Kidney injury protein 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 renal injury, in particular to a renal injury protein marker and application thereof in preparation of a composition for diagnosing early renal injury. The invention provides a novel protein marker combination, which can more accurately and comprehensively judge the degree of renal injury, provides more powerful support for clinical diagnosis and treatment, and fills the blank in the aspect in the current field of renal injury diagnosis.

Description

Technical Field

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

[0002] Currently, the incidence of renal injuries is increasing year by year. Due to the rich blood supply to the kidneys, injury easily leads to bleeding, urine extravasation, and subsequently, critical conditions such as shock and infection. Common complications include infection, perirenal abscess, and ureteral stricture. Accurately assessing the degree of renal injury is crucial for developing treatment plans and evaluating prognosis. Protein biomarkers play an indispensable role in the diagnosis of renal injury.

[0003] Currently, various protein biomarkers are used in the diagnostic research of kidney injury. For example, microalbumin (mALB) is a widely used clinical biomarker for glomerular lesions, especially significant for the early diagnosis of diabetic-induced glomerular microvascular disease; transferrin (TRF) can be used to assess the degree of glomerular damage; the presence of immunoglobulin G (IgG) often indicates severe damage and potential disease progression. Alpha-1 microglobulin (α1-MG), β2-microglobulin (β2-MG), retinol-binding protein (RBP), and N-acetyl-β-D-glucosidase (NAG) reflect kidney damage from different perspectives, including renal tubular reabsorption function, glomerular filtration rate, proximal tubular function, and renal parenchymal lesions.

[0004] In addition, neutrophil gelatinase-associated lipocalin (NGAL) is considered the most promising biomarker for acute kidney injury, detectable in urine in the early stages of acute kidney injury and more sensitive than serum creatinine; kidney injury molecule 1 (KIM-1) can serve as a marker of proximal tubular injury; and the products of tissue inhibitor of metalloproteinases-2 (TIMP-2) and insulin-like growth factor binding protein 7 (IGFBP-7) have been established as early biomarkers for acute kidney injury.

[0005] While the aforementioned protein biomarkers have some value in the diagnosis of kidney injury, current technology still lacks a comprehensive and accurate combination of protein biomarkers that can reflect the degree of kidney damage. Currently, single or partially existing biomarker combinations are insufficient to comprehensively and accurately assess the various stages and severity of kidney injury from its onset to its progression. For example, serum creatinine, as a traditional indicator of kidney injury, suffers from poor specificity and a delayed reflection of kidney damage; its elevation does not accurately provide substantial evidence of the location, cause, and treatment of the injury. Other traditional biomarkers, such as urinalysis and blood urea nitrogen, are also affected by various factors, resulting in poor sensitivity and specificity. When these indicators become abnormal, kidney injury is often already at a relatively severe stage.

[0006] Therefore, there is an urgent need to obtain a new combination of protein biomarkers that can more accurately and comprehensively assess the degree of kidney damage, provide stronger support for clinical diagnosis and treatment, and fill the current gap in the field of kidney damage diagnosis. Summary of the Invention

[0007] This application provides a kidney injury protein biomarker and its use in the preparation of compositions for diagnosing early kidney injury.

[0008] In a first aspect, this application provides a kidney injury protein biomarker, employing the following technical solution:

[0009] A kidney injury protein biomarker, characterized in that the kidney injury protein biomarker comprises 108 proteins, as shown in the table below:

[0010]

[0011]

[0012] Secondly, this application provides a gene encoding the aforementioned kidney injury protein markers, as shown in the table above.

[0013] Serial Number Protein name The name of the gene encoding this protein Serial Number Protein name The name of the gene encoding this protein Serial Number Protein name The name of the gene encoding this protein 1 A8K2U0 A2ML1 37 P49768 PSEN1 73 Q8TB61 SLC35B2 2 O00559 EBAG9 38 P50416 CPT1A 74 Q8TCJ2 STT3B 3 O43169 CYB5B 39 P51572 BCAP31 75 Q8WVV4 POF1B 4 O43292 GPAA1 40 P51798 CLCN7 76 Q8WY22 BRI3BP 5 O60725 ICMT 41 P54252 ATXN3 77 Q92685 ALG3 6 O75352 MPDU1 42 P55060 CSE1L 78 Q96G23 CERS2 7 O76024 WFS1 43 P60660 MYL6 79 Q96GC9 VMP1 8 O76062 TM7SF2 44 P62341 SELENOT 80 Q96P63 SERPINB12 9 O95373 IPO7 45 Q01546 KRT76 81 Q96P70 IPO9 10 O95562 SFT2D2 46 Q02413 DSG1 82 Q99808 SLC29A1 11 P00374 DHFR 47 Q04695 KRT17 83 Q9BT22 ALG1 12 P02533 KRT14 48 Q07812 BAX 84 Q9BTX1 NDC1 13 P02538 KRT6A 49 Q08554 DSC1 85 Q9C0D9 SELENOI 14 P03886 MT-ND1 50 Q13454 TUSC3 86 Q9GZP9 DERL2 15 P04264 KRT1 51 Q14134 TRIM29 87 Q9H3K6 BOLA2B 16 P05089 ARG1 52 Q15041 ARL6IP1 88 Q9H4G4 GLIPR2 17 P05109 S100A8 53 Q15125 EBP 89 Q9H6U8 ALG9 18 P06702 S100A9 54 Q15392 DHCR24 90 Q9H8H3 TMT1A 19 P08779 KRT16 55 Q15517 CDSN 91 Q9HD20 ATP13A1 20 P09972 ALDOC 56 Q15828 CST6 92 Q9NR77 PXMP2 21 P0DOY2 IGLC2 57 Q16270 IGFBP7 93 Q9NRC1 ST7 22 P0DP23 CALM1 58 Q16822 PCK2 94 Q9NS69 TOMM22 23 P13645 KRT10 59 Q5D862 FLG2 95 Q9NWW5 CLN6 24 P13647 KRT5 60 Q5JS54 PSMG4 96 Q9NXE4 SMPD4 25 P16615 ATP2A2 61 Q5SNT2 TMEM201 97 Q9NYP7 ELOVL5 26 P20930 FLG 62 Q5T4F4 ZFYVE27 98 Q9NYZ1 TVP23B 27 P22532 SPRR2D 63 Q5T749 KPRP 99 Q9NZ01 TECR 28 P24390 KDELR1 64 Q6KB66 KRT80 100 Q9NZJ7 MTCH1 29 P27449 ATP6V0C 65 Q6NUQ4 TMEM214 101 Q9NZT1 CALML5 30 P29373 CRABP2 66 Q6UX53 TMT1B 102 Q9P003 CNIH4 31 P29508 SERPINB3 67 Q6ZT21 TMPPE 103 Q9P035 HACD3 32 P31151 S100A7 68 Q6ZVX7 NCCRP1 104 Q9UGT4 SUSD2 33 P31944 CASP14 69 Q7KZN9 COX15 105 Q9UI42 CPA4 34 P36952 SERPINB5 70 Q8IW92 GLB1L2 106 Q9Y487 ATP6V0A2 35 P47929 LGALS7B 71 Q8N1N4 KRT78 107 Q9Y679 AUP1 36 P48651 PTDSS1 72 Q8NHP6 MOSPD2 108 Q9Y6M5 SLC30A1 .

[0014] Thirdly, this application provides the use of the above-mentioned kidney injury protein marker in the preparation of a composition for diagnosing early kidney injury.

[0015] Fourthly, this application provides a kit for the early diagnosis of kidney injury. The kit includes components for detecting the aforementioned kidney injury protein biomarkers.

[0016] In summary, this application includes at least one of the following beneficial technical effects:

[0017] This application provides a novel combination of protein biomarkers that can more accurately and comprehensively assess the degree of kidney damage, providing stronger support for clinical diagnosis and treatment, and filling a gap in the current field of kidney damage diagnosis. Attached Figure Description

[0018] Figure 1 This refers to the cellular state of renal tubular organoids after resuscitation.

[0019] Figure 2 The cellular state of renal tubular organoids 24 hours after resuscitation.

[0020] Figure 3 This is the cell state of renal tubular organoids one week after resuscitation.

[0021] Figure 4 The results of LDH cytotoxicity assay in organoid models of different types of kidney injury.

[0022] Figure 5 The results show the fluorescence viability of organoid models with different types of kidney injury.

[0023] Figure 6 Western blotting results for organoid models of different types of kidney injury. Detailed Implementation

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] The present application will be further described in detail below with reference to the embodiments and test results.

[0031] Example 1

[0032] 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:

[0033] (I) Preparatory work

[0034] Pre-cool the refrigerated centrifuge (Beckman, Microfuge) to 4°C.

[0035] Pre-cool the pipette tip in a -20°C freezer before adding the sample.

[0036] Preheat the 24-well plate in a 37°C incubator.

[0037] Thaw the matrix adhesive (Danwang Medical, model: D23016-0010) on ice or in a 4°C refrigerator beforehand.

[0038] Rinse 15mL sterile centrifuge tubes with rinsing solution (Danwang Medical, model: D23025-0050) and then pre-cool them on ice.

[0039] Remove the complete culture medium (Danwang Medical, model: 108-A101-0100) from the refrigerator and allow it to equilibrate to room temperature.

[0040] (II) Recovery Process

[0041] (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.

[0042] (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.

[0043] (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.

[0044] (4) Centrifuge at 4℃ and 250xg for 5 min in a refrigerated centrifuge; discard the supernatant and keep the precipitate (organoid suspension) after centrifugation.

[0045] (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.

[0046] (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.

[0047] (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.

[0048] (8) After incubation, remove the culture plate and add 500 μL of complete culture medium to each well.

[0049] (9) Observe the organoid resuscitation under a microscope and place the culture plate in a 37°C, 5% CO2 incubator for further culture.

[0050] (10) Organoid medium replacement: Replace with fresh culture medium every 2-3 days.

[0051] (III) Organoid resuscitation status

[0052] The cellular state of organoids after resuscitation, such as Figure 1 As shown.

[0053] Cellular status of organoids 24 hours after resuscitation as follows Figure 2 As shown.

[0054] Depend on Figure 1 and Figure 2 It can be seen that the organoid cells were in good condition after resuscitation.

[0055] (iv) Organoid Culture Status

[0056] One week after organoid resuscitation and culture, the cell state is as follows: Figure 3 As shown.

[0057] Depend on Figure 3 It can be seen that the revived organoids showed good cell growth one week after culture.

[0058] Example 2

[0059] 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.

[0060] The specific methods for constructing the aforementioned organoid model of kidney injury are as follows:

[0061] 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.

[0062] (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.

[0063] (2) Two kidney injury organoid models were obtained by inducing kidney injury for 24h and 48h with 100μg / ml gentamicin (MCE, model HY-K1050).

[0064] (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.

[0065] (4) After 72 hours of hypoxia culture, the kidney was reoxygenated for 6 hours to construct an organoid model of hypoxia-reoxygenation kidney injury.

[0066] 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.

[0067] Example 3

[0068] 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).

[0069] 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).

[0070] The measurement process is as follows:

[0071] a. Add 60 μL of LDH detection working solution to each well.

[0072] 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.

[0073] c. Calculate cytotoxicity.

[0074] Cytotoxicity is calculated as follows:

[0075] Cytotoxicity (%) = (Experimental group LDH activity - Negative control LDH activity) / Negative control LDH activity × 100%.

[0076] Test results as follows Figure 4 As shown in the figure. The control group represents the results of the negative control group.

[0077] 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.

[0078] 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%.

[0079] Example 4

[0080] 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).

[0081] 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).

[0082] 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%.

[0083] Test results as follows Figure 5 As shown.

[0084] 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%.

[0085] Example 5

[0086] 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:

[0087] The detection method is as follows: The renal tubular organoids were stimulated using the four methods provided in Example 2. After stimulation, the supernatant was aspirated, and the samples were prepared.

[0088] (I) Sample Preparation

[0089] (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;

[0090] (2) Digest cells with 0.25% EDTA trypsin, collect the cell pellet, and wash once with PBS;

[0091] (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.

[0092] 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;

[0093] (4) On-ice lysis for 30 min (or ultrasonic disruption);

[0094] (5) Centrifuge at 15000×g, 4℃ for 15 min and collect the supernatant to obtain the Western Blot sample.

[0095] (II) Protein concentration determination (BCA method)

[0096] (1) Take 2 mg / mL BSA from -20℃, thaw it on ice, and set it aside for later use;

[0097] (2) Take several 1.5mL centrifuge tubes and label them as 0, 1, 2, 3, 5, 7, protein 1, protein 2, protein 3, etc.

[0098] (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.

[0099] (4) Add the following reagents to each tube as shown in Table 1;

[0100] Table 1 shows the specific reagents added.

[0101]

[0102] (5) Vortex to mix, then momentarily separate to remove the liquid from the tube wall;

[0103] (6) Incubate in a 37℃ water bath in the dark for 30 minutes;

[0104] (7) Dispense the tubes into 96-well microplates in duplicate, taking care to avoid generating air bubbles;

[0105] (8) The multi-functional microplate reader detects OD at 562nm and calculates the concentration of different proteins according to the standard curve.

[0106] (III) SDS-PAGE

[0107] Select an appropriate separating gel concentration based on the molecular weight of the separated protein and prepare 10 mL of gel (for example, a 12% gel formulation is shown in Table 2).

[0108] 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.

[0109] 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.

[0110] Table 2 12% Gel Formulation

[0111] 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

[0112] 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.

[0113] 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.

[0114] Table 3. Formulation of 5% Concentrated Gum

[0115] Serial Number type volume 1 <![CDATA[ddH2O]]> 2.7mL 2 30% Acrylamide solution 0.67mL 3 1.0 mol / L Tris-HCl (pH 6.8) 0.03mL 4 10% SDS 0.04mL 5 10% ammonium persulfate solution 0.04mL 6 TEMED 0.001mL

[0116] 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.

[0117] 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.

[0118] The electrophoresis buffer (Running Buffer) is as follows: Tris 0.125M; Glycine 1.25M; SDS 0.5% (w / v).

[0119] (iv) Western Blot

[0120] (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.

[0121] The transfer buffer consisted of: Tris 0.25M; Glycine 0.2M; and Methanol 20% (V / V).

[0122] (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.

[0123] 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 at room temperature after dissolution.

[0124] 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.

[0125] The blocking solution (5% BSA) is prepared as follows: Weigh 2g of BSA and dissolve it in 40mL of 1×TBST buffer.

[0126] (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.

[0127] Table 4 Types of Primary Antibodies

[0128] 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

[0129] (4) Washing the membrane: Rinse the PVDF membrane three times with 1×TBST for 5 minutes each time, and wash it once with ddH2O.

[0130] (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.

[0131] (6) Washing the membrane: Rinse the PVDF membrane 5 times with 1×TBST for 5 minutes each time, and wash it once with ddH2O.

[0132] (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.

[0133] (V) Test Results

[0134] Test results as follows Figure 6 As shown.

[0135] 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.

[0136] Example 6

[0137] This embodiment utilizes proteomics to analyze the kidney injury organoid model obtained above.

[0138] The analysis of common differentially expressed proteins in the aforementioned kidney injury models requires three core steps: proteolysis, LC-MS mass spectrometry analysis, and data analysis. The final step is to screen out the common differentially expressed proteins across different models. The specific process is described below:

[0139] (1) Proteolytic digestion: converting proteins into detectable peptides.

[0140] Total protein was extracted from the aforementioned kidney injury models, and the protein concentration was determined. Subsequently, the proteins were denatured and enzymatically digested to obtain peptide mixtures from each kidney injury model. The peptide purity and concentration met the requirements for LC-MS detection (typically 0.1-1 μg / μL).

[0141] (2) LC-MS mass spectrometry analysis: peptide separation and qualitative and quantitative analysis

[0142] Liquid chromatography (LC) separation and mass spectrometry (MS) detection were used to obtain raw mass spectrometry data (.raw format), which includes information such as peptide retention time, mass-to-charge ratio (m / z), and ionic intensity, and can be used for subsequent protein identification and quantification.

[0143] (3) Data analysis: Screening for common differentially expressed proteins

[0144] The obtained data were preprocessed to identify proteins and their relative expression levels. Statistical analysis (e.g., t-test) was performed on the protein expression levels of each kidney injury model, setting thresholds (Fold Change > 1.2 or < 0.8, P < 0.05) to screen for differentially expressed proteins (upregulated or downregulated) in each group. Venn diagrams or Volcano plots were used to compare the differentially expressed protein lists of different models, and the intersection was identified as the "common differentially expressed proteins."

[0145] Based on the above analysis, 108 common differentially expressed proteins were obtained from the cells of the aforementioned organoid models of kidney injury. These are detailed in Table 5.

[0146] Table 5. 108 common differentially expressed proteins

[0147]

[0148]

[0149] Example 7

[0150] In this embodiment, 108 common differentially expressed proteins obtained in Example 6 were used as the detection targets to detect two groups of organoid samples, with three replicates in each group. The detection results are shown in Table 6.

[0151] (I) Detection Method

[0152] The detection method is the same as that in Example 6.

[0153] (II) Test Results

[0154] The test results are shown in Table 6.

[0155] Table 6 Test Results

[0156]

[0157]

[0158] As shown in Table 6, the expression levels of all 108 common differentially expressed proteins in Sample 1 differed significantly from those in the normal samples, indicating that Sample 1 was a kidney-damaged sample. In contrast, the expression levels of all 108 common differentially expressed proteins in Sample 2 showed only minor differences from those in the normal samples, indicating that Sample 2 was a normal sample.

[0159] Therefore, the 108 common differential proteins 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 in the early stages.

[0160] 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.

[0161] 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 protein biomarker for kidney injury, characterized in that, The kidney injury protein biomarkers include 108 proteins, as shown in the table below: 。 2. A gene encoding the kidney injury protein marker of claim 1, characterized in that, The specific genes are shown in the table below: 。 3. The use of the kidney injury protein marker of claim 1 in the preparation of a composition for diagnosing early kidney injury.

4. A kit for early diagnosis of kidney injury, characterized in that, The kit includes components for detecting the kidney injury protein biomarkers of claim 1.

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