Kidney injury exosome microRNA marker and application thereof in preparation of composition for diagnosing early kidney injury

Abstract

The invention relates to the technical field of early diagnosis of kidney injury, in particular to a kidney injury exosome microRNA marker and application thereof in preparation of a composition for diagnosing early kidney injury. The invention provides a kidney injury exosome microRNA marker, which can more accurately and comprehensively judge the degree of kidney injury, provides more powerful support for clinical diagnosis and treatment, and fills the blank of the current kidney injury diagnosis field in the aspect.

Description

Technical Field

[0001] This application relates to the technical field of early diagnosis of kidney injury, and more particularly to a kidney injury exosomal microRNA marker and its use in the preparation of compositions for diagnosing early kidney injury. Background Technology

[0002] Kidney injury is a common clinical emergency, and its early and accurate diagnosis and assessment of the extent of damage are crucial for treatment decisions and prognosis improvement. In recent years, microRNAs (miRNAs), as a class of endogenous non-coding small RNA molecules, have gradually become a research hotspot in the field of kidney injury diagnosis due to their key regulatory roles in the physiological and pathological processes of the kidneys, as well as their advantages such as high tissue specificity, strong stability, and convenient detection.

[0003] Numerous studies have confirmed the diagnostic value of miRNAs in kidney injury. For example, miR-126 is significantly downregulated in the plasma of patients with acute kidney injury, reflecting the degree of vascular endothelial damage; miR-21 is persistently highly expressed in chronic kidney disease models and is closely related to the progression of renal tubular interstitial fibrosis; miR-155 participates in renal ischemia-reperfusion injury by regulating the inflammatory response, and its urinary level is positively correlated with the severity of injury. Furthermore, the miR-29 family, due to its targeting and regulation of collagen synthesis, has been shown to serve as a potential biomarker for renal fibrosis; and miR-30e, by affecting renal tubular epithelial cell apoptosis, exhibits high sensitivity in the early detection of drug-induced kidney injury. Summary of the Invention

[0004] This application provides a renal injury exosomal microRNA marker and its use in the preparation of compositions for diagnosing early renal injury.

[0005] In a first aspect, this application provides a microRNA biomarker for exosomal renal injury, employing the following technical solution:

[0006] A kidney injury exosomal microRNA marker, the kidney injury exosomal microRNA marker comprising the sequence shown in SEQ ID NO: 1.

[0007] Secondly, this application provides the use of the above-mentioned kidney injury exosomal microRNA marker in the preparation of a composition for diagnosing early kidney injury.

[0008] Thirdly, this application provides a kit for the early diagnosis of kidney injury. The kit includes components for detecting the aforementioned kidney injury exosome microRNA biomarkers.

[0009] In summary, this application includes at least one of the following beneficial technical effects:

[0010] This application provides a renal injury exosomal microRNA biomarker that can more accurately and comprehensively assess the degree of renal injury, providing stronger support for clinical diagnosis and treatment, and filling the current gap in the field of renal injury diagnosis. Attached Figure Description

[0011] Figure 1 This refers to the cellular state of renal tubular organoids after resuscitation.

[0012] Figure 2 The cellular state of renal tubular organoids 24 hours after resuscitation.

[0013] Figure 3 This is the cell state of renal tubular organoids one week after resuscitation.

[0014] Figure 4 The results of LDH cytotoxicity assay in organoid models of different types of kidney injury.

[0015] Figure 5 The results show the fluorescence viability of organoid models with different types of kidney injury.

[0016] Figure 6 Western blotting results for organoid models of different types of kidney injury. Detailed Implementation

[0017] Before describing the embodiments of this application in detail, it should be understood that the terminology used herein is for the purpose of describing a particular embodiment only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term pertains.

[0018] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0019] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and such ranges or values ​​should be understood to include values ​​close to such ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0020] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0021] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0022] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0023] The present application will be further described in detail below with reference to the embodiments and test results.

[0024] Example 1

[0025] This embodiment demonstrates the resuscitation of renal tubular organoids. The renal tubular organoids were purchased from Danwang Medical, model number: D23046-0050. The specific process is as follows:

[0026] (I) Preparatory work

[0027] Pre-cool the refrigerated centrifuge (Beckman, Microfuge) to 4°C.

[0028] Pre-cool the pipette tip in a -20°C freezer before adding the sample.

[0029] Preheat the 24-well plate in a 37°C incubator.

[0030] Thaw the matrix adhesive (Danwang Medical, model: D23016-0010) on ice or in a 4°C freezer beforehand.

[0031] Rinse 15mL sterile centrifuge tubes with rinsing solution (Danwang Medical, model: D23025-0050) and then pre-cool them on ice.

[0032] Remove the complete culture medium (Danwang Medical, model: 108-A101-0100) from the refrigerator and allow it to equilibrate to room temperature.

[0033] (II) Recovery Process

[0034] (1) Remove the renal tubular organoids from the liquid nitrogen storage tube, place them in a -80℃ freezer for 30 minutes, and then perform the resuscitation operation.

[0035] (2) When thawing, quickly place the cryovial into a 37°C water bath and shake it rapidly for 1-2 minutes. Remove it after most of the ice in the cryovial has melted.

[0036] (3) Place the cryopreservation tubes on ice and bring them into the intercellular space. After disinfecting the tube walls with an alcohol swab, transfer the renal tubular organoid suspension to a pre-cooled 15mL sterile centrifuge tube in a biosafety cabinet (ESCO, model AC2-4S1). Add 5mL of organoid resuscitation solution (Danwang Medical, model: D23040-0010) and gently pipette 10 times with a rinsed P1000 pipette tip to mix.

[0037] (4) Centrifuge at 4℃ and 250xg for 5 min in a refrigerated centrifuge; discard the supernatant and keep the precipitate (organoid suspension) after centrifugation.

[0038] (5) Add 400 μL of pre-cooled matrix gel to the centrifuge tube and gently shake it 10-15 times to mix the matrix gel with the organoid suspension.

[0039] (6) Add the matrix gel and organoid suspension to the center of the 24-well plate in a hemispherical shape at a rate of 50 μL / well.

[0040] (7) Place the 24-well plate in a cell culture incubator (ESCO, model CCL-170B-8) at 37°C for 10 min. Avoid shaking the culture plate during placement.

[0041] (8) After incubation, remove the culture plate and add 500 μL of complete culture medium to each well.

[0042] (9) Observe the organoid resuscitation under a microscope and place the culture plate in a 37°C, 5% CO2 incubator for further culture.

[0043] (10) Organoid medium replacement: Replace with fresh culture medium every 2-3 days.

[0044] (III) Organoid resuscitation status

[0045] The cellular state of organoids after resuscitation, as follows Figure 1 As shown.

[0046] Cellular status of organoids 24 hours after resuscitation as follows Figure 2 As shown.

[0047] Depend on Figure 1 and Figure 2 It can be seen that the organoid cells were in good condition after resuscitation.

[0048] (iv) Organoid Culture Status

[0049] One week after organoid resuscitation and culture, the cell state is as follows: Figure 3As shown.

[0050] Depend on Figure 3 It can be seen that the revived organoids showed good cell growth one week after culture.

[0051] Example 2

[0052] This embodiment provides a method for constructing an organoid model of kidney injury. The organoid used is the renal tubular organoid resuscitated in Example 1.

[0053] The specific methods for constructing the aforementioned organoid model of kidney injury are as follows:

[0054] 96-well plates were seeded with 5 μL of matrix gel and 3000 cells per well, and then organoid models of kidney injury were constructed using the following modeling methods.

[0055] (1) Two kidney injury organoid models were obtained by inducing kidney injury for 72 hours with 10 μM and 100 μM cisplatin (Sigma, model P4394) respectively.

[0056] (2) Two kidney injury organoid models were obtained by inducing kidney injury for 24h and 48h with 100μg / ml gentamicin (MCE, model HY-K1050).

[0057] (3) Kidney injury organoid models were induced for 24 hours by using 50 μg / ml, 100 μg / ml and 200 μg / ml LPS (lipopolysaccharide, Sigma, model L4130) respectively, and three kidney injury organoid models were obtained respectively.

[0058] (4) After 72 hours of hypoxia culture, the kidney was reoxygenated for 6 hours to construct an organoid model of hypoxia-reoxygenation kidney injury.

[0059] After the above treatments, cisplatin 10 μM, cisplatin 100 μM, gentamicin 24h, gentamicin 48h, LPS 50 μg / ml, LPS 100 μg / ml, LPS 200 μg / ml and hypoxic injury organoid models were obtained respectively.

[0060] Example 3

[0061] In this embodiment, kidney injury organoid models were constructed according to the method provided in Example 2, and LDH cytotoxicity was detected using an LDH detection kit (Beyotime, model C0016).

[0062] The detection method is as follows: Renal tubular organoids were stimulated according to the four methods provided in Example 2. After stimulation, the supernatant was aspirated, and 150 μL of LDH release reagent (1 volume of LDH release reagent added to 10 volumes of PBS) diluted 10-fold with PBS was added and mixed well. The culture plate was shaken appropriately to mix, and then incubated in a cell culture incubator for 1 hour. Subsequently, 120 μL of the supernatant from each well was taken and added to the corresponding well of a new 96-well plate, and the samples were immediately measured. The negative control group consisted of normally cultured organoid cells without modeling treatment (i.e., the resuscitated renal tubular organoids from Example 1).

[0063] The measurement process is as follows:

[0064] a. Add 60 μL of LDH detection working solution to each well.

[0065] b. Mix well and incubate at room temperature (approximately 25°C) in the dark for 30 minutes (you can wrap it in aluminum foil and place it on a horizontal shaker or a side-swing shaker with gentle shaking). Then, measure the absorbance at 490 nm using a microplate reader (Thermo Fisher Scientific). Use any wavelength of 600 nm or greater as a reference wavelength for dual-wavelength measurement.

[0066] c. Calculate cytotoxicity.

[0067] Cytotoxicity is calculated as follows:

[0068] Cytotoxicity (%) = (Experimental group LDH activity - Negative control LDH activity) / Negative control LDH activity × 100%.

[0069] Test results as follows Figure 4 As shown in the figure. The control group represents the results of the negative control group.

[0070] Depend on Figure 4 It can be seen that the cytotoxicity of the kidney injury organoid model constructed using 100 μM cisplatin and 200 μg / ml LPS reached 80% and 58%, respectively, indicating that most of the cells in the kidney injury organoid model constructed using the above method were damaged, and 100 μM cisplatin and 200 μg / ml LPS have strong cytotoxicity.

[0071] Furthermore, the cytotoxicity of the kidney injury organoid model constructed using 10 μM cisplatin reached 48%, indicating that the cells in this model suffered significant damage, demonstrating the cytotoxicity of 10 μM cisplatin. In contrast, the cytotoxicity of kidney injury organoid models constructed using other methods was all below 20%.

[0072] Example 4

[0073] In this embodiment, kidney injury organoid models were constructed according to the method provided in Example 2, and fluorescence activity was detected using an organoid fluorescence activity detection kit (Bozhen, model E238004).

[0074] The detection method is as follows: Renal tubular organoids were stimulated according to the four methods provided in Example 2. After stimulation, the supernatant was aspirated, and 100 μl of the prepared 1× organoid viability detection working solution was added (component B organoid viability detection buffer was used to dilute component A 10× live cell fluorescent dye to 1×). The culture plate was shaken appropriately to mix, and then incubated in a cell culture incubator for 1 hour. Cell viability was then detected using a fluorescence microplate reader. The control group consisted of normally cultured organoid cells without modeling treatment (i.e., the resuscitated renal tubular organoids from Example 1).

[0075] Cell viability is calculated as follows: Cell viability = Excitation wavelength Ex / Emission wavelength Em × 100%; that is, Cell viability = 560(±10)Ex / 590(±10)Em × 100%.

[0076] Test results as follows Figure 5 As shown.

[0077] Depend on Figure 5 It can be seen that the cell viability of the kidney injury organoid model constructed using 100 μM cisplatin and hypoxia injury decreased to below 50%, while the cytotoxicity of the kidney injury organoid model constructed using other methods was above 60%.

[0078] Example 5

[0079] In this embodiment, kidney injury organoid models were constructed according to the method provided in Example 2, and proteins were extracted from them and detected by Western blotting (WB). The specific process is as follows:

[0080] The detection method is as follows: The renal tubular organoids were stimulated according to the four methods provided in Example 2. After stimulation, the supernatant was aspirated, and the samples were prepared.

[0081] (I) Sample Preparation

[0082] (1) Collecting cells: Observe the cell state under a microscope, remove the culture medium, add PBS pre-cooled at 4°C and gently shake to wash the cells, then discard the washing solution;

[0083] (2) Digest cells with 0.25% EDTA trypsin, collect the cell pellet, and wash once with PBS;

[0084] (3) Add an appropriate amount of RIPA lysis buffer containing PI (protease inhibitor, MCE, catalog number: HY-K0011) and PMSF (phenylmethylsulfonyl fluoride, Beyotime, catalog number: ST507-10ml); wherein, the final concentration of PI is: take one PI tablet and add it to 10mL of sample and mix well to dissolve; the final concentration of PMSF is 1mM.

[0085] The RIPA lysis buffer is as follows: Tris 50mM; NaCl 150mM; Triton X-100 1%; sodium deoxycholate 0.1-1%; SDS 0.1%; adjust pH to 7.5, store at 4℃; add PI before use;

[0086] (4) On-ice lysis for 30 min (or ultrasonic disruption);

[0087] (5) Centrifuge at 15000×g, 4℃ for 15 min and collect the supernatant to obtain the Western Blot sample.

[0088] (II) Protein concentration determination (BCA method)

[0089] (1) Take 2 mg / mL BSA from -20℃, thaw it on ice, and set it aside for later use;

[0090] (2) Take several 1.5mL centrifuge tubes and label them as 0, 1, 2, 3, 5, 7, protein 1, protein 2, protein 3, etc.

[0091] (3) Determine the weight of BCA working solution according to 400μL per tube, mix solution A and solution B evenly at a volume ratio of 50:1, and store in the dark for later use.

[0092] (4) Add the following reagents to each tube as shown in Table 1;

[0093] Table 1 shows the specific reagents added.

[0094]

[0095]

[0096] (5) Vortex to mix, then momentarily separate to remove the liquid from the tube wall;

[0097] (6) Incubate in a 37℃ water bath in the dark for 30 minutes;

[0098] (7) Dispense the tubes into 96-well microplates in duplicate, taking care to avoid generating air bubbles;

[0099] (8) The multi-functional microplate reader detects OD at 562nm and calculates the concentration of different proteins according to the standard curve.

[0100] (III) SDS-PAGE

[0101] Select an appropriate separating gel concentration based on the molecular weight of the separated protein and prepare 10 mL of gel (taking a 12% gel formulation as an example, as shown in Table 2).

[0102] The preparation method of 10% SDS is as follows: accurately weigh 10g of SDS powder and dissolve it in 100mL of distilled water. Dissolve in a water bath at 55℃, adjust the pH to 7.2, and store at room temperature. If precipitation occurs, dissolve in a water bath before use.

[0103] The preparation method of 1.5 mol / L Tris-HCl (pH 8.8) is as follows: accurately weigh 18.671 g Tris (MW121.14) and dissolve it in 100 mL of deionized water, adjust the pH to 8.8, and store at room temperature or 4 °C.

[0104] Table 2 12% Gel Formulation

[0105] Serial Number type volume 1 <![CDATA[ddH2O]]> 3.3mL 2 30% Acrylamide solution 4.0mL 3 1.5 mol / L Tris-HCl (pH 8.8) 2.5mL 4 10% SDS 0.1mL 5 10% ammonium persulfate solution 0.1mL 6 TEMED 0.004mL

[0106] After adding the above reagents in sequence, mix well and pour the gel. Take an appropriate amount of ddH2O and cover the gel surface to keep the gel surface flat. After the separating gel solidifies, remove the ddH2O, prepare a 5% stacking gel according to the formula shown in Table 3, spread it on the separating gel, and insert a comb.

[0107] The preparation method of 1.0 mol / L Tris-HCl (pH 6.8) is as follows: accurately weigh 12.114 g Tris (MW121.14) and dissolve it in 100 mL of deionized water, adjust the pH to 6.8, and store at room temperature or 4 °C.

[0108] Table 3. Formulation of 5% Concentrated Gum

[0109]

[0110]

[0111] Sample loading and electrophoresis: Remove the protein sample, boil it in a constant temperature metal bath for 10 minutes, and centrifuge. Add the protein sample and protein marker to the wells of the electrophoresis gel in the required order using a pipette or sample loading needle. Cover the tank, turn on the power, and run the gel at 80V until the sample exits the stacking gel. Then, switch to 100V and run the gel until the bromophenol blue reaches the bottom of the gel.

[0112] The loading buffer (5×Loading Buffer) is as follows: 1.0 mol / L Tris.HCl (pH 6.8) 250 mmol / L; SDS 10% (w / v); bromophenol blue 0.5% (w / v); glycerol 50% (v / v). Dissolve in deionized water and aliquot into 1.5 mL centrifuge tubes (1 mL / tube). Store at room temperature. Add 50 μL of β-mercaptoethanol before use. It can be stored at room temperature for one month.

[0113] The electrophoresis buffer (Running Buffer) is as follows: Tris 0.125M; Glycine 1.25M; SDS 0.5% (w / v).

[0114] (iv) Western Blot

[0115] (1) Transfer: After electrophoresis, transfer the protein onto a PVDF membrane. Trim the size of the gel, trim the edges and excess parts, and cut the PVDF membrane to the appropriate size according to the size of the trimmed gel. Activate the membrane in an appropriate amount of methanol for 20 seconds, and then soak it in pre-cooled transfer buffer. Cut the filter paper into 8cm×10cm pieces and soak it in pre-cooled transfer buffer as well. Prepare the transfer "sandwich" in the order of sponge-filter paper-electrophoresis gel-membrane-filter paper-sponge. Add the transfer buffer to the tank, assemble the transfer apparatus, add floating ice, bury the apparatus in an ice bath or place it in a refrigerator, turn on the power, and transfer at 300mA. The transfer time is determined according to the size of the protein to be detected, and the general rule is 1kd = 1min.

[0116] The transfer buffer consisted of: Tris 0.25M; Glycine 0.2M; and Methanol 20% (V / V).

[0117] (2) Blocking: Wash the membrane after protein transfer with 1×TBST at a rate of 0.1 mL / cm. 2 Add the amount of sealing solution and seal at room temperature for 1 hour.

[0118] The preparation method of 1×TBST buffer is as follows: Take 100mL of 10×TBS buffer, dissolve it in 900mL of distilled water, add 1mL of Tween-20, and store it at room temperature after dissolution.

[0119] The preparation method of 10×TBS buffer is as follows: accurately weigh 24.2g of Tris (MW121.14) and 80.0g of NaCl, dissolve them in 1L of distilled water, adjust the pH to 7.6, and store at room temperature.

[0120] The blocking solution (5% BSA) is prepared as follows: Weigh 2g of BSA and dissolve it in 40mL of 1×TBST buffer.

[0121] (3) Add primary antibody: After blocking, rinse the PVDF membrane once with 1×TBST for 5 min each time. Dilute the primary antibody with 5% BSA (as shown in Table 4) to the working concentration, place it in the PVDF membrane, and react at 37°C for 1 h or at 4°C overnight.

[0122] Table 4 Types of Primary Antibodies

[0123] name Working concentration Band size Secondary antibody selection Anti-KIM-1 (Abclonal, model: A2831) 1:1000 50KD GAR Anti-NGAL (Abclonal, model: A2092) 1:1000 22KD GAR Anti-GAPDH (Abclonal, model AC002) 1:1000 36KD GAM

[0124] (4) Washing the membrane: Rinse the PVDF membrane three times with 1×TBST for 5 minutes each time, and wash it once with ddH2O.

[0125] (5) Add enzyme-labeled secondary antibody: Dilute horseradish peroxidase-labeled secondary antibody with 5% BSA, place it in a PVDF membrane, and react at room temperature for 1 hour.

[0126] (6) Washing the membrane: Rinse the PVDF membrane 5 times with 1×TBST for 5 minutes each time, and wash it once with ddH2O.

[0127] (7) Color development: Take out the PVDF membrane, drain the water, and lay it flat on plastic wrap with the protein side facing up. Add an equal volume of the ECL and A / B solution mixture onto the membrane to react in the dark. Clamp the membrane and transfer it into a plastic sealer, keeping the protein side facing up. Place the membrane in the imaging system, set the parameters, and start the exposure. Adjust the brightness and contrast, and save the image.

[0128] (V) Test Results

[0129] Test results as follows Figure 6 As shown.

[0130] Depend on Figure 6 It was found that all three types of primary antibodies showed bands in the kidney injury organoid models constructed using 100 μM cisplatin and the hypoxic injury organoid models, while no NGAL bands were observed in the kidney injury organoid models constructed using other methods. This indicates that the kidney injury organoid models constructed using 100 μM cisplatin and the hypoxic injury organoid models exhibit a clear kidney injury phenotype.

[0131] Example 6

[0132] In this embodiment, exosomes were extracted from the kidney injury organoid model constructed in Example 2 using ultracentrifugation.

[0133] The specific process is as follows:

[0134] (1) Samples were melted at 37°C.

[0135] (2) Transfer the sample to a new centrifuge tube and centrifuge at 2000×g and 4℃ for 30 min.

[0136] (3) Transfer the supernatant to a new centrifuge tube and centrifuge again at 10,000×g and 4℃ for 45 min to remove larger vesicles.

[0137] (4) Take the supernatant, filter it through a 0.45μm filter membrane, and collect the filtrate.

[0138] (5) Transfer the filtrate to a new centrifuge tube, select an ultracentrifuge rotor, and centrifuge at 4°C and 100,000×g for 70 min.

[0139] (6) Remove the supernatant, resuspend in 10 mL of pre-cooled 1×PBS, select an ultracentrifuge rotor, and centrifuge again at 4℃ and 100,000×g for 70 min.

[0140] (7) Remove the supernatant and resuspend in 200 μL of pre-cooled 1×PBS to obtain exosomes of the kidney injury organoid model. Store at -80℃ for later use.

[0141] Example 7

[0142] This embodiment utilizes transcriptomics to analyze the exosomes of the kidney injury organoid model obtained above.

[0143] Screening for common differential microRNAs in exosomes from the aforementioned kidney injury models requires RNA extraction, library construction, sequencing, and data analysis to ultimately identify common differential microRNAs from different models.

[0144] The core of exosomal differential microRNA (miRNA) screening is to "extract high-purity RNA from exosomals → convert the RNA into a sequenceable library → obtain miRNA sequence and abundance information through high-throughput sequencing → screen for differential miRNAs shared by multiple models through bioinformatics".

[0145] The following is a detailed explanation of the process:

[0146] (1) RNA extraction

[0147] The particle size distribution and purity of exosomes from the aforementioned kidney injury model were determined to ensure that subsequent RNA samples were high-purity exosomes. RNA was extracted from the exosomes to obtain total RNA derived from them.

[0148] (2) Database creation

[0149] Since miRNAs are small RNA fragments of approximately 20-24 nt in length, a specialized small RNA library preparation kit (such as the Illumina Small RNA Library Prep Kit) is required. The core steps are "adapter addition → reverse transcription → PCR amplification". Through PCR amplification, the miRNA cDNA fragments with ligated adapters are enriched, forming a sufficient sequencing library.

[0150] (3) Sequencing

[0151] Using Illumina high-throughput sequencing platforms (such as NovaSeq and MiSeq), and employing sequencing-by-synthesis (SBS) technology, the sequence and abundance information of miRNAs in the library were read. Raw sequencing data files (such as .fastq format) were generated.

[0152] (3) Data Analysis

[0153] The workflow followed "raw data quality control → miRNA alignment and quantification → differential miRNA screening → common differential miRNA enrichment analysis," using software such as FastQC (quality control), Bowtie (alignment), DESeq2 (differential analysis), and miRBase (database annotation). The differential miRNA lists from each model were imported into Venny 2.1 or TBtools software, and Venn diagrams were plotted. The intersection of differential miRNAs from all kidney injury models was taken as the "common differential miRNAs in kidney injury exosomes."

[0154] Based on the above analysis and comparison, one common differential microRNA marker was obtained from the exosomes of the above-mentioned kidney injury organoid models, specifically the sequence shown in SEQ ID NO: 1.

[0155] Example 8

[0156] In this embodiment, a common differential microRNA biomarker obtained in Example 7 was used as the detection target to detect two groups of organoid samples, with three replicates in each group. The detection results are shown in Table 6.

[0157] (I) Testing Process

[0158] The detection method is the same as that in Example 7.

[0159] (II) Test Results

[0160] The test results are shown in Table 6.

[0161] Table 6 Test Results

[0162]

[0163] As shown in Table 6, the expression levels of common differentially expressed microRNA markers in Sample 1 differed significantly from those in normal samples, indicating that Sample 1 was a kidney-damaged sample. In contrast, the expression levels of common differentially expressed microRNA markers in Sample 2 showed only minor differences from those in normal samples, indicating that Sample 2 was a normal sample.

[0164] Therefore, the common differential microRNA biomarker provided in this application can be used for the diagnosis of early kidney injury with higher accuracy, enabling effective monitoring and timely treatment of kidney injury at an early stage.

[0165] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0166] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A biomarker of exosomal microRNA for kidney injury, characterized in that, The kidney injury exosomal microRNA markers include sequences as shown in SEQ ID NO:

1.

2. The use of the kidney injury exosome microRNA marker of claim 1 in the preparation of a composition for diagnosing early kidney injury.

3. A reagent kit for early diagnosis of kidney injury, characterized in that, The kit includes components for detecting the kidney injury exosomal microRNA biomarkers of claim 1.

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