Novel diagnosis and treatment marker CD36 for acute kidney injury caused by sepsis and application of novel diagnosis and treatment marker CD36

Abstract

The invention provides an application of a novel biomarker CD36 of acute kidney injury caused by sepsis (SAKI) in the detection, diagnosis and treatment of the SAKI, and provides an application of the novel biomarker CD36 of the acute kidney injury caused by the sepsis (SAKI) in the detection, diagnosis and treatment of the SAKI. According to the invention, bioinformatics analysis and exploration are utilized to confirm that kidney CD36 and SAKI have good correlation in people, and animal models are also verified, so that a new diagnosis and treatment marker is provided for clinical diagnosis and treatment of SAKI, and the application has important clinical significance.

Description

Technical Field

[0001] The present invention specifically relates to a novel diagnostic and therapeutic marker for sepsis-induced acute kidney injury and application thereof. Background Art

[0002] Sepsis-associated acute kidney injury (SAKI) is a serious clinical pathological state, often accompanied by systemic inflammatory response and multiple organ failure. The current treatment of sepsis-associated acute kidney injury is limited to supportive options, including antibiotic use, fluid resuscitation and early hemodynamic optimization; however, the morbidity and mortality of sepsis-associated acute kidney injury remain high; although recent studies have shown that the pathophysiology of sepsis-associated acute kidney injury involves inflammation, microvascular dysfunction, oxidative stress and apoptosis, its exact mechanism is still unclear.

[0003] As a fatty acid transport receptor, CD36 is expressed in proximal tubular cells, podocytes, mesangial cells, etc. in the kidney, and is involved in fatty acid metabolism, immune response, and inflammation regulation. Through in-depth research on the mechanism of SAKI, the present invention finds that CD36 is a potential new diagnostic and therapeutic marker that can improve the efficiency and accuracy of SAKI diagnosis and treatment, and has important clinical value. Summary of the invention

[0004] One object of the present invention is to provide the use of CD36 in preparing SAKI diagnosis or prognosis prediction products.

[0005] To achieve the above technical objectives, the present invention first studied the expression of CD36 in the renal tissue of AKI population and healthy control population, and found that the expression of CD36 in the renal tissue of AKI population was increased; further, we collected serum from clinical healthy population and sepsis patients for testing, and found that the expression of CD36 in the serum of patients with acute kidney injury caused by sepsis was also significantly increased.

[0006] Therefore, we used CRISPR-Cas9 technology to construct a CD36 knockout human renal tubular epithelial cell line (HK-2 cells), treated HK-2 cells with 10ug / ml lipopolysaccharide (LPS) to construct an in vitro model of acute kidney injury caused by sepsis, and observed the degree of inflammatory damage after 2h. The mRNA expression of intracellular inflammatory factors after LPS treatment was detected. The results showed that the expression of IL-6, TNF-α and KIM-1 in HK-2 cells increased after LPS treatment, but the inflammatory response of CD36 knockout cells was significantly lower than that of NC cells. The above results show that CD36 deficiency has a significant alleviating effect on LPS-induced acute kidney injury and can significantly improve the inflammatory symptoms during SAKI. Based on the above research results, CD36 can be preliminarily used as a marker for SAKI for the diagnosis or prognosis prediction of SAKI.

[0007] The present invention provides the use of CD36 in preparing SAKI diagnosis or prognosis prediction products.

[0008] Furthermore, the present invention provides the use of a reagent for detecting CD36 expression in the preparation of a SAKI diagnosis or prognosis prediction product.

[0009] The reagent includes a reagent for detecting the expression amount of CD36, the reagent includes a nucleic acid that binds to CD36 or its DNA sequence, and the nucleic acid includes a PCR amplification primer used when using SYBR Green, TaqMan probe, molecular beacon, double hybridization probe, or composite probe to detect the expression amount of CD36.

[0010] Furthermore, the SAKI diagnosis or prognosis prediction product includes a chip, a test paper or a kit.

[0011] The chip comprises a reagent for detecting the expression amount of CD36, wherein the reagent comprises a nucleic acid that binds to CD36 or its DNA sequence, and the nucleic acid comprises a probe that can detect the expression amount of CD36.

[0012] The kit includes an antibody against a certain peptide segment when using Western blot, ELISA, chemiluminescence or immunofluorescence to detect the expression amount of CD36 protein.

[0013] The present invention also provides the use of CD36 in screening and preparing SAKI therapeutic pharmaceutical compositions.

[0014] The present invention also provides a pharmaceutical composition for treating SAKI, wherein the pharmaceutical composition comprises an agent or compound that inhibits CD36 expression.

[0015] The pharmaceutical composition of the present invention can be administered as a medicine alone or together with other drugs.

[0016] The pharmaceutical composition of the present invention can be prepared into various preparations as required, including but not limited to tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral administration.

[0017] The administration route of the pharmaceutical composition of the present invention is not limited, as long as it can exert the desired therapeutic effect or preventive effect. In some cases, it can be administered systemically, and in some cases it can be administered locally.

[0018] The dosage of the pharmaceutical composition of the present invention is not limited as long as the desired therapeutic effect or preventive effect is obtained, and can be appropriately determined based on symptoms, gender, age, etc.

[0019] The dosage of the therapeutic pharmaceutical composition or preventive pharmaceutical composition of the present invention can be determined using, for example, the therapeutic effect or preventive effect on a disease as an indicator.

[0020] The present invention discovers a marker for diagnosing and treating SAKI, provides a new use of CD36 in preparing a SAKI diagnosis or prognosis prediction method or product, and provides a new target for the diagnosis and treatment of clinical SAKI. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Analysis of CD36 expression in renal tissue of AKI population, *P<0.05.

[0022] Figure 2 Analysis of the expression of CD36 in the serum of SAKI patients, *P<0.05.

[0023] Figure 3 Figure 2 is the result of the effect of CD36 knockout on SAKI; A is the identification result of the successful construction of the CD36 knockout HK-2 cell line, and B is the mRNA expression result of the inflammatory factors IL-6, TNF-α and kidney injury factor KIM-1 in HK-2 cells after LPS treatment, *P<0.05. DETAILED DESCRIPTION

[0024] The present invention is further described below by specific embodiments. The following examples are specific embodiments of the present invention, but the embodiments of the present invention are not limited by the following examples. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0025] Example 1: Analysis of the relationship between AKI and CD36 in the human population using bioinformatics methods

[0026] Gene chip GSE139061 was collected from the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ), and data from 9 healthy subjects and 39 AKI patients were collected. GraphPad Prism v8.0 software was used to analyze the expression of CD36 in renal tissues of the two groups of people.

[0027] Example 2: ELISA method to detect CD36 expression in the serum of SAKI and control populations

[0028] according to Wuhan Cloud Clone The seb530hu-CD36 (homo) reagent instructions were used for detection.

[0029] (1) Prepare all reagents, samples, and standards.

[0030] (2) Add 100 μl of standard or sample to each well and incubate at 37°C for 1 hour.

[0031] (3) Aspirate and add 100 μl of the prepared detection reagent A and incubate at 37°C for 1 hour.

[0032] (4) Aspirate and wash three times.

[0033] (5) Add 100 μl of the prepared detection reagent B and incubate at 37°C for 30 minutes.

[0034] (6) Aspirate and wash 5 times.

[0035] (7) Add 90 μl of substrate solution and incubate at 37°C for 10-20 minutes.

[0036] (8) Add 50 μl of stop solution and read immediately at 450 nm.

[0037] Example 3: HK-2 cell experiment

[0038] 1) Cell culture.

[0039] HK-2 cells were cultured in RPMI1640 medium, to which 10% fetal bovine serum and 1% penicillin-streptomycin mixed solution were added, and the cell culture conditions were set at 37° C. in an incubator with saturated humidity and a carbon dioxide concentration of 5%.

[0040] 2) Construction of CD36 knockout cell line.

[0041] CRISPR-Cas9 lentivirus was provided by Shanghai GeneCare Gene Medical Technology Co., Ltd. (Shanghai, China). To construct the CD36 knockout HK-2 cell line, the virus and virus infection enhancement reagent were co-transfected into the target cells, and positive cells were selected with 0.3ug / ml puromycin. CD36 knockout expression cell lines can be confirmed by Western blotting.

[0042] 3) Cell protein extraction.

[0043] (1) Pour off the cell culture medium and wash once with PBS.

[0044] (2) RIPA lysis buffer was mixed with phosphatase inhibitor A solution, phosphatase inhibitor B solution and protease inhibitor in a ratio of 100:1, and 200 μl of the prepared solution was added to each well of a six-well plate.

[0045] (3) Use a scraper to scrape off the cells and mix them thoroughly with the lysis buffer.

[0046] (4) Shake the cells at 4°C on a Ferris wheel shaker for 30 min to allow for complete lysis.

[0047] (5) Centrifuge at 12,000 × g and 4°C for 15 min.

[0048] (6) Transfer the supernatant to a clean 1.5 ml EP tube.

[0049] (7) Prepare loading buffer solution by mixing bromophenol blue and β-mercaptoethanol in a ratio of 9:1.

[0050] (8) Add 1 / 3 volume of the supernatant to each tube and mix well.

[0051] (9) Boil in a constant temperature metal bath at 100°C for 10 minutes to denature the protein and then store in a -20°C refrigerator.

[0052] 4) Extraction of total cellular RNA.

[0053] (1) Add 200 μl RNAiso lysis buffer to the cell culture dish covering the six-well plate and transfer to a 1.5 ml EP tube.

[0054] (2) Add chloroform (1 / 5 the volume of the lysate) to the EP tube, mix by inversion for 15 seconds, let stand at room temperature for 10 minutes, and then centrifuge at 12,000 × g and 4°C for 15 minutes.

[0055] (3) Add the upper aqueous phase to a new RNase-free EP tube, then add an equal volume of isopropanol to the EP tube, gently invert and mix, let stand at room temperature for 10 min, and centrifuge at 12,000 × g and 4°C for 10 min.

[0056] (4) Discard the liquid in the EP tube, add 1 ml of 75% ethanol, turn the EP tube upside down, wash thoroughly, and then centrifuge at 12,000 × g and 4°C for 10 min.

[0057] (5) Discard the supernatant and place at room temperature for 10 min to dry.

[0058] (6) Add an appropriate amount of RNase-free water to fully dissolve the RNA.

[0059] (7) The purity and concentration of RNA were tested by nanodrop2000c ultra-micro spectrophotometer. OD260 / OD280 should be between 1.8 and 2.0. The extracted RNA should be immediately reverse transcribed into cDNA and stored in a -20℃ refrigerator.

[0060] Example 4: QRT-PCR detection of the expression of IL-6, TNF-α and KIM-1 in HK-2 cells

[0061] (1) RT-PCR (reverse transcription-PCR).

[0062] After extraction, RNA reverse transcription and other further operations must be performed on ice. In this experiment, the TAKARA one-step method was used for RNA reverse transcription. The experimental reaction system is as follows:

[0063] 5X PrimeScript RT Master Mix 4μl

[0064] Total RNA 1μl

[0065] Make up to 20 μl with RNase Free ddH2O

[0066] The reaction conditions were as follows: 37°C for 15 min, 85°C for 5 s, and storage at 4°C. After RNA reverse transcription, the cDNA was diluted 1-fold and then Real-Time PCR was performed.

[0067] (2) Real-Time PCR analysis was used to detect the expression of IL-6, TNF-α and KIM-1.

[0068] Real-Time PCR analysis was performed using the fluorescent quantitative PCR instrument from Bio-Rad, USA, according to the CFX-linked Real-time System.

[0069] Primer sequences:

[0070] IL-6 forward primer sequence F: 5′-ACTCACCTCTTCAGAACGAATTG-3′

[0071] IL-6 reverse primer sequence R: 5′-CCATCTTTGGAAGGTTCAGGTTG-3′

[0072] TNF-α forward primer sequence F: 5′-GAGGCCAAGCCCTGGTATG-3′

[0073] TNF-α reverse primer sequence R: 5′-CGGGCCGATTGATCTCAGC-3′

[0074] KIM-1 forward primer sequence F: 5′-TGGCAGATTCTGTAGCTGGTT-3′

[0075] KIM-1 reverse primer sequence R: 5′-AGAGAACATGAGCCTCTATTCCA-3′

[0076] The Real-Time PCR reaction system is as follows:

[0077] 2x SP qPCR Mix 10μl

[0078] PCR forward primer 0.25 μl

[0079] PCR reverse primer 0.25 μl

[0080] DNA template 1μl

[0081] ddH2O (sterile distilled water) 8.5μl

[0082] Total 20μl

[0083] The reaction conditions are as follows:

[0084] Pre-denaturation: 50℃ for 2 min, 95℃ for 30 s.

[0085] PCR: 95°C for 20 s, 60°C for 20 s, 39 cycles.

[0086] 95℃1min, 55℃1min.

[0087] Melting curve: 55℃ to 95℃, temperature gradient is 0.5℃, time is 5s.

[0088] Statistical methods

[0089] The results were analyzed using Graphpad Prism 8.0 statistical analysis software, and t-test was used for comparison between samples.

[0090] result

[0091] 1. Analysis of CD36 expression in renal tissue of AKI population.

[0092] Bioinformatics analysis showed that the expression of CD36 in the kidney tissue of the AKI population was higher than that in the normal control group. Figure 1 .

[0093] 2. Analysis of serum CD36 expression in SAKI patients.

[0094] The experimental results showed that the expression of CD36 in the serum of SAKI patients was higher than that in the normal control group. Figure 2 .

[0095] 3. The impact of CD36 deficiency on SAKI.

[0096] 3.1 Construction of CD36 knockout cell line.

[0097] The experimental results showed that CD36 expression was significantly lost in the KO cell line, indicating that CD36 was successfully knocked out in HK-2 cells.

[0098] 3.2 Effect of CD36 knockout on LPS-induced inflammatory response of HK-2 cells.

[0099] Compared with the NC group, the RNA expression of IL-6, TNF-α and KIM-1 in HK-2 cells in the LPS-treated NC group was significantly increased. However, in the CD36KO cell line, the expression of inflammatory factors and damage factors KIM-1 in LPS-treated cells was lower than that in the LPS-treated NC group. The results showed that there was a significant difference in the expression of inflammatory factors and damage factors between the two groups (P < 0.05). The experimental results are shown in Figure 3 .

Claims

1. Use of reagents for detecting CD36 expression in the preparation of SAKI diagnosis, treatment, and prognosis products.

2. The use according to claim 1, wherein the reagent or kit is used for SAKI diagnosis or prognosis detection of CD36 expression in serum or blood samples or urine samples.

3. Application of CD36 in screening drug compositions for the treatment of SAKI.

4. Application of CD36 in the preparation of pharmaceutical compositions for the treatment of SAKI.

5. A pharmaceutical composition for treating SAKI, characterized in that: The pharmaceutical composition includes an agent or compound that inhibits CD36 expression.

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