CN118858505A - Application of itaconic acid as a marker in the diagnosis of chronic kidney disease - Google Patents

Abstract

The invention discloses an application of itaconic acid as a marker in chronic kidney disease diagnosis, which quantitatively detects the content of itaconic acid in urine samples of patients through LC-MS/MS, can early warn and/or diagnose whether the patients suffer from chronic kidney disease, and provides an effective means for improving the early diagnosis rate of the chronic kidney disease and evaluating the progress of the chronic kidney disease.

Description

Application of itaconic acid as a marker in the diagnosis of chronic kidney disease

Technical Field

The present invention relates to the technical field of chronic kidney disease diagnosis, and specifically to the application of itaconic acid as a marker in the diagnosis of chronic kidney disease.

Background Art

Chronic kidney disease (CKD) is a common and serious health problem characterized by a gradual decline in renal function, which eventually leads to end-stage renal disease (ESRD). With the aging of the population and the prevalence of chronic diseases such as hypertension and diabetes, the incidence of CKD is increasing rapidly worldwide. Therefore, ESRD and its complications will further increase the global public health burden. CKD usually has no obvious symptoms in the early stages, so many patients are often diagnosed only when their renal function is already severely impaired.

Currently, the diagnosis of CKD mainly relies on renal function tests, such as the determination of serum creatinine and urea nitrogen levels, and renal imaging examinations. However, these indicators have certain limitations in the diagnosis of early CKD and monitoring of disease progression. Therefore, finding new biomarkers has become an important direction in the research field of early diagnosis of CKD and evaluation of CKD progression.

Current studies have shown that chronic inflammation of the kidney is a key factor leading to the progression of CKD, and that kidney inflammation is significantly positively correlated with the progression of CKD, indicating that kidney inflammation can be used as an indicator for the early diagnosis of CKD and the assessment of CKD progression. In recent years, researchers have paid more and more attention to the application of metabolites as biomarkers. Currently, there are no reports of non-invasive metabolites as markers of kidney inflammation. The application of advanced technologies such as high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) and metabolomics has enabled researchers to discover new biomarkers, thereby improving the ability to diagnose and monitor the disease. Among them, urine has become a non-invasive biological sample that is rich in metabolites and is therefore potentially important for the diagnosis and monitoring of CKD.

In view of the lack of biomarkers for early diagnosis of CKD and assessment of CKD progression, it is necessary to propose a biomarker.

Summary of the invention

The present invention proposes the use of itaconic acid as a marker in the diagnosis of chronic kidney disease. Quantitative detection of itaconic acid content in patient urine samples by LC-MS/MS can be used for kidney disease early warning and/or diagnosis, thereby solving the problem of the lack of biomarkers for early diagnosis of CKD and evaluation of CKD progression in the prior art.

In view of this, the scheme of the present invention is:

The present invention proposes the use of itaconic acid as a marker in the preparation of a chronic kidney disease early warning and/or diagnosis kit; the kit is used to detect the content of itaconic acid in urine.

In a preferred embodiment, the kit further comprises an itaconic acid standard, wherein the itaconic acid content in the itaconic acid standard is taken from the itaconic acid content in urine of healthy people.

In some embodiments, when the kit is used for detection, the results obtained can determine the difference in itaconic acid content compared with healthy people, thereby distinguishing chronic kidney disease patients from healthy people. When the detection value of itaconic acid in urine is greater than the concentration of the itaconic acid standard, it is determined to be chronic kidney disease.

In a preferred embodiment, the kit comprises a reagent combination for detecting the content of itaconic acid using liquid chromatography and tandem mass spectrometry, and a standard working curve of itaconic acid for liquid chromatography.

Since the test sample is human urine, the itaconic acid content is low, and the sample retention time is inconsistent when using triple quadrupole liquid mass spectrometry. Therefore, we use the accurate mass number of high-resolution liquid mass spectrometry for qualitative analysis. The resolution of high-resolution mass spectrometry is 140,000, which can exclude false positives.

In a more preferred embodiment, the reagent combination includes detection reagents for liquid chromatography: a chromatographic column, a mobile phase A, and a mobile phase B.

In a more preferred embodiment, the chromatographic column is Hypersil GOLD C18, the mobile phase A is a 0.1% formic acid aqueous solution, and the mobile phase B is a methanol solution.

In a preferred embodiment, the itaconic acid standard working curve is obtained by gradiently diluting the itaconic acid standard reagent with artificial urine to obtain itaconic acid working solutions with a certain concentration range, and the peak areas at each gradient concentration are obtained based on liquid chromatography analysis and combined with the concentration values to draw the curve; the concentration range of the itaconic acid working solution is 0.2-500 μg/mL, preferably 0.2-200 μg/mL, and more preferably 0.2-150 μg/mL.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention proposes itaconic acid as a diagnostic marker for chronic kidney disease. By quantitatively detecting the content of itaconic acid in a patient's urine sample, it is possible to warn and/or diagnose whether a patient has chronic kidney disease, thereby providing an effective means for improving the early diagnosis rate of chronic kidney disease and evaluating the progression of chronic kidney disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is the metabolomics results of urine of healthy controls and CKD patients in Example 1 of the present invention.

FIG. 2 is the result of detecting the itaconic acid content in urine of healthy controls and CKD patients using LC-MS/MS in Example 3 of the present invention.

FIG3 shows the correlation between the itaconic acid content in urine of CKD patients and the levels of blood creatinine, urea nitrogen and glomerular filtration rate in Example 3 of the present invention.

FIG. 4 shows the correlation between itaconic acid content in urine of CKD patients and renal inflammation in Example 3 of the present invention.

FIG. 5 is a research result showing that knocking out IRG1 promotes CKD progression in Example 3 of the present invention.

DETAILED DESCRIPTION

The technical solution of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

In one embodiment, the content of itaconic acid in the patient's urine sample was quantitatively detected by LC-MS/MS, and it was found that the level of itaconic acid in the urine of CKD patients was significantly increased, and the degree of renal inflammation and the levels of blood creatinine, urea nitrogen and eGFR were correlated, and it was found that the content of itaconic acid in urine was positively correlated with the levels of blood creatinine and urea nitrogen, and negatively correlated with the level of glomerular filtration rate; through the correlation analysis of the content of itaconic acid in urine and the level of renal inflammation, it was found that the positive staining area of CD68 was positively correlated with the content of itaconic acid in urine, the positive staining area of CD206 was positively correlated with the content of itaconic acid in urine, and the positive staining area of IL-1β was positively correlated with the content of itaconic acid in urine. This fully proves that itaconic acid can be used as a diagnostic marker for chronic kidney disease. Therefore, the content of itaconic acid in the patient's urine sample can be quantitatively detected by LC-MS/MS to achieve early diagnosis of CKD and evaluate the progression of CKD.

In the above embodiment, quantitative detection of itaconic acid content in patient urine sample by LC-MS/MS includes using a reagent combination for detecting itaconic acid content by liquid chromatography and tandem mass spectrometry, and a standard working curve of itaconic acid for liquid chromatography. The liquid chromatography method can select reagents for detecting itaconic acid content in liquid samples in the art, and related setting parameters; the reagents used in the liquid chromatography method include commonly used chromatographic columns, mobile phase A, and mobile phase B.

In a preferred embodiment, the chromatographic column is Hypersil GOLD C18, the mobile phase A is a 0.1% formic acid aqueous solution, and the mobile phase B is a methanol solution.

In a preferred embodiment, the itaconic acid standard working curve is a standard curve commonly used in liquid chromatography, and the itaconic acid standard reagent is gradiently diluted with artificial urine to obtain an itaconic acid working solution with a certain concentration range, and the peak area under each gradient concentration is obtained based on liquid chromatography analysis and combined with the concentration value to draw the standard curve. The concentration range of the itaconic acid working solution can be selected from 0.2-500 μg/mL, preferably 0.2-200 μg/mL, and more preferably 0.2-150 μg/mL.

The following are preferred implementation examples. Unless otherwise specified, the reagents used in the examples are commonly used commercially available reagents, and the means used are methods known in the art.

Example 1 Metabolomics Detection

After collecting urine samples from clinical CKD patients (excluding patients with hepatitis B nephropathy), sample extraction was performed and LC-MS/MS was selected as the analysis platform for sample detection and data acquisition to obtain the mass spectra or chromatograms of metabolites. The collected data were preprocessed, including data calibration, peak extraction, peak matching, and quantitative analysis. Statistical and bioinformatics analyses were performed on the processed data to identify differences between samples and the biological significance of metabolites. The OPLS-DA graph showed that the urine samples of CKD patients and healthy controls were significantly different in metabolic characteristics (Figure 1A), and the level of itaconic acid in the urine of CKD patients was significantly increased (Figure 1B).

Example 2 LC-MS/MS detection

The first morning urine samples were collected from CKD patients and healthy controls and centrifuged at 4°C, 1,500 rpm to remove the precipitate (Figure 2A). Itaconic acid in urine was quantitatively analyzed by LC-MS/MS, and the corresponding liquid chromatography parameters and mass spectrometry parameters were set to improve the sensitivity and accuracy of the detection (Figure 2B-C).

(1) Liquid chromatography parameters

Chromatographic column: Hypersil GOLD C18 (100x 2.1mm, 1.9μm)

The mobile phase composition and gradient elution conditions are shown in Table 1.

Table 1: Mobile phase composition and gradient elution table

(2) Mass spectrometry parameters are shown in Tables 2 and 3.

Table 2: Mass spectrometry parameter settings

Table 3: Mass spectrometry scanning parameter settings

(3) Drawing of standard curve

Preparation of artificial urine: 10mL ultrapure water, add 180mg urea, 5mg uric acid, 110mg NaCl, ultrasonic for 10min, mix for 3min, let stand for 5min, and take the supernatant solution for use. Use artificial urine to dilute the itaconic acid standard in a gradient to obtain an itaconic acid working solution with a concentration of 0.2-150μg/mL. Draw a standard curve based on the peak areas obtained by the liquid chromatography parameter analysis of the working solution at the concentration value.

(4) Detection

The sample was loaded onto the machine, and the sample concentration was calculated by the standard curve according to the peak area of the sample under the liquid chromatography parameters. The qualitative analysis of itaconic acid in the sample was performed by the retention time and accurate mass number of high-resolution liquid chromatography-mass spectrometry, and the accurate mass number in the negative ion mode was 129.01933.

Example 3 Analysis of itaconic acid levels and correlation in urine of CKD patients

The itaconic acid concentration data obtained by LC-MS/MS were used to compare the expression levels of itaconic acid in the urine of CKD patients and healthy controls, and it was found that the level of itaconic acid in the urine of CKD patients was significantly increased (Figure 2D). The correlation analysis was also performed with the degree of renal inflammation and the levels of serum creatinine, urea nitrogen and eGFR.

3) Correlation between urine itaconic acid content and renal function index levels: Serum creatinine, urea nitrogen and glomerular filtration rate of CKD patients were detected, and correlation analysis was performed with urine itaconic acid content. It was found that urine itaconic acid content was positively correlated with serum creatinine and urea nitrogen levels (Figure 3A, B), and negatively correlated with glomerular filtration rate levels (Figure 3C).

4) Correlation between urine itaconic acid content and renal inflammation level: Immunohistochemical staining of CD68 was performed on renal pathological sections of CKD patients (Figure 4A), and quantitative analysis was performed, and it was found that the positive staining area of CD68 was positively correlated with the urine itaconic acid content (Figure 4B). Immunohistochemical staining of CD206 was performed on renal pathological sections of CKD patients (Figure 4C), and quantitative analysis was performed, and it was found that the positive staining area of CD206 was positively correlated with the urine itaconic acid content (Figure 4D). Immunohistochemical staining of IL-1β was performed on renal pathological sections of CKD patients (Figure 4E), and quantitative analysis was performed, and it was found that the positive staining area of IL-1β was positively correlated with the urine itaconic acid content (Figure 4F).

5) Knockout of IRG1 promotes CKD progression: Itaconic acid is produced by the conversion of cis-aconitic acid catalyzed by aconitate decarboxylase 1 encoded by the IRG1 gene. Knockout of IRG1 can block the production of itaconic acid. A bilateral ischemia-reperfusion (bIRI) model was constructed using IRG1 knockout transgenic mice (Figure 5A). By detecting renal function indicators such as blood creatinine and blood urea nitrogen (Figure 5B, C), it was found that IRG1 knockout promoted further deterioration of renal function in bIRI mice.

Through the above implementation method, a new method for CKD diagnosis and progression assessment based on urinary itaconic acid is provided, which provides an effective means to improve the early diagnosis rate of CKD and evaluate the progression of CKD.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims (7)

1. The itaconic acid is used as a marker in the preparation of a chronic kidney disease early warning and/or diagnosis kit; the kit is used for detecting the content of the itaconic acid in urine.

2. The use of claim 1, wherein the kit further comprises an itaconic acid standard, the itaconic acid content of which is derived from the content of itaconic acid in urine of a healthy population.

3. The use according to claim 2, wherein the kit is for detection, and chronic kidney disease is determined when the detection value of itaconic acid in urine is greater than the concentration of itaconic acid standard.

4. The use according to claim 1, wherein the kit comprises a combination of reagents for detecting itaconic acid content using a liquid chromatography and tandem mass spectrometry technique, and an itaconic acid standard working curve for liquid chromatography.

5. The use according to claim 4, wherein the combination of reagents comprises a detection reagent for liquid chromatography: a chromatographic column, a mobile phase A and a mobile phase B.

6. The use according to claim 5, wherein the chromatographic column is Hypersil GOLD C18, the mobile phase a is 0.1% formic acid in water and mobile phase B is methanol in solution.

7. The application of claim 4, wherein the itaconic acid standard working curve is obtained by gradient dilution of itaconic acid standard reagent by artificial urine to obtain itaconic acid working solution with a certain concentration range, and is obtained by drawing peak area combined concentration values under each gradient concentration based on liquid chromatography analysis; the concentration range of the itaconic acid working solution is 0.2-500 mug/mL.