Phenylacetylglutamine and combination of phenylacetylglutamine and trimethylamine oxide as diagnostic marker for diabetic nephropathy

Abstract

The invention discloses phenylacetyl glutamine and a combination of phenylacetyl glutamine and trimethylamine oxide as a diagnostic marker for diabetic nephropathy. The marker provided by the invention has higher sensitivity, specificity and AUC (area under curve) value, and the excellent performance indicates that the marker has wide application potential in the fields of early diagnosis and illness monitoring of diabetic nephropathy. The invention provides a novel diagnostic tool, the morbidity of diabetes and complications thereof is increased year by year along with the change of social aging and lifestyle, the life quality of DKD patients is improved, and medical and social economic burdens are reduced.

Description

Technical Field

[0001] The present invention relates to the technical field related to the diagnosis of diabetic nephropathy, and in particular to phenylacetylglutamine and its combination with trimethylamine oxide as a diagnostic marker for diabetic nephropathy. Background Art

[0002] With the increasing prevalence of diabetes mellitus (DM), the number of patients with complications caused by DM has increased significantly. Diabetic kidney disease (DKD) is one of the chronic microvascular complications of DM and the main cause of end-stage renal disease (ESRD), which seriously threatens the life safety of patients. DKD is often hidden and progresses rapidly. Most patients have already suffered severe and irreversible renal damage when diagnosed. And the overall prognosis is poor. Even if blood sugar, blood pressure and urine protein are actively controlled, 20% to 30% of patients still have progressive decline in renal function and develop ESRD. Therefore, early diagnosis and monitoring are of great significance for delaying the progression of DKD, improving the survival rate of patients, and reducing the medical and socioeconomic burden.

[0003] Renal biopsy is considered the gold standard for diagnosing DKD, but it is not widely used due to its invasiveness. Urinary albumin-to-creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR) are currently the most commonly used markers for diagnosing and predicting the progression of DKD. The American Kidney Foundation's Kidney Disease Prognosis Quality Initiative and the Chinese Diabetic Kidney Disease Prevention and Control Guidelines both propose that eGFR < 60 ml / min / 1.73 m 2 and (or) UACR>30mg / g are the main criteria for clinical diagnosis of DKD. However, the levels of eGFR and albuminuria are affected by many factors, and there are deficiencies in sensitivity and specificity. Since biomarkers have great value in diagnosis and prediction of disease progression, and the existing markers of DKD have limitations, there is an urgent need to find new, safe, easy-to-use, highly sensitive and specific markers.

[0004] Trimethylamine N-oxide (TMAO) is metabolized by intestinal microorganisms from choline, L-carnitine, betaine and other substances into trimethylamine (TMA), which is then oxidized by flavin monooxygenase in the liver. It has been identified as a risk factor for cardiovascular disease, type 2 diabetes (T2DM) and chronic kidney disease (CKD). Two recent studies in T2DM patients have shown that the plasma TMAO level of T2DM patients with CKD or confirmed DKD is higher than that of T2DM patients alone. Elevated TMAO levels are closely related to the increased risk of DKD in T2DM patients and are one of the independent risk factors for the development of DKD. Mechanistic studies have shown that TMAO can aggravate DKD by promoting renal inflammation and fibrosis and is a pathogenic factor of DKD. Some scholars have proposed that because TMAO is closely related to the development of DKD, it has a certain clinical predictive value for DKD and may become a potential target for the early diagnosis and treatment of diabetic nephropathy. However, although it has a high specificity as a diagnostic marker for DKD, its sensitivity and AUC value are relatively low. Therefore, it is still necessary to find new diagnostic markers for diabetic nephropathy. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a new diagnostic marker for diabetic nephropathy.

[0006] The present invention also proposes the application of the above kidney disease diagnostic marker.

[0007] According to one aspect of the present invention, a use of a diabetic nephropathy diagnostic marker in the preparation of a diabetic nephropathy diagnostic reagent, a kit or a detection device is provided, wherein the diabetic nephropathy diagnostic marker comprises phenylacetylglutamine (PAGln).

[0008] According to a preferred embodiment of the present invention, at least the following beneficial effects are achieved: the markers of the present invention have high sensitivity, specificity and AUC (area under the curve) values, and these excellent properties indicate that they have broad application potential in the field of early diagnosis and disease monitoring of diabetic nephropathy. The present invention provides a new diagnostic tool. With the aging of society and changes in lifestyle, the incidence of diabetes and its complications is increasing year by year, which helps to improve the quality of life of DKD patients and reduce medical and socioeconomic burdens.

[0009] Phenylacetylglutamine (PAGln) is a metabolite of intestinal flora, which is formed by the metabolism of phenylalanine in the diet into phenylacetic acid by intestinal flora and then combined with glutamine in the liver. PAGln is mainly cleared by the kidneys, and its clearance rate is closely related to the eGFR. This characteristic makes PAGln an ideal indicator for evaluating changes in renal function and monitoring the progression of DKD. By regularly testing PAGln levels, clinicians can more accurately grasp the dynamics of DKD patients' condition and adjust treatment plans in a timely manner, thereby achieving precise management and effective control of DKD.

[0010] In some embodiments of the present invention, the diabetic nephropathy diagnostic marker also includes trimethylamine oxide. The combination of PAGln and TMAO can better achieve early diagnosis and effective monitoring of DKD. DKD is one of the main complications of type 2 diabetes (T2DM), and its early diagnosis plays a vital role in delaying disease progression and improving patient prognosis.

[0011] In some embodiments of the present invention, the diagnosis includes early diagnosis. Given that DKD is one of the main microvascular complications of diabetes, its early detection and intervention are crucial to delaying disease progression and improving patient prognosis. The present invention provides an innovative and efficient tool for the early diagnosis of DKD, which is expected to significantly reduce the disease burden of DKD patients through early intervention, improve the quality of life of patients, and indirectly reduce the overall pressure on the medical system and socio-economy.

[0012] In some embodiments of the present invention, the diagnosis also includes monitoring. The scheme of the present invention not only enriches the diagnostic means of DKD by introducing phenylacetylglutamine and trimethylamine oxide as biomarkers for early diagnosis and monitoring of DKD, but also opens up a new way for the comprehensive management of diabetes and its complications. By regularly testing the levels of markers, clinicians can more accurately grasp the disease dynamics of DKD patients and adjust the treatment plan in time, thereby achieving accurate management and effective control of DKD.

[0013] In some embodiments of the present invention, the kit includes a standard substance and an internal standard substance of the marker.

[0014] In some embodiments of the invention, the internal standard comprises phenylacetylglutamine-d5.

[0015] In some embodiments of the present invention, the internal standard further comprises deuterated trimethylamine oxide.

[0016] In some embodiments of the invention, the kit comprises a protein precipitation agent.

[0017] In some embodiments of the present invention, the protein precipitant is acetonitrile. According to another aspect of the present invention, a diabetic nephropathy marker combination is provided, comprising phenylacetylglutamine and trimethylamine oxide.

[0018] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0020] Figure 1 It is the TMAO and PAGln levels of T2DM and DKD groups detected in the specific embodiment of the present invention.

[0021] Figure 2 This is a correlation analysis between TMAO and renal function indicators of patients with type 2 diabetes in a specific embodiment of the present invention.

[0022] Figure 3 This is a correlation analysis between PAGln and renal function indicators of type 2 diabetes patients in a specific embodiment of the present invention.

[0023] Figure 4 It is a graph showing the analysis result of the ROC curve in evaluating the predictive value of TMAO and PAGln for DKD in a specific embodiment of the present invention. DETAILED DESCRIPTION

[0024] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, other embodiments obtained by technicians in this field without creative work are all within the scope of protection of the present invention. The test methods used in the embodiments are conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can be reagents and materials obtained from commercial channels. Unless otherwise specified, the same parameter in each embodiment has the same value. The embodiments described below are exemplary and are only used to explain the present invention, and cannot be understood as limitations on the present invention.

[0025] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0026] Example 1

[0027] This example provides a diagnostic marker for diabetic nephropathy, specifically phenylacetylglutamine.

[0028] Example 2

[0029] This example provides a diagnostic marker combination for diabetic nephropathy, specifically phenylacetylglutamine and trimethylamine oxide.

[0030] Comparative Example

[0031] This example provides a diagnostic marker for diabetic nephropathy, specifically trimethylamine oxide.

[0032] The markers or marker combinations of the above embodiments and comparative examples are used for diagnosing diabetic nephropathy, specifically comprising the following steps:

[0033] I. Correlation analysis between the above markers or marker combinations and diabetic nephropathy

[0034] S1. Preparation of experimental group samples and control group samples:

[0035] Experimental group: 3-5 mL of fasting venous blood was collected from patients (102 cases) diagnosed with diabetic nephropathy (DKD) and stored at 4°C for later use.

[0036] Control group: 3-5 mL of fasting venous blood was collected from patients with simple type 2 diabetes (T2DM) (63 cases) and stored at 4°C for later use.

[0037] The plasma portion was collected from the venous blood of the experimental group and the control group.

[0038] Among them, the inclusion and exclusion criteria of patients in the above experimental group and control group are as follows:

[0039] Inclusion criteria:

[0040] ① Age ≥ 18 years old; ② Patients clinically diagnosed with type 2 diabetes according to the "Guidelines for the Prevention and Treatment of Type 2 Diabetes in China (2020 Edition)", with eGFR>60 and no fundus lesions; or patients clinically diagnosed with diabetic nephropathy according to the "Guidelines for the Prevention and Treatment of Diabetic Kidney Disease in China (2021 Edition)"; ③ Voluntarily sign the informed consent form.

[0041] Exclusion criteria:

[0042] ① Patients with other important organ damage such as heart, liver, and lung; ② Patients with severe immunodeficiency, blood system diseases, severe infectious diseases, malignant tumors, etc.; ③ Patients with kidney disease that occurred earlier than the diagnosis of diabetes or primary or other secondary glomerular diseases; ④ Patients in pregnancy and lactation period; ⑤ Patients who have used antibacterial drugs or probiotic preparations within the past 4 weeks; ⑥ Patients with a history of diarrhea or currently need to use drugs to control diarrhea; ⑦ Patients with a history of surgery within the past 4 weeks; ⑧ Patients who have undergone lower gastrointestinal examination by enema within the past 4 weeks; ⑨ Patients who may not be able to complete this trial due to other reasons or who the researchers believe should not be included.

[0043] The information collected included age, gender, ethnicity, place of birth, height, weight, medical history, medication history, surgical history; systolic blood pressure (SBP), diastolic blood pressure (DSP), fasting blood glucose (GLU), glycosylated hemoglobin (HbA1c), urine microalbumin (MAU), urine creatinine (UCr), urine microalbumin / creatinine ratio (ACR), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), uric acid (UA), urea nitrogen (BUN), blood creatinine (SCr), and estimated glomerular filtration rate (eGFR, calculated using the "CKD-EPI" formula).

[0044] S2. Detection of marker concentration: The following experiments were performed on the samples of the experimental group and the control group:

[0045] A: Phenylacetylglutamine concentration test

[0046] 1) Solution preparation:

[0047] Precipitant: acetonitrile containing 20 ng / mL internal standard (phenylacetylglutamine-d5);

[0048] Standards: solutions with phenylacetylglutamine concentrations of 5, 15, 60, 300, and 600 ng / mL.

[0049] 2) Sample detection: Take an appropriate amount of plasma or standard, add a precipitant, vortex mix, and centrifuge. Transfer the supernatant to a sample injection bottle and detect it by liquid chromatography-mass spectrometry. The retention time and mass spectrum are used for qualitative analysis, and the internal standard method is used for quantitative analysis.

[0050] Chromatographic and mass spectrometric conditions were:

[0051] Instruments: Liquid phase system: ExionLC AD (Sciex, USA), mass spectrometry system: Triple Quad 6500+ (Sciex, USA);

[0052] Mobile phase: A phase is acetonitrile, B phase is 2mM ammonium acetate (0.1vol% FA) aqueous solution, chromatographic column: WatersACQUITY UPLC HSS T3 1.8; elution gradient: 0-0.5min 5.0vol%B, 0.5-1.8min 5.0vol%B-40.0vol%B, 1.8-2.2min 40.0vol%B, 2.2-2.21min 40.0vol%B-5.0vol%B, 2.21-3min5.0vol%B; flow rate: 0.3mL / min; injection volume: 2μL; column temperature: 40℃;

[0053] Ion source: Turbo VTM electrospray ion source; positive ion mode; drying gas temperature: 500°C; multiple reaction monitoring mode scanning.

[0054] B: TMAO concentration detection

[0055] 1. Take 50 μL of the sample to be tested, calibrator (5, 10, 20, 50, 100, 200, 500 or 1000 ng / mL) or quality control (low quality control (LQC), medium quality control (MQC), high quality control (HQC) are 15, 150, 800 ng / mL respectively) into a deep well plate or 1.5 mL centrifuge tube, and add 150 μL of internal standard working solution (deuterated TMAO, 50 ng / mL) to each well. (The deep well plate needs to be sealed with aluminum foil; the 1.5 mL centrifuge tube needs to be tightly covered).

[0056] 2. Vortex and oscillate for 120 seconds, and then centrifuge (deep well plate at 4000 rpm, centrifuge for 15 minutes; centrifuge tube at 12000 rpm, centrifuge for 10 minutes).

[0057] 3. Carefully transfer 50μL of supernatant to a new deep-well plate or 1.5mL centrifuge tube, and add 450μL of purified water to each well. (The deep-well plate needs to be sealed with aluminum foil; the 1.5mL centrifuge tube needs to be tightly covered)

[0058] 4. Vortex and oscillate for 120 seconds, and then centrifuge. (Deep well plate at 4000 rpm, centrifuge for 10 minutes; centrifuge tube at 12000 rpm, centrifuge for 10 minutes)

[0059] 5. Detection conditions: Chromatographic conditions: collection time is 0-2 min; flow rate is 0.3 mL / min; isocratic conditions are 30% mobile phase A; column temperature is 40°C; chromatographic column: HILIC column (or chromatographic column with equivalent performance). Mass spectrometry conditions: ionization mode is ESI+, TMAO parent-daughter ion pair is 75.95→58.09; TMAO-9 parent-daughter ion pair is 84.95→66.05.

[0060] 6. Place the centrifuged sample in the liquid phase sample tray and conduct sample testing.

[0061] 7. Method for drawing the standard curve: With the labeled concentration of TMAO in the calibrator as the horizontal axis (X), and the ratio of the measured peak area of ​​TMAO in the calibrator to the measured peak area of ​​TMAO-d9 internal standard as the vertical axis (Y), draw the standard curve and perform linear least squares regression to obtain the regression equation: Y = a + bx, weight 1 / x 2 , where a is the intercept and b is the slope, and the correlation coefficient (r) is calculated, requiring r ≥ 0.990.

[0062] 8. Calculation of test sample results: Substitute the ratio of the measured peak area of ​​TMAO in the sample to be tested to the measured peak area of ​​TMAO-9 internal standard into the above standard curve equation, and calculate the measured content of IMAO in the sample to be tested by mass spectrometry software fitting.

[0063] S3. Data Analysis

[0064] The basic clinical information and laboratory test results of patients with T2DM and DKD were compared and analyzed. The results are shown in Table 1.

[0065] Table 1

[0066]

[0067] Note: SBP: systolic blood pressure, DSP: diastolic blood pressure, GLU: fasting blood glucose, HbA1c: glycosylated hemoglobin, MAU: urine microalbumin, UCr: urine creatinine, ACR: urine microalbumin / creatinine ratio, TC: total cholesterol, TG: triglycerides, HDL: high-density lipoprotein cholesterol, LDL: low-density lipoprotein cholesterol, UA: uric acid, BUN: urea nitrogen, SCr: serum creatinine, eGFR: estimated glomerular filtration rate. * P < 0.05; ** P < 0.01; *** P<0.001.

[0068] As can be seen from Table 1, the systolic blood pressure, diastolic blood pressure, fasting blood glucose, glycosylated hemoglobin, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, blood urea, blood creatinine, urine creatinine, blood uric acid, urine microalbumin, urine microalbumin / creatinine ratio, estimated glomerular filtration rate, and TMAO level in the DKD group were significantly different from those in the T2DM group (P < 0.05).

[0069] The levels of TMAO and PAGln in T2DM and DKD groups Figure 1 As shown in the figure, the TMAO level in the DKD group was significantly higher than that in the T2DM group [623.1 (257.0, 1418.6) vs 182.5 (100.1, 289.3) ng / mL, P < 0.001], and the PAGln level in the DKD group was also significantly higher than that in the T2DM group [1558.0 (419.2, 3790.2) vs 231.0 (105.1, 446.4) ng / mL, P < 0.001].

[0070] Correlation analysis between TMAO, PAGln and renal function indexes Figures 2-3 As shown in Tables 2-3.

[0071] Table 2

[0072]

[0073] Note: SBP: systolic blood pressure; DSP: diastolic blood pressure; GLU: fasting blood glucose; HbA1c: glycosylated hemoglobin; MAU: urine microalbumin; UCr: urine creatinine; ACR: urine microalbumin / creatinine ratio; TC: total cholesterol; TG: triglycerides; HDL: high-density lipoprotein cholesterol; LDL: low-density lipoprotein cholesterol; UA: uric acid; BUN: urea nitrogen; SCr: serum creatinine; eGFR: estimated glomerular filtration rate; CAD: coronary artery disease; HTN: hypertension. *P<0.05; **P<0.01; ***P<0.001.

[0074] from Figure 2 As can be seen from Table 2, TMAO was positively correlated with blood urea (BUN), serum creatinine (SCr), serum uric acid (UA), urine microalbumin (MAU), and urine microalbumin / creatinine ratio (ACR) (r=0.610, 0.617, 0.379, 0.303, and 0.323, P<0.001), and negatively correlated with eGFR (r=-0.616, P<0.001).

[0075] Table 3

[0076]

[0077] Note: SBP: systolic blood pressure; DSP: diastolic blood pressure; GLU: fasting blood glucose; HbA1c: glycosylated hemoglobin; MAU: urine microalbumin; UCr: urine creatinine; ACR: urine microalbumin / creatinine ratio; TC: total cholesterol; TG: triglycerides; HDL: high-density lipoprotein cholesterol; LDL: low-density lipoprotein cholesterol; UA: uric acid; BUN: urea nitrogen; SCr: serum creatinine; eGFR: estimated glomerular filtration rate; CAD: coronary artery disease; HTN: hypertension. * P < 0.05; ** P < 0.01; *** P<0.001.

[0078] from Figure 3 As can be seen from Table 3, PAGln was positively correlated with blood urea (BUN), serum creatinine (SCr), serum uric acid (UA), urine microalbumin (MAU), and urine microalbumin / creatinine ratio (ACR) (r=0.657, 0.649, 0.35, 0.402, and 0.419, P<0.001), and negatively correlated with eGFR (r=-0.682, P<0.001).

[0079] Spearman correlation analysis was used to evaluate the correlation between TMAO and PAGln and other indicators. Regression analysis was used to determine the adjusted odds ratios (OR) and their 95% confidence intervals (CI) of risk factors for DKD.

[0080] Table 4

[0081]

[0082] Note: Model 1: no adjustment; Model 2: adjusted for age, sex, BMI, systolic blood pressure, diastolic blood pressure, coronary heart disease, total cholesterol, triglycerides, and low-density lipoprotein cholesterol; Model 3: adjusted for age, sex, BMI, systolic blood pressure, diastolic blood pressure, coronary heart disease, total cholesterol, triglycerides, low-density lipoprotein cholesterol, eGFR, urea, and uric acid.

[0083] Table 5

[0084]

[0085] Note: Model 1: no adjustment; Model 2: adjusted for age, sex, BMI, systolic blood pressure, diastolic blood pressure, coronary heart disease, total cholesterol, triglycerides, and low-density lipoprotein cholesterol; Model 3: adjusted for age, sex, BMI, systolic blood pressure, diastolic blood pressure, coronary heart disease, total cholesterol, triglycerides, low-density lipoprotein cholesterol, eGFR, urea, and uric acid.

[0086] As can be seen from Tables 4 and 5, regression analysis showed that after correction for age, gender, BMI, blood pressure, blood lipids, eGFR, urea, uric acid and other factors, high levels of TMAO (OR = 8.685, 95% CI, 2.368-31.854, P = 0.001) and PAGln (OR = 4.384, 95% CI, 1.049-18.330, P = 0.04) were independent risk factors for DKD patients.

[0087] II. Validation Analysis

[0088] Another batch of the same number of experimental and control groups were selected to measure the TMAO and PAGln content in plasma through similar operations as above. The receiver operating characteristic (ROC) curve was drawn to analyze the diagnostic efficacy of TMAO and PAGln alone and in combination in DKD patients, and the specificity, sensitivity, AUC and cutoff value were calculated.

[0089] The receiver operating characteristic (ROC) curve analysis results were combined with the test results. Figure 4 As shown in the figure, the correlation between each substance and chronic kidney disease is determined according to the size of the area under the curve (AUC) (between 0.5 and 1.0). The larger the AUC, the greater the diagnostic efficacy of the indicator (i.e., the higher the diagnostic accuracy). The AUC values ​​of various indicators and related statistical information are shown in Table 6 below:

[0090] Table 6

[0091]

[0092]

[0093] As can be seen from the above table, the area under the ROC curve (AUC) of TMAO for predicting DKD is only 0.7951. The plasma TMAO level at the maximum Youden index is 394.822 ng / mL, with a sensitivity of 65.69% and a specificity of 92.06%; the area under the ROC curve (AUC) of PAGln for predicting DKD can reach 0.8319, and the plasma PAGln level at the maximum Youden index is 960.91 ng / mL, with a sensitivity of 68.29% and a specificity of 88.52%. The area under the ROC curve (AUC) of TMAO combined with PAGln for predicting DKD can reach 0.8465, reaching the maximum predictive value. At the same time, the sensitivity is significantly improved, and the specificity is also maintained at a high level.

[0094] The embodiments of the present invention are described in detail above, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. Use of a diabetic nephropathy diagnostic marker in the preparation of a diabetic nephropathy diagnostic reagent, kit or detection device, characterized in that: The diabetic nephropathy diagnostic marker includes phenylacetylglutamine.

2. The use according to claim 1, characterized in that: The diabetic nephropathy diagnostic marker also includes trimethylamine oxide.

3. The use according to claim 1 or 2, characterized in that: The diagnosis includes early diagnosis.

4. The use according to claim 1 or 2, characterized in that: The diagnosis also includes monitoring.

5. The use according to claim 1 or 2, characterized in that: The kit includes a standard substance and an internal standard substance of the marker.

6. The use according to claim 5, characterized in that: The internal standard includes phenylacetylglutamine-d5.

7. The use according to claim 6, characterized in that: The internal standard also includes deuterated trimethylamine oxide.

8. The use according to claim 5, characterized in that: The kit includes a protein precipitation agent.

9. The use according to claim 8, characterized in that: The protein precipitant is acetonitrile.

10. A diabetic nephropathy marker combination, characterized in that: These include phenylacetylglutamine and trimethylamine oxide.

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