Amino acid kit for auxiliary diagnosis of early stage of diabetic nephropathy

Abstract

The invention discloses application of amino acid in a kit for auxiliary diagnosis of early-stage diabetic nephropathy, and belongs to the technical field of biological medicine. The application is realized by detecting the content change or the concentration ratio of specific amino acid in serum or urine, the amino acid is a differential metabolite screened through metabonomics of the serum and the urine, the prediction efficiency is excellent, and the AUC value is close to 1 or even reaches 1. The invention not only provides a new idea for early diagnosis of diabetic nephropathy, but also provides a technical means for prognosis evaluation of treatment of diabetic nephropathy.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a kit for assisting in diagnosing pre-diabetic nephropathy. Background Art

[0002] Diabetic Kidney Disease (DKD) is one of the important complications of diabetic patients. It is caused by long-term diabetes and leads to a progressive decrease in proteinuria and glomerular filtration rate. At present, diabetic nephropathy is the second leading cause of end-stage renal disease in my country, and it can be reversed after early treatment.

[0003] The early diagnosis of DKD mainly depends on two indicators: estimated glomerular filtration rate (eGFR) and urine albumin / creatinine ratio (UACR). Prior art CN110987835A discloses a kit for early diagnosis of diabetic nephropathy. The test results of trace element content in the urine of the subject show that the zinc content is less than 150 μg / L and the chromium content is less than 8 μg / L, which is judged as early diabetic nephropathy. Prior art CN107091927B also discloses a kit for early diagnosis of diabetic nephropathy, including a urine microalbumin test strip, a haptoglobin test strip and a creatinine test strip. The present invention can realize the simultaneous detection of three indicators of urine microalbumin, haptoglobin and creatinine in one sample, and can increase the detection rate of early diabetic nephropathy to more than 95%. However, the use of creatinine concentration to evaluate GFR lacks sensitivity. Creatinine can only be detected after renal function has significantly decreased, and it often underestimates the GFR of DKD patients. In addition, the diagnosis using biochemical indicators such as trace urine protein 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 diabetic nephropathy, and then develop a practical amino acid detection kit based on this model.

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

[0006] In one aspect, the present invention provides the use of amino acids in a kit for diagnosing prediabetic nephropathy.

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

[0008] Specifically, the test sample is selected from any one of serum or urine.

[0009] Specifically, the sample is serum, and the contents of serine (Serine, Ser), methionine (Methionine, Met), proline (Proline, Pro), lysine (Lysine, Lys), histidine (Histidine, His), alanine (Alanine, Ala), threonine (Threonine, Thr), glutamine (Glntamine, Gln), aspartic acid (Aspartic acid, Asp) or the ratios of proline / ornithine (Ornithine, Orn), tyrosine (Tyrosine, Tyr) / phenylalanine (Phenylalanine, Phe), alanine / branched-chain amino acids (Branched-Chain AminoAcids, BCAA), ornithine / arginine (Arginine, Arg) in serum are detected; the sample is urine, and the contents of glycine (Glycine, Cre), lysine, tryptophan (Tryptophan, Trp), histidine, glutamine or the ratio of tryptophan / phenylalanine in urine are detected.

[0010] In one embodiment, the sample is serum, and diabetic nephropathy is determined when the Ser content in the serum is <10.53 mg / mL, the Met content is <3.46 mg / mL, the Pro content is <5.02 mg / mL, the Lys content is <37.00 mg / mL, the His content is <6.25 mg / mL, the Ala content is <11.27 mg / mL, the Thr content is <7.70 mg / mL, the Gln content is <31.06 mg / mL, the Asp content is <2.58 mg / mL, or 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.

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

[0012] Furthermore, the serum or urine was collected from mice at 13 weeks and 17 weeks of age.

[0013] On the other hand, the present invention provides an amino acid metabolism model for diagnosing diabetic nephropathy, wherein the model is predicted by the ratio and content changes of specific amino acids.

[0014] Specifically, the model also includes the determination of important renal injury indicators.

[0015] In some embodiments, the renal injury indicators include any one or more of urinary albumin (ALB), urinary protein (UP), urinary creatinine (UCr), serum creatinine (SCr), 24h urinary protein excretion, urinary albumin to 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 the ratio and content changes of specific amino acids, the model determines diabetic nephropathy as follows: urine albumin content>911.80ug / L, urine protein content>87.86ug / L, urine creatinine content<306.32umol / L, blood creatinine content>24.91umol / L, 24h urine protein excretion content>1464.27pg, urine albumin to creatinine ratio>27.47mg / g, triglyceride content>4.17mmol / L, total cholesterol content>1.96mmol / L, and low-density lipoprotein cholesterol content>1.33mmol / L.

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

[0018] The present invention adopts a dual model of amino acid metabolism of diabetes and amino acid metabolism of diabetic nephropathy, and the prediction results are safer and more reliable. The present invention also provides a new amino acid kit for diagnosing diabetic nephropathy, and the predicted AUC of specific amino acids is close to 1 or even reaches 1, which provides a new technical means for early diagnosis and prognosis evaluation of diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is a graph showing the measurement results of important indicators of renal injury.

[0021] Figure 2 These are the results of hematoxylin-eosin staining (HE), Masson staining (Masson), and periodic acid-Schiff staining (PAS) of mice.

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

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

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

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

[0026] Figure 7 It is the area under the ROC curve of serum in the control group and model group of 17-week-old mice.

[0027] Figure 8 It is the area under the ROC curve of serum in the 13-week-old and 17-week-old mouse model groups.

[0028] Fig. 9 It is the area under the ROC curve of urine in the 13-week-old and 17-week-old mouse model groups. DETAILED DESCRIPTION

[0029] Now describe in detail various exemplary embodiments of the present invention, this detailed description should not be considered as limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing a particular embodiment, and are not used to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in 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 have typical clinical symptoms of diabetes, such as morbid obesity, polyphagia, polydipsia, and polyuria, and are an ideal animal model for type 2 diabetes.

[0032] 1. Determination of important indicators of renal damage in diabetic nephropathy

[0033] (1) After one week of adaptive feeding of db / db mice, the animals were randomly divided into three groups according to their body weight: model group (db / db), control group (db / m) and drug-treated group (db / db+yang). The control group and model group were fed with a standard diet and free access to drinking water, and drug-treated animals were continuously administered for 4 weeks.

[0034] (2) During the experiment, the weight, food intake, calorie intake, and water intake of mice were measured every week;

[0035] (3) Blood was collected from the control and model groups by orbital capillary sampling at the first and last week to measure the levels of random blood glucose (RBG), fasting blood glucose (FBG) and glycosylated hemoglobin (HbA1c). The results are shown in the figure.

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

[0037] (5) Urine was collected from fasting mice on the last day of dosing to 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) After isoflurane anesthesia, mice were killed by cervical dislocation, and kidney tissue samples were obtained and stained with hematoxylin-eosin, Masson's staining, and periodic acid-Schiff staining. The results were as follows: Figure 2 As shown, focal atrophy of renal tubules was observed in many places in the cortex of renal tissue of the model group, and the structure was unclear. Calcification foci were observed in a small number of renal tubules, and a large number of inflammatory cell infiltrations were observed locally. Renal tubular atrophy was observed in many places, and the arrangement was irregular. 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 the amino acid metabolism model of diabetic nephropathy

[0040] C57BL / Ks mice homozygous for spontaneous mutations in diabetes show symptoms of morbid obesity, chronic hyperglycemia, pancreatic β-cell atrophy, and hypoinsulinemia. Related literature shows that db / db mice are a genetic diabetic rodent model that develops hyperglycemia 5 to 8 weeks after birth, accompanied by insulin resistance and obesity. After 10 to 20 weeks of sustained hyperglycemia, significant renal pathology is shown, similar to that observed in human diabetes. With the expansion of the mesangium, the accumulation of mesangial matrix and the thickening of the glomerular basement membrane. According to the dynamic tracking of UCr, UP, 24h urine output, ALB, and ACR after the increase in blood glucose in mice, various renal injury indicators in the blood and urine of mice continued to increase from 13 to 17 weeks of age. At 17 weeks of age, the kidney tissue of mice was taken for pathological observation and it was found that the kidneys of mice had a large number of inflammatory cell infiltrations, tubular atrophy and other mild renal injuries. The onset time points of diabetes and early diabetic nephropathy in mice were determined to be 13 weeks and 17 weeks of age, respectively; secondly, targeted metabolomics was used to detect the changes in amino acid levels in the 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 diabetes to the early stages of diabetic nephropathy, and to identify potential biomarkers for early diabetic nephropathy.

[0041] Figure 3 QXM in the figure represents the serum of mice in the model group at 13 weeks of age, and HXM represents the serum of mice in the model group at 17 weeks of age. The threshold was set as FC>1.2 or FC<0.833 and P-value<0.05 to screen differential metabolites. Figure 3 Serum metabolome and Figure 4The results of content detection showed that compared with the control group, the amino acid metabolism characteristics of db / db mice (13 weeks old) in the diabetic stage were 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 in db / db mice (17 weeks old) in the early stage of diabetic nephropathy were significantly reduced, and the levels of Arg and Cre were significantly increased. 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 reduced, the ratios of Pro / Orn, Tyr / Phe, and Ala / BCAA were reduced, and the ratio of Orn / Arg was increased. The above results indicate that the levels of Ser, Met, Pro, Lys, His, Ala, Thr, Gln, and Asp in serum, and the ratios of Pro / Orn, Tyr / Phe, Ala / BCAA, and Orn / Arg can be identified as potential biomarkers for early diabetic nephropathy.

[0042] Figure 5 QNM in the figure 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 The urine metabolome and Figure 6 The results of content detection showed that compared with the control group, the amino acid metabolism characteristics of db / db mice (13 weeks old) in the diabetic stage were reduced 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 in the early stage of diabetic nephropathy in db / db mice (17 weeks old) were reduced. During the progression of the db / db mouse model, the levels of Lys, Try, His, and Gln increased significantly, the level of Cre decreased significantly, the ratio of Tyr / Phe increased, and the ratio of Ala / BCAA decreased. The above results indicate that the levels of Cre, Lys, Trp, His, and Gln in the fluid, and the ratios of Tyr / Phe and Ala / BCAA can be used as predictors of the progression of diabetic nephropathy.

[0043] From the above, it can be concluded that Ser, Met, Pro, Lys, His, Ala, Thr, Gln, Asp in serum and Cre, Lys, Trp, His, Gln in urine are potential biomarkers for early diabetic nephropathy, while the ratios of Pro / Orn, Tyr / Phe, Ala / BCAA, Orn / Arg in serum and the ratios of Tyr / Phe, Ala / BCAA in urine can be used 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 evaluate the sensitivity and specificity of biomarkers in predicting the occurrence of events. The sensitivity and specificity of each metabolite are determined by the optimal threshold of the ROC curve. The single-factor ROC curve is drawn, and the area under the ROC curve of the serum control group and the model group is shown in the figure. 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 It shows that metabolites such as Ser, Met, Pro, Arg, and Ala have higher prediction accuracy for the progression of renal injury. However, the area under the ROC curve of urine is Fig. 9 As shown, it was shown that glycine Cre, Lys, Try, His, and Gln had 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, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Application of amino acids in diagnostic kits for prediabetic nephropathy.

2. The use according to claim 1, characterized in that: The application is achieved by detecting the changes in the ratio and content of specific amino acids in the sample.

3. The use according to claim 2, characterized in that: The test sample is selected from any one of serum or urine.

4. The use according to claims 1-3, characterized in that: The sample is serum, and the content of Ser, Met, Pro, Lys, His, Ala, Thr, Gln, Asp or the ratio of Pro / Orn, Tyr / Phe, Ala / BCAA, Orn / Arg in the serum is detected; the sample is urine, and the content of Cre, Lys, Trp, His, Gln or the ratio of Tyr / Phe in the urine is detected.

5. The use according to claim 4, characterized in that: The sample is serum, and diabetic nephropathy is determined when the Ser content in the serum is <10.53 mg / mL, the Met content is <3.46 mg / mL, the Pro content is <5.02 mg / mL, the Lys content is <37.00 mg / mL, the His content is <6.25 mg / mL, the Ala content is <11.27 mg / mL, the Thr content is <7.70 mg / mL, the Gln content is <31.06 mg / mL, the Asp content is <2.58 mg / mL, or 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.

6. The use according to claim 4, characterized in that: The sample is urine. When the Cre content in urine is <32.65 mg / mL, the Lys content is >6.89 mg / mL, the Try content is >1.87 mg / mL, the His content is >8.83 mg / mL, the Gln content is >6.89 mg / mL, or the Tyr / Phe ratio is >209.77, and the Ala / BCAA ratio is <6.0, it is determined to be diabetic nephropathy.

7. The use according to claims 1-6, characterized in that: The serum or urine was collected from mice at 13 weeks and 17 weeks of age.

8. An amino acid metabolism model for diagnosing diabetic nephropathy, characterized in that: Predicted by changes in ratios and content of specific amino acids.

9. The amino acid metabolism model of diabetic nephropathy according to claim 8, characterized in that: It also includes the determination of important indicators of mouse kidney damage.

10. The amino acid metabolism model of diabetic nephropathy according to claim 9, characterized in that: The renal injury indicators include any one or more of urine albumin, urine protein, urine creatinine, blood creatinine, 24h urine protein excretion, urine albumin to creatinine ratio, triglycerides, total cholesterol, and low-density lipoprotein cholesterol.

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