential lipid medium (BPEL medium), which includes basal medium (IMDM), Ham's F-12 nutrient mixture, PFHM-II, polyvinyl alcohol (PVA), GlutaMAX, and Lipid concentrate. The specific components and concentrations are shown in Table 1.

[0038] Table 1 Formulation of the First Culture Medium

[0039] To achieve the desired concentration, the IMDM basal liquid volume percentage was 40.13%; Ham's F-12 Nutrient Mix basal liquid volume percentage was 45.38%; protein-free hybridoma medium (PFHM-II) basal liquid volume percentage was 5%; GlutaMAX was diluted from 100×stock to a final concentration of 1×; Lipid concentrate was diluted from 100×stock to a final concentration of 1×; and ITS-X was diluted from 100×stock to 1:1000.

[0040] (2) Second stage culture medium (second culture medium) The optimized culture medium (second medium) includes basal medium (DMEM #11965), KOSR, polyvinyl alcohol (PVA), NEAA, GlutaMAX, VEGF-A, BMP7, retinoic acid and HEPES. The specific components and concentrations are shown in Table 2.

[0041] Table 2 Optimized culture medium formulation

[0042] To achieve the desired concentration, non-essential amino acids (NEAA) are diluted from 100× stock to a final concentration of 1×; GlutaMAX is diluted from 100× stock to a final concentration of 1×. In the P / S, a 1× concentration includes 100 U / mL penicillin and 100 μg / mL streptomycin.

[0043] (3) Third culture medium The third culture medium formulation is shown in Table 3.

[0044] Table 3 Third Culture Medium Formulation

[0045] Note: The third culture medium also includes additives. Different experimental groups add different reagents according to experimental needs. For example, the Ctrl group also adds glucose, the DN model group needs to add a combination of (glucose, PA, LA), and the isotonic group needs to add mannitol and glucose.

[0046] It should be noted that KOSR (KnockOut Serum Replacement) is a serum substitute with a clearly defined chemical composition that replaces fetal bovine serum. It is particularly suitable for serum-free culture of stem cells (such as iPSCs and ESCs), providing the proteins, lipids, and trace elements required to maintain the growth and undifferentiated state of pluripotent stem cells. It is often used in the early differentiation stage to support cell survival and proliferation during mesodermal induction and plays an important role in the induction of mesoderm and renal progenitor cells. AA2P is ascorbate-2-phosphate; ITS-X is an insulin-transferrin-selenoethanolamine supplement; GlutaMAX (L-Alanyl-L-Glutamine) replaces L-glutamine in cell culture, providing essential amino acids for cell growth. Unlike traditional L-glutamine, GlutaMAX is L-alanyl-L-glutamine. Dipeptides, more stable and less prone to degradation, provide glutamine, essential for energy metabolism and protein synthesis in kidney organoids, while avoiding the toxic accumulation of ammonia; NEAAs are non-essential amino acids, supplementing amino acids that cells can synthesize but may be insufficient in high-density cultures (such as kidney organoids); Penicillin-Streptomycin (P / S) is penicillin-streptomycin used to prevent bacterial contamination during culture; HEPES is a biological buffer used to maintain pH stability in the culture medium, enhancing the buffering capacity of the medium against CO2 fluctuations in the later stages of kidney organoid differentiation (such as the formation of glomerular structures), and preventing changes in the medium pH. These components are fundamental supporting elements for cell growth, playing a crucial auxiliary role in the formation and quality of kidney organoids; Plasmocin is a commercially available antibiotic combination specifically designed to eliminate mycoplasma contamination in cell culture; Lipid Concentrate is a lipid mixture containing essential lipid components required for cell growth; PFHM-II is a second-generation protein-free hybridoma medium.

[0047] II. Culture of Human Embryonic Stem Cells (ESCs) Cells were cultured using mTeSR (StemCell, #05850) and Matrigel medium in a humidified incubator at 37°C and 5% CO2, with the mTeSR medium replaced daily to ensure adequate nutrients and factors. Subculturing was necessary when cell confluence reached 70-80%. When the cell number exceeded 1×10⁶ cells / year... 6 At that time, differentiation can be carried out to construct kidney organoids.

[0048] III. Construction of Kidney Organoid Models 1. Construction Method On day 0, H9 cells were isolated using dispersase II (1 mg / mL, Gibco, 17105041). Cell clones were gently scraped off with a cell scraper and transferred to medium I supplemented with 3.3 μM Y27632 (BD, #562822), 8 μM CHIR99021 (Selleck, #S2924), and 100 μM β-mercaptoethanol (Gibco, #21985023) for resuscitation. The cell suspension was then cultured in 6-well ultra-low concentration attachment plates (Corning, #3471). On day 2, half the volume of medium I supplemented with 3.3 μM Y27632, 8 μM CHIR99021, and 100 μM β-mercaptoethanol was replaced with medium I supplemented with 8 μM CHIR99021. On day 3, embryoid bodies (EBs) formed. The EBs were then transferred to a second culture medium, with half the volume of the second culture medium replaced every two days. Differentiation lasted for 14 days to obtain kidney organoids.

[0049] The cells can be human embryonic stem cell line H9, and the components of the second culture medium include DMEM medium (Gibco, #11965), 100~200 ng / mL VEGF-A, 50~100 ng / mL BMP7 and 1~2 μM retinoic acid (RA).

[0050] 2. Optimization of the differentiation culture system Kidney organoids were cultured and differentiated using both conventional and optimized culture media as secondary culture media for 14 days to obtain kidney organoids.

[0051] The formulation of the optimized medium is shown in Table 2, which includes DMEM medium, 200 ng / mL VEGF-A, 100 ng / mL BMP7 and 2 μM retinoic acid.

[0052] The difference between traditional medium and optimized medium is that in traditional medium, the concentration of VEGF-A is 100 ng / mL, the concentration of BMP7 is 50 ng / mL, and the concentration of retinoic acid (RA) is 1 μM.

[0053] Immunofluorescence staining results of kidney organoids obtained from conventional culture media and optimized culture media are as follows: Figure 1 As shown, the qPCR analysis and detection data are as follows: Figure 2 As shown.

[0054] Compared to traditional culture media, the optimized culture medium increased the concentrations of VEGF-A, BMP7, and Retinoic Acid. The resulting kidney organoids showed more mature glomerular structures in immunofluorescence data, exhibiting denser cells and larger volumes. Figure 1 As shown in (A), NPHS1 (red) has a higher fluorescence intensity and is more fully expressed. Figure 1 (B) Quantitative statistical data on NPHS1 also showed that the optimized culture medium resulted in higher NPHS1 fluorescence intensity in kidney organoids compared to the conventional culture medium (n=3, * P<0.05 indicates that the expression of NPHS1 (renal podocyte-specific protein) was significantly increased in the optimized culture medium compared to the conventional culture medium. Figure 2 qPCR data showed that, compared with traditional culture medium, the optimized culture medium significantly increased the expression of podocyte-specific genes (NPHS1, WT1), glomerular endothelial cell marker (PECAM1), glomerular mesangial cell marker (ACTA2), and glomerular basement membrane (GBM) marker gene (COL4A3) in kidney organoids (n=3, * P<0.05, ** (P<0.01) indicates that the glomeruli and basement membrane of the kidney organoids obtained by the increased cytokines are more mature, providing ideal kidney organoids for the construction of DN kidney organoid models.

[0055] 3. Differentiation of H9 cell line into kidney organoids Using the optimized culture medium as the second culture medium, the H9 cell line was differentiated into kidney organoids using the above construction method.

[0056] Immunofluorescence experiments were performed on kidney organoids differentiated from H9 cell lines. First, the kidney organoids were fixed with 4% paraformaldehyde and then dehydrated using 30% sucrose solution. Next, the kidney organoids were frozen sectioned using OCT. The sections were incubated at a 1:200 ratio in primary antibody solutions containing LRP2 (Proteintech, #19700-1-AP), MEIS1 / 2 / 3 (abcam, #ab222246), GATA3 (Proteintech, 66400-1), NPHS1 (Zen Bio, 503048), and LTL. LRP2 / MEIS1 / 2 / 3, LRP2 / GATA3, and NPHS1 / LTL were co-incubated separately. After incubation overnight at 4°C, the primary antibodies were removed by washing with PBST (PBS + 0.1% Tween 20). The corresponding fluorescent secondary antibodies (anti-rabbit IgG, anti-mouse IgG, Alexa Fluor-488) were then used. The slides were incubated with CST#4412, Anti-rabbit IgG Alexa Fluor-555, CST#4413, Anti-mouse IgG Alexa Fluor-488, CST#4408, and Anti-mouse IgG Alexa Fluor-555, CST#4409) and DPAI for 2 hours at room temperature. After washing with PBST to remove the secondary antibody and DPAI, anti-fluorescence quenching mounting medium was added, and the slides were mounted and then immunofluorescence images were acquired using a laser confocal microscope.

[0057] Immunofluorescence staining results as follows Figure 3 As shown in the figure, the kidney organoids differentiated from the H9 cell line in optimized culture medium possess structures such as renal tubules (LRP2, LTL), glomeruli (NPHS1), renal interstitium (MEIS1 / 2 / 3), and collecting ducts (GATA3), and express their specific proteins. It can be seen that the differentiated kidney organoids have structures such as renal tubules, renal interstitium, glomeruli, and collecting ducts, and express specific proteins, indicating that the kidney organoids were successfully constructed.

[0058] IV. Commonly Used Inducing Factors to Construct Diabetic Organ Models Since there are currently no reported methods for constructing kidney organoid models of diabetic nephropathy, this invention attempts to introduce commonly used inducing factors (such as glucose, palmitic acid, and AGEs) currently used to construct cell models of diabetic nephropathy into the kidney organoid system.

[0059] A blank control group, a high glucose group, a palmitic acid group, an AGEs group, a high glucose combined with palmitic acid (Glu + PA) group, and a high glucose combined with palmitic acid and AGEs group were set up. After constructing kidney organoids according to the above method on day 14, the culture medium was replaced with the third culture medium shown in Table 3, and the additives were added to the third culture medium of each group as shown in Table 4. The culture was carried out from day 14 to day 17.

[0060] Table 4. Culture environment for each group

[0061] Immunofluorescence was used to analyze the glomerular status of the blank control group, high glucose group, palmitic acid group, AGEs group, high glucose combined with palmitic acid group, high glucose combined with palmitic acid group, and AGEs group. Figure 4 As shown in (A), the statistical analysis of the kidney podocyte-specific gene NPHS1 was quantified, and the results are as follows: Figure 4 (B) shows that the Ctrl, Glucose, Palmitic Acid, AGEs, and Glu+PA groups showed obvious glomerular and tubular structures, and the fluorescence value of NPHS1 showed no statistical difference. This indicates that the use of high glucose, palmitic acid, AGEs, or high glucose combined with palmitic acid alone could not significantly induce podocyte damage and destroy glomerular structure. However, the Glu+PA+AGEs group showed severe destruction of glomerular and tubular structures, and the expression levels of NPHS1 and LRP2 were extremely low. The cell nuclei stained with DAPI showed many small dots, indicating the presence of apoptosis, suggesting severe damage to the kidney organoids, affecting their cell survival and structural integrity. Therefore, the above additives either failed to induce the typical pathological phenotype of diabetic nephropathy or caused excessive damage to the kidney organoids, affecting their survival and structural integrity, and could not effectively induce the pathological phenotype of DN kidney organoids. It is evident that the reagents and combinations of common cell line inducing factors used for cell modeling are not suitable for kidney organoid modeling.

[0062] VI. Construction of a kidney organoid model for diabetic nephropathy After obtaining kidney organoids on day 14, pathological stimulation was administered from day 14 to day 17. Specifically, the high-glucose basal medium DMEM#11965 in the second culture medium was replaced with a glucose-free basal medium DMEM#11966-025 to remove the influence of glucose in the original medium. The third culture medium was prepared according to the formula shown in Table 3, and the combination (25-35 mM glucose, 0.15-0.25 mM palmitic acid and 0.15-0.25 mM linoleic acid) was added. The induction time was 3 days (72 hours) to obtain a kidney organoid model of diabetic nephropathy.

[0063] Experimental groups: normal control group (Control, Ctrl), isotonic control group (Osmotic Control, OC), and diabetic nephropathy kidney organoid model group (DN-Model).

[0064] Among them, the Ctrl group, OC group and DN-Model group prepared the third culture medium according to the formula shown in Table 3, and replaced the second culture medium used to construct kidney organoids with the third culture medium. Then, the additives were added to the culture medium of each group according to Table 5, and induced for 72 hours to obtain the kidney organoid models of each group.

[0065] In the Ctrl group, only 6 mM glucose was added to the culture medium as a low-glucose medium. In the OC group, 24 mM mannitol was added to the culture medium to achieve isotonicity with 30 mM glucose, in order to eliminate the influence of high glucose osmotic pressure.

[0066] Table 5. Additives used in the third culture medium for each group of kidney organoids

[0067] 1. Pathological observation of kidney organoids in each group (1) Immunofluorescence assay Immunofluorescence experiments were performed on the various groups of kidney organoids obtained from the culture.

[0068] Immunofluorescence assay results as follows Figure 5 As shown, Figure 5 (A) shows the immunofluorescence staining results of kidney organoids in each group; Figure 5 (B) shows the fluorescence intensity of NPHS1 in each group of kidney organoids. From... Figure 5 (A) It can be seen that the expression of green fluorescence in the kidney organoids of the DN-Model group was significantly lower than that of the Ctrl and OC groups; Figure 5 (B) Immunofluorescence intensity quantification data showed that the green fluorescence intensity corresponding to NPHS1 in the kidney organoids of the DN-Model group was significantly lower than that of the Ctrl group and the OC group (n=3, *** P < 0.001 indicates that the expression of NPHS1 in the kidney organoids of this group was significantly lower than that in the Ctrl and OC groups, suggesting severe damage to kidney podocytes.

[0069] (2) Masson staining Masson staining (Masson's Trichrome Staining) is a classic histological staining method, mainly used to detect collagen fibers in tissues. It is often used to assess the degree of renal fibrosis and extracellular matrix (ECM) accumulation. Masson staining can stain collagen fibers blue or green (depending on the dye combination) and can clearly show the accumulation of type I and type III collagen in kidney tissue. In kidney disease, extracellular matrix (such as collagen and fibronectin) accumulates abnormally. Masson staining, as a visual detection method, is helpful in studying the dynamic changes of ECM.

[0070] Masson staining was performed on kidney organoids from the Ctrl, OC, and DN-Model groups. The Masson staining method for kidney organoids was the same as for ordinary tissue samples. First, the kidney organoids fixed in 4% paraformaldehyde were dehydrated, embedded in paraffin, and sectioned. After dewaxing, Masson staining was performed according to the instructions of the Masson staining kit (LeaGene, #DC0033). Finally, after dehydration, clearing, and mounting, collagen deposition was observed under a microscope to assess the degree of fibrosis and ECM accumulation in the organoids.

[0071] Masson staining results are shown in […]. Figure 6 . Figure 6 (A) shows the white light images of each group after Masson staining. Figure 6 (B) is Figure 6 (A) ECM statistical quantification plots obtained from the blue area. As shown in the figure, compared with the OC group kidney organoids, the blue area in the Masson staining results of the DN-Model group kidney organoids was significantly increased (n=3, * P < 0.05 indicates that ECM accumulation in kidney organoids was significantly increased in the DN-Model group.

[0072] (3) qPCR analysis Fibronectin, Collagen IV, α-SMA, and TGF-β1 are commonly used marker genes in extracellular matrix (ECM) accumulation and renal fibrosis, reflecting pathological changes such as collagen deposition, matrix remodeling, and myofibroblast activation. qPCR analysis of the mRNA expression of these genes in renal organoids from the Ctrl, OC, and DN-Model groups yielded the following results: Figure 7 As shown. From Figure 7 It was observed that the expression of ECM-related genes in the kidney organoids of the DN-Model group was significantly increased, specifically, mRNA expression was significantly upregulated, such as fibronectin (…). * P<0.05), type IV collagen (Collagen IV) **P<0.01), α-SMA ( ** P<0.01) and TGF-β1 ( * P<0.05).

[0073] (4) Protein immunoblotting analysis Western blotting analysis was performed on kidney organoids from the Ctrl, OC, and DN-Model groups. The Western blotting method for kidney organoids was the same as that for ordinary tissue samples. First, total protein was extracted from the organoids using RIPA lysis buffer. After quantification, an equal amount of protein sample was added to SDS loading buffer and boiled for denaturation. After separating the proteins by SDS-PAGE electrophoresis, the samples were transferred to PVDF membranes. Non-specific sites were blocked, and the samples were incubated with primary antibody and HRP-labeled secondary antibody in sequence. The expression level of the target protein was detected by chemiluminescent substrate (ECL) imaging.

[0074] Results of Western blot analysis of proteins as follows Figure 8 As shown. The results indicated that the expression of ECM-related proteins in the kidney organoids of the DN-Model group was significantly increased. Specifically, compared to the OC group, the expression of fibronectin (…) in the DN-Model group was significantly higher. * P<0.05), type IV collagen ( * P<0.05), α-SMA ( ** Protein expression was significantly elevated (P<0.01). Compared to gene expression, protein expression is more critical. Proteins are key molecules that directly perform biological functions; therefore, protein expression levels more accurately reflect changes in cellular state and function than gene expression. Figure 8 ) and genes ( Figure 7 The results expressed are consistent with those of the DN-Model group ECM accumulation, further confirming the accumulation of ECM.

[0075] (5) Transmission electron microscopy characterization The microstructure of each group of kidney organoids was observed using transmission electron microscopy, and the results are as follows: Figure 9 As shown in the figure. Transmission electron microscopy characterization of the kidney organoids in each group revealed the presence of the glomerular basement membrane (GBM) in all groups. ImageJ software was used to measure GBM thickness, and further quantitative statistical results showed that the GBM thickness of the kidney organoids in the DN-Model group was significantly higher than that in the Ctrl and OC groups. ** P<0.01), confirming the pathological phenotype of GBM thickening in the kidney organoids of the DN-Model group.

[0076] The above experimental results show that the kidney organoids in the DN-Model group exhibited severe damage to renal podocytes, significantly increased ECM accumulation, significantly increased expression of ECM-related genes and proteins, and significantly increased GBM thickness. This indicates that the kidney organoids in the DN-Model group exhibited the pathological phenotype of diabetic nephropathy. This kidney organoid model of diabetic nephropathy was successfully constructed by inducing kidney organoids with glucose, palmitic acid, and linoleic acid.

[0077] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A kidney organoid model for diabetic nephropathy, characterized in that, Kidney organoids were obtained by inducing renal organoids with a composition comprising glucose, palmitic acid, and linoleic acid.

2. The kidney organoid model for diabetic nephropathy according to claim 1, characterized in that, The concentration of glucose is 25-35 mM; the concentration of palmitic acid is 0.15-0.25 mM; and the concentration of linoleic acid is 0.15-0.25 mM.

3. The renal organoid model for diabetic nephropathy according to claim 1 or 2, characterized in that, The time required to induce kidney organoids using the composition is 72–96 hours.

4. The method for constructing a renal organoid model of diabetic nephropathy according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Human embryonic stem cells were resuspended in a first culture medium containing ROCK inhibitor, CHIR 99021 and antioxidant. The cell suspension was cultured, and on the second day, the first culture medium containing CHIR 99021 was replaced with the first culture medium containing ROCK inhibitor, CHIR 99021 and antioxidant. The cells were cultured until embryoids were formed, and the embryoids were transferred to a second culture medium to continue differentiation to obtain kidney organoids. (2) Replace the second culture medium with the third culture medium containing the composition to induce diabetic nephropathy kidney organoids, and thus obtain the product; The components of the first culture medium include IMDM, Ham's F-12 nutrient mixture, protein-free hybridoma medium, polyvinyl alcohol, GlutaMAX, lipids, ITS-X, 1-thioglycerol, AA2P and the antibiotic Plasmocin. The components of the second culture medium include VEGF-A, BMP7, retinoic acid, DMEM high glucose medium, KOSR, polyvinyl alcohol, non-essential amino acids NEAA, GlutaMAX, HEPES and the antibiotic Plasmocin. The components of the third culture medium include: DMEM sugar-free medium, KOSR, polyvinyl alcohol, non-essential amino acids NEAA, GlutaMAX, HEPES, a combination of ingredients, and the antibiotic Plasmocin.

5. The method for constructing a kidney organoid model for diabetic nephropathy according to claim 4, characterized in that, The concentration of VEGF-A was 100-200 ng / mL; the concentration of BMP7 was 50-100 ng / mL; and the concentration of retinoic acid was 1-2 μM.

6. The method for constructing a kidney organoid model for diabetic nephropathy according to claim 4, characterized in that, In the first culture medium, the volume percentage of polyvinyl alcohol is 0.20%~0.30%; the volume percentage of 1-thioglycerol is 0.0037%~0.0042%; the concentration of AA2P is 45~55 μg / mL; and the concentration of the antibiotic Plasmocin is 2.0~3.0 μg / mL. The second culture medium contains the following components: KOSR (volume percentage) 10%–20%; polyvinyl alcohol (volume percentage) 0.20%–0.30%; HEPES (concentration) 8–15 mM; VEGF-A (concentration) 100–200 ng / mL; BMP-7 (concentration) 50–100 ng / mL; retinoic acid (concentration) 1–2 μM; and the antibiotic Plasmocin (concentration) 2.0–3.0 μg / mL. The components of the third culture medium include KOSR (volume percentage) of 10%–20%, polyvinyl alcohol (volume percentage) of 0.20%–0.30%, HEPES (concentration) of 8–15 mM, and the antibiotic Plasmocin (concentration) of 2.0–3.0 μg / mL.

7. The method for constructing a kidney organoid model for diabetic nephropathy according to claim 4, characterized in that, The first culture medium also includes BSA and penicillin-streptomycin; the second and third culture media also include penicillin-streptomycin.

8. The method for constructing a kidney organoid model for diabetic nephropathy according to claim 4, characterized in that, The concentration of the ROCK inhibitor is 3.1~3.5 mM; the concentration of CHIR 99021 is 6~9 μM; and the concentration of the antioxidant is 0.7~1.2 mM.

9. The method for constructing a kidney organoid model for diabetic nephropathy according to claim 4, characterized in that, In step (1), on the second day, half the volume of the first culture medium containing ROCK inhibitor, CHIR 99021 and antioxidant was replaced with the first culture medium containing CHIR 99021. In step (1), the embryoids are transferred from the first culture medium to the second culture medium to continue differentiation, and half the volume of the second culture medium is replaced every 2 days; Step (1) takes 14 days.

10. The application of the renal organoid model of diabetic nephropathy as described in any one of claims 1 to 3 in pathological mechanism research, drug screening, and regenerative medicine research.

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