Use of secreted lctl in the preparation of a biomarker for the diagnosis of chronic kidney disease

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to the use of a secreted lactase-like protein (LCTL) as a diagnostic marker in the preparation of a chronic kidney disease diagnostic kit. The present application first discovers that LCTL exists in a secreted form, first detects the content of secreted LCTL in human serum by using an existing commercial ELISA kit, further research finds that the content of secreted LCTL in serum of patients with chronic kidney disease is significantly lower than that of normal people, and the content of LCTL in serum is significantly related to the degree of kidney function damage and the degree of proteinuria of patients, and therefore the use of secreted LCTL in the preparation of a biomarker for the diagnosis of chronic kidney disease is first proposed. The present application provides a new method for the diagnosis of chronic kidney disease, and has great clinical significance and commercial value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to the use of a secreted lactase-like protein (LCTL) as a diagnostic marker in the preparation of a chronic kidney disease diagnostic kit. BACKGROUND

[0002] Chronic kidney disease (CKD) is a common chronic disease characterized by structural damage or functional decline of the kidney. Epidemiological studies have shown that the incidence of chronic kidney disease in Guangdong Province is as high as 10.1% (in cities) to 13.6% (in rural areas). With the aging of society, the incidence of diabetes and hypertension is increasing year by year, and the incidence of CKD is also increasing. However, existing research still cannot find effective intervention targets, and finding new biomarkers and treatment targets for kidney damage is a major demand of the state and society.

[0003] LCTL (Lactase-like protein, lactase-like protein) is also known as γ-Klotho (KLγ) or Klotho / Lactase-phlorizin hydrolase-related protein (KLPH), and belongs to the Klotho family. The family includes α-Klotho (KLα), β-Klotho (KLβ), and LCTL protein. Among the three members of the Klotho protein family, KLα is the most extensively and thoroughly studied kidney-protective protein, and thus KLα is also directly referred to as Klotho protein. KLα was originally defined as an anti-aging gene, and is mainly expressed in the kidney, parathyroid gland, brain, and heart. Most studies have shown that membrane and secreted KLα play a protective role in kidney disease (see, for example, Lin Y, Kuro-o M, Sun Z. Genetic deficiency of anti-aging gene klotho exacerbates early nephropathy in STZ-induced diabetes in male mice. Endocrinology 2013; 154(10): 3855-63). LCTL is highly homologous to KLα, and is mainly expressed in the kidney, skin, and lens. LCTL consists of 14 exons, and translation thereof expresses a protein consisting of 567 amino acids, and has high homology between humans and mice. Similar to KLα, LCTL consists of a large extracellular domain, which includes an extracellular segment consisting of 519 amino acid residues, followed by a single transmembrane domain and an intracellular segment consisting of 5 amino acid residues. LCTL is a new member of the Klotho protein family discovered in recent years based on homology analysis of KLα (see, for example, Ito S, Kinoshita S, Shiraishi N, Nakagawa S, Sekine S, Fujimori T, Nabeshima YI. Molecular cloning and expression analyses of mouse beta klotho, which encodes a novel Klotho family protein. Mech Dev 2000; 98(1-2): 115-9), and since it is a recently discovered protein, there is very little research on LCTL, and its function is not yet clear, and there is no research on kidney disease diagnostic markers. SUMMARY

[0004] In order to fill the gap in the prior art, the present application first discovers that LCTL exists in a secreted form, first detects the content of secreted LCTL in human serum, and confirms that LCTL is significantly correlated with renal function damage and the degree of proteinuria of patients, and can be used as a novel biomarker reflecting renal damage.

[0005] To solve the above technical problems, the technical scheme adopted by the present application is:

[0006] The present application provides the use of secreted lactase-like protein (LCTL) as a diagnostic marker in the preparation of a chronic kidney disease diagnostic kit.

[0007] As an optional mode, in the above use, the kit is a human secreted LCTL ELISA detection kit.

[0008] Preferably, the ELISA kit is a commercial OKEH06555 kit.

[0009] As an optional mode, in the above use, the detection sample of the kit is peripheral blood.

[0010] As an optional mode, in the above use, the detection sample of the kit is plasma or serum.

[0011] Preferably, the detection sample of the kit is serum.

[0012] As an optional mode, in the above use, the content of secreted LCTL in the peripheral blood of a chronic kidney disease patient is significantly lower than that of a normal person.

[0013] As an optional mode, in the above use, the content of secreted LCTL in the peripheral blood of a chronic kidney disease patient is significantly negatively correlated with the degree of renal function damage.

[0014] As an optional mode, in the above use, the content of secreted LCTL in the peripheral blood of a chronic kidney disease patient is significantly negatively correlated with the serum creatinine and blood urea nitrogen of the patient, and the content of secreted LCTL in the peripheral blood of a chronic kidney disease patient is significantly positively correlated with the eGFR level of the patient.

[0015] As an optional mode, in the above use, the chronic kidney disease is selected from one or more of the following: chronic kidney damage caused by ischemia-reperfusion, chronic kidney damage caused by drug toxicity, chronic kidney damage caused by bacterial infection, or chronic kidney damage caused by immune stress.

[0016] As an optional mode, in the above use, the chronic kidney disease is selected from one or more of the following: diabetic nephropathy, hypertensive nephropathy, lupus nephritis, IgA nephropathy, or end-stage renal disease.

[0017] Compared with the prior art, the present application has the following advantages:

[0018] The present application firstly discovers the existence of secretory LCTL, firstly detects the content of secretory LCTL in human serum by using the existing commercial ELISA kit, and further researches find that the content of secretory LCTL in serum of patients with chronic kidney disease is significantly lower than that of normal people, and the same conclusion is obtained in the verification queue, and the content of LCTL in serum is significantly related to the degree of kidney function damage and proteinuria of patients, so the use of secretory LCTL in preparing a biomarker for diagnosing chronic kidney disease is firstly proposed. BRIEF DESCRIPTION OF DRAWINGS

[0019] The accompanying drawings are included to provide a further understanding of the present application, and constitute a part of the specification, and are used together with embodiments of the present application to explain the present application, and do not constitute a limitation on the present application. In the drawings:

[0020] Figure 1 : Detection results of serum LCTL content of different groups in the screening queue.

[0021] Figure 2 : Correlation analysis of serum LCTL of CKD patients and clinical indicators in the screening queue.

[0022] Figure 3 : ROC curve of LCTL as CKD diagnosis in the screening queue.

[0023] Figure 4 : Detection results of serum LCTL content of different groups in the verification queue.

[0024] Figure 5 : Correlation analysis of serum LCTL of CKD patients and clinical indicators in the verification queue.

[0025] Figure 6 : ROC curve of LCTL as CKD diagnosis in the verification queue. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application. The materials, reagents, etc. used, if not specially specified, are commercially available reagents and materials. Embodiments:

[0027] 1. Main experimental materials

[0028] Human Secreted LCTL ELISA Kit: OKEH06555, Aviva Systems Biology, USA.

[0029] 2. Experimental Methods

[0030] 2.1 Screening cohort of CKD patients

[0031] (1) Peripheral blood serum samples of CKD patients (including diabetic nephropathy (DN, n=30), lupus nephritis (LN, n=15) and IgA nephropathy (IgAN, n=10) diagnosed by kidney biopsy in the First Affiliated Hospital of Sun Yat-Sen University, disease control of minimal change disease (MCD, n=28) and healthy controls (NC, n=39) were collected, and relevant clinical data were collected.

[0032] (2) The content of LCTL in serum of CKD patients, disease controls and healthy controls was detected by ELISA kit (OKEH06555, Aviva Systems Biology, USA).

[0033] (3) The content of LCTL in serum of CKD patients, disease controls and healthy controls was compared, and the differences between groups were compared by analysis of variance.

[0034] (4) Pearson correlation analysis was used to analyze the correlation between the content of LCTL in serum of CKD patients and the patient's serum creatinine, blood urea nitrogen, eGFR, CKD stage and proteinuria degree (patient clinical indicators were detected by automatic biochemical detector in the First Affiliated Hospital of Sun Yat-Sen University).

[0035] (5) The serum LCTL curve was drawn for the ROC curve for diagnosing CKD in patients and the area under the curve (AUC) was calculated.

[0036] 2.2 Verification cohort of CKD patients

[0037] (1) Peripheral blood serum samples of CKD patients (including diabetic nephropathy (DN, n=30), lupus nephritis (LN, n=15) and IgA nephropathy (IgAN, n=10) diagnosed by kidney biopsy in the First Affiliated Hospital of Sun Yat-Sen University, disease control of minimal change disease (MCD, n=28) and healthy controls (NC, n=39) were collected, and relevant clinical data were collected.

[0038] (2) The content of LCTL in serum of CKD patients, disease controls and healthy controls was compared, and the differences between groups were compared by analysis of variance.

[0039] (3) Pearson correlation analysis was used to analyze the correlation between the serum LCTL content of CKD patients and the patients' serum creatinine, blood urea nitrogen, eGFR, CKD stage and degree of proteinuria (patient clinical indicators were detected by the fully automated biochemical analyzer of the First Affiliated Hospital of Sun Yat-sen University).

[0040] (4) Draw the ROC curve of serum LCTL for the diagnosis of CKD in patients and calculate the area under the curve (AUC).

[0041] 3. Experimental Results

[0042] 3.1 CKD Filtering Queue

[0043] (1) Serum LCTL levels are significantly reduced in CKD patients.

[0044] like Figure 1 As shown, compared with normal controls (NC) and disease controls (MCD), the serum LCTL levels in CKD patients (IgAN, LN, DN) were significantly reduced.

[0045] (2) Serum LCTL levels in CKD patients were significantly negatively correlated with the degree of renal function impairment.

[0046] like Figure 2 As shown, further correlation analysis was performed between serum LCTL levels and clinical indicators (serum creatinine, blood urea nitrogen, and eGFR) in CKD patients. The results revealed a significant negative correlation between serum LCTL levels and serum creatinine (r = -0.4897, P = 0.0003) and blood urea nitrogen (r = -0.5004, P = 0.0002), and a significant positive correlation with eGFR levels (r = 0.3643, P = 0.0093). This indicates that serum LCTL levels in CKD patients are significantly negatively correlated with the degree of renal impairment.

[0047] (3) ROC curve of serum LCTL in the screening cohort for diagnosing CKD

[0048] like Figure 3 As shown, ROC curves were calculated by further using secretory LCTL as a diagnostic biomarker for CKD, and it was found that LCTL can be used as a biomarker for diagnosing CKD (AUC = 0.850, P < 0.0001).

[0049] 3.2 CKD Verification Queue

[0050] (1) Serum LCTL levels are significantly reduced in CKD patients.

[0051] like Figure 4As shown, the content of serum LCTL in CKD patients (DN) was significantly lower than that in normal controls (NC) and disease controls (DM).

[0052] (2) The content of LCTL in serum of CKD patients was significantly negatively correlated with the degree of renal function damage

[0053] As shown, the content of LCTL in serum of CKD patients was significantly negatively correlated with the degree of renal function damage Figure 5 As shown, the content of LCTL in serum of CKD patients was significantly negatively correlated with the degree of renal function damage

[0054] (3) ROC curve of serum LCTL in verification cohort for diagnosing CKD

[0055] As shown, the content of LCTL in serum of CKD patients was significantly negatively correlated with the degree of renal function damage Figure 6 As shown, the content of LCTL in serum of CKD patients was significantly negatively correlated with the degree of renal function damage

[0056] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, the technical solutions can also be modified or replaced by equivalents, and these modifications or equivalent replacements should also be considered as the protection scope of the present application.

Claims

1. The use of secreted lactase-like protein (LCTL) as a diagnostic marker in the preparation of a diagnostic kit for chronic kidney disease, characterized in that: The detection sample of the kit is peripheral blood, the content of secretory LCTL in the peripheral blood of the patient with chronic kidney disease is significantly lower than that of normal people, the content of secretory LCTL in the peripheral blood of the patient with chronic kidney disease is significantly negatively correlated with the degree of kidney function damage, the content of secretory LCTL in the peripheral blood of the patient with chronic kidney disease is significantly negatively correlated with the blood creatinine and blood urea nitrogen of the patient, and the content of secretory LCTL in the peripheral blood of the patient with chronic kidney disease is significantly positively correlated with the eGFR level of the patient.

2. Use according to claim 1, characterized in that: The kit is a human secretory LCTL ELISA detection kit.

3. Use according to claim 2, characterized in that: The ELISA kit is a commercial OKEH06555 kit.

4. Use according to claim 1, characterized in that: The detection sample of the kit is plasma or serum.

5. Use according to any one of claims 1 to 4, characterized in that: The chronic kidney disease is selected from one or more of the following: chronic kidney injury caused by ischemia-reperfusion, chronic kidney injury caused by drug toxicity, chronic kidney injury caused by bacterial infection, or chronic kidney injury caused by immune stress.

6. Use according to any one of claims 1 to 4, characterized in that: The chronic kidney disease is selected from one or more of the following: diabetic nephropathy, hypertensive nephropathy, lupus nephritis, IgA nephropathy, or end-stage renal disease.

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