Amino acid kit for assisting diagnosis of pre-diabetic nephropathy

Abstract

The application discloses application of amino acids in a kit for assisting in diagnosing prediabetic nephropathy, and belongs to the technical field of biological medicine. The application is realized by detecting content changes or concentration ratios of specific amino acids in serum or urine. The amino acids are differential metabolites screened through serum and urine metabolomics, and have high prediction efficiency and an AUC value close to 1 or even reaching 1. The application provides a new thought for early diagnosis of diabetic nephropathy and a technical means for prognosis evaluation of treatment of diabetic nephropathy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a reagent kit for the auxiliary diagnosis of prediabetic nephropathy. Background Technology

[0002] Diabetic kidney disease (DKD) is a significant complication of diabetes, caused by prolonged diabetes leading to proteinuria and a progressive decrease in glomerular filtration rate. Currently, DKD is the second leading cause of end-stage renal disease in my country, and its progression is often reversible with early treatment.

[0003] Early diagnosis of diabetic kidney disease (DKD) primarily relies on two indicators: estimated glomerular filtration rate (eGFR) and urine albumin / creatinine ratio (UACR). Existing technology CN110987835A discloses a kit for early diagnosis of diabetic nephropathy, which uses urine trace element content detection; zinc levels less than 150 μg / L and chromium levels less than 8 μg / L are considered early-stage DKD. Existing technology CN107091927B also discloses a kit for early diagnosis of diabetic nephropathy, including urine microalbumin test strips, haptoglobin test strips, and creatinine test strips. This invention enables simultaneous detection of urine microalbumin, haptoglobin, and creatinine in a single sample, increasing the detection rate of early-stage DKD to over 95%. However, using creatinine concentration to assess GFR lacks sensitivity; creatinine can only be detected after a significant decline in renal function, and it often underestimates the GFR in DKD patients. In addition, the use of biochemical indicators such as microalbuminuria for diagnosis has a high false-positive rate, and further development of biomarkers with high sensitivity and specificity for diagnosing diabetic nephropathy is needed. Summary of the Invention

[0004] This study aims to explore the development of an amino acid model that can prevent and diagnose early-stage diabetic nephropathy, and then, based on this model, to develop a practical amino acid detection kit.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] On the one hand, the present invention provides the application of amino acids in a diagnostic kit for prediabetic nephropathy.

[0007] Specifically, the application is achieved by detecting changes in the ratio and content of specific amino acids in a sample.

[0008] Specifically, the test sample is selected from either serum or urine.

[0009] Specifically, the sample is serum, and the content of serine (Ser), methionine (Met), proline (Pro), lysine (Lys), histidine (His), alanine (Ala), threonine (Thr), glutamine (Gln), and aspartic acid (Asp) in serum is detected, or the ratios of proline / ornithine (Orn), tyrosine (Tyr) / phenylalanine (Phe), alanine / branched-chain amino acids (BCAA), and ornithine / arginine (Arg) in serum is detected; the sample is urine, and the content of glycine (Cre), lysine, tryptophan (Trp), histidine, and glutamine in urine is detected, or the ratio of tryptophan / phenylalanine is detected.

[0010] In one embodiment, the sample is serum. Diabetic nephropathy is defined as follows: Ser <10.53 mg / mL, Met <3.46 mg / mL, Pro <5.02 mg / mL, Lys <37.00 mg / mL, His <6.25 mg / mL, Ala <11.27 mg / mL, Thr <7.70 mg / mL, Gln <31.06 mg / mL, Asp <2.58 mg / mL, or a Pro / Orn ratio <0.66, a Tyr / Phe ratio <102.00, an Ala / BCAA ratio <0.12, or an Orn / Arg ratio >0.58.

[0011] In one embodiment, the sample is urine. Diabetic nephropathy is diagnosed when the urine contains Cre <32.65 mg / mL, Lys >6.89 mg / mL, Try >1.87 mg / mL, His >8.83 mg / mL, Gln >6.89 mg / mL, or a Tyr / Phe ratio >209.77 and an Ala / BCAA ratio <6.0.

[0012] Furthermore, the serum or urine samples were collected from 13-week-old and 17-week-old mice.

[0013] On the other hand, the present invention provides an amino acid metabolism model for diagnosing diabetic nephropathy, which predicts the disease by means of changes in the ratio and content of specific amino acids.

[0014] Specifically, the model also includes the measurement of important indicators of kidney injury.

[0015] In some embodiments, the kidney injury indicators include any one or more of the following: urinary albumin (ALB), urinary protein (UP), urinary creatinine (UCr), serum creatinine (SCr), 24-hour urinary protein excretion, urinary albumin to urinary creatinine ratio (ACR), triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C).

[0016] Specifically, based on the risk assessment of changes in the ratio and content of specific amino acids, the model diagnoses diabetic nephropathy as follows: urinary albumin content >911.80 ug / L, urinary protein content >87.86 ug / L, urinary creatinine content <306.32 umol / L, serum creatinine content >24.91 umol / L, 24-hour urinary protein excretion >1464.27 pg, urinary albumin to creatinine ratio >27.47 mg / g, triglyceride content >4.17 mmol / L, total cholesterol content >1.96 mmol / L, and low-density lipoprotein cholesterol content >1.33 mmol / L.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention employs a dual model of amino acid metabolism in diabetes and diabetic nephropathy, resulting in safer and more reliable predictions. It also provides a novel amino acid kit for diagnosing diabetic nephropathy, with predicted AUCs for specific amino acids approaching or even reaching 1. This offers a new technical approach for the early diagnosis and prognostic assessment of diabetic nephropathy. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The graph shows the measurement results of important indicators of kidney damage.

[0021] Figure 2 The images show the results of hematoxylin-eosin (HE), Masson, and iodate-Schiff (PAS) staining in mice.

[0022] Figure 3 This is a graph showing the results of blood metabolomics.

[0023] Figure 4 This is a graph showing the determination of biomarker levels in blood.

[0024] Figure 5 This is a graph showing the metabolomics results of urine.

[0025] Figure 6 This is a graph showing the determination of biomarker levels in urine.

[0026] Figure 7 The area under the ROC curve for serum in the control and model groups of 17-week-old mice is shown.

[0027] Figure 8 The area under the ROC curve for serum samples from 13-week-old and 17-week-old mouse model groups is given.

[0028] Figure 9 The area under the ROC curve for urine in 13-week-old and 17-week-old mouse model groups is given. Detailed Implementation

[0029] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, regarding numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in the invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Example 1: Construction of an amino acid metabolism model for diabetic nephropathy

[0031] This study used db / db mice with a BKS(D)-Leprdb / J background as an animal model. db / db mice exhibit typical clinical symptoms of diabetes, such as morbid obesity, polyphagia, thirst, and polyuria, making them an ideal animal model for type 2 diabetes.

[0032] 1. Determination of key indicators of kidney damage in diabetic nephropathy

[0033] (1) After one week of adaptive feeding, the db / db mice were randomly divided into three groups according to their body weight according to the experimental design: model group (db / db), control group (db / m) and drug administration group (db / db+positive). The control group and model group were fed standard diet and free drinking water, and the drug administration was continued for 4 weeks.

[0034] (2) During the experiment, the mice’s weight, food intake, calorie intake, and water intake were measured weekly;

[0035] (3) Blood was collected from mice in the control group and model group by capillary orbital sampling at the first and last weeks. Random blood glucose (RBG), fasting blood glucose (FBG), and glycated hemoglobin (HbA1c) levels were measured. The results are shown in the figure.

[0036] (4) Mice were placed in metabolic cages at fixed times each week to collect 24-hour urine samples. Changes in UCr, UP, 24-hour urine output, ALB, and ACR were measured. At the end of the week, changes in SCr, blood urea nitrogen (BUN), and creatinine clearance were measured. The results are as follows: Figure 1 As shown;

[0037] (5) Collect urine from mice that had been fasted on the last day of drug administration, and measure changes in triglycerides, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol. The results are as follows: Figure 1 As shown;

[0038] (6) Mice were euthanized by cervical dislocation after isoflurane anesthesia. Kidney tissue samples were collected and stained with hematoxylin-eosin, Masson's stain, and iodos-Schiff stain. The results are as follows: Figure 2 As shown, focal atrophy of renal tubules was observed in multiple locations within the renal cortex of the model group, with unclear structure. Calcifications were observed in a small number of renal tubules, and a large number of inflammatory cells were infiltrated locally. Multiple renal tubules showed atrophy and irregular arrangement. A small amount of collagen fiber proliferation was observed in the interstitium, indicating that db / db mice progressed from diabetes to early diabetic nephropathy between 13 and 17 weeks of age.

[0039] 2. Construction of an amino acid metabolism model for diabetic nephropathy

[0040] C57BL / Ks mice, homozygous for a spontaneous mutation leading to diabetes, exhibit morbid obesity, chronic hyperglycemia, pancreatic β-cell atrophy, and hypoinsulinemia. Related literature indicates that db / db mice are a genetically inherited diabetic rodent model, developing hyperglycemia, insulin resistance, and obesity 5–8 weeks after birth. After 10–20 weeks of sustained hyperglycemia, significant renal pathology is observed, similar to that seen in human diabetes. This includes mesangial expansion, mesangial matrix accumulation, and thickening of the glomerular basement membrane. Dynamic monitoring of UCr, UP, 24-hour urine output, ALB, and ACR in mice after blood glucose elevation revealed persistently elevated levels of various renal damage indicators in blood and urine between 13 and 17 weeks of age. Pathological examination of kidney tissue taken at 17 weeks of age revealed extensive inflammatory cell infiltration and mild renal tubular atrophy. The onset time points of diabetes and early diabetic nephropathy in mice were determined to be 13 weeks and 17 weeks of age, respectively. Then, targeted metabolomics was used to detect the changes in amino acid levels in serum and urine of 13-week-old and 17-week-old mice, respectively, to obtain the specific changes in amino acids in db / db mice from the progression of diabetes to the early stage of diabetic nephropathy, and to identify potential biomarkers for early diabetic nephropathy.

[0041] Figure 3 In the syllabuschert group, QXM represents serum from 13-week-old model mice, and HXM represents serum from 17-week-old model mice. Differential metabolites were screened by setting thresholds of FC > 1.2 or FC < 0.833 with a P-value < 0.05. Figure 3 serum metabolome and Figure 4The results of amino acid content analysis showed that, compared with the control group, the amino acid metabolism characteristics of db / db mice (13 weeks old) in the diabetic stage were characterized by increased levels of Met, Pro, Leu, Phe, Lys, Tyr, His, Valine (Val), Orn, Ala, Tau, Iso, Thr, Gln, and Asn. Compared with the control group, the levels of Lys, Val, and Gln amino acids were significantly decreased, while the levels of Arg and Cre were significantly increased in the early stage of diabetic nephropathy in db / db mice (17 weeks old). During the progression of the db / db mouse model (comparison between 13-week-old and 17-week-old db / db model groups), the levels of Ser, Met, Pro, Lys, His, Ala, Thr, Gln, and Asp were significantly decreased, the Pro / Orn, Tyr / Phe, and Ala / BCAA ratios were decreased, and the Orn / Arg ratio was increased. The above results indicate that the serum levels of Ser, Met, Pro, Lys, His, Ala, Thr, Gln, and Asp, as well as the Pro / Orn, Tyr / Phe, Ala / BCAA, and Orn / Arg ratios, can be identified as potential biomarkers for early diabetic nephropathy.

[0042] Figure 5 QNM represents urine from mice in the 13-week-old model group, and HNM represents urine from mice in the 17-week-old model group. Figure 5 urinary metabolome and Figure 6 The results of amino acid content analysis showed that, compared with the control group, the amino acid metabolism characteristics of db / db mice (13 weeks old) in the diabetic stage were characterized by decreased levels of Leu, Cre, Gln, Phe, Ala, Gly, Ser, Arg, Trp, Tyr, Iso, Asp, Thr, and Asn. Compared with the control group, the levels of Ser, Pro, Cre, Gln, Lys, Asp, Thr, Gln, and Asn were decreased in the early stage of diabetic nephropathy in db / db mice (17 weeks old). During the progression of the db / db mouse model, the levels of Lys, Try, His, and Gln were significantly increased, the level of Cre was significantly decreased, the Tyr / Phe ratio was increased, and the Ala / BCAA ratio was decreased. These results indicate that the levels of Cre, Lys, Trp, His, and Gln in the amniotic fluid, as well as the Tyr / Phe and Ala / BCAA ratios, can serve as predictors of the progression of diabetic nephropathy.

[0043] The conclusion drawn from the above is that Ser, Met, Pro, Lys, His, Ala, Thr, Gln, and Asp in serum, and Cre, Lys, Trp, His, and Gln in urine are potential biomarkers for early diabetic nephropathy. Furthermore, the ratios of Pro / Orn, Tyr / Phe, Ala / BCAA, and Orn / Arg in serum, and the ratios of Tyr / Phe and Ala / BCAA in urine, can serve as predictors of the progression of diabetic nephropathy.

[0044] Example 2: Accuracy assessment of potential biomarkers for early diabetic nephropathy

[0045] AUC is used to assess the sensitivity and specificity of biomarkers in predicting events. The sensitivity and specificity of each metabolite are determined by the optimal threshold of the ROC curve. Single-factor ROC curves are plotted, and the area under the ROC curve for the serum control group and model group is shown below. Figure 7 As shown, the area under the curve (AUC) values ​​of Cre, Lys, Arg, Val, and Gln metabolites are very close to or even reach 1, indicating that these metabolites have excellent predictive efficacy. Further analysis from the perspective of disease progression in the model group... Figure 8 The results showed that metabolites such as Ser, Met, Pro, Arg, and Ala had higher predictive accuracy for the progression of kidney injury. However, the area under the ROC curve for urine was... Figure 9 As shown, glycine Cre, Lys, Try, His, and Gln have certain predictive efficacy.

[0046] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of amino acid detection reagents in the preparation of diagnostic kits for prediabetic nephropathy, characterized in that, The kit enables diagnosis by detecting changes in the ratio or content of specific amino acids in serum samples; This kit detects the levels of Ser, Met, Pro, Lys, His, Ala, Thr, Gln, and Asp in serum, or the ratios of Pro / Orn, Tyr / Phe, Ala / BCAA, and Orn / Arg.

2. The application according to claim 1, characterized in that, Diabetic nephropathy is diagnosed when serum levels are <10.53 mg / mL, Met <3.46 mg / mL, Pro <5.02 mg / mL, Lys <37.00 mg / mL, His <6.25 mg / mL, Ala <11.27 mg / mL, Thr <7.70 mg / mL, Gln <31.06 mg / mL, and Asp <2.58 mg / mL, or when the Pro / Orn ratio is <0.66, the Tyr / Phe ratio is <102.00, the Ala / BCAA ratio is <0.12, and the Orn / Arg ratio is >0.

58.

3. The application according to any one of claims 1-2, characterized in that, The serum was collected from 13-week-old and 17-week-old mice.

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