KR102691358B1 - Biomarker composition for diagnosing susceptibility to nephrotoxicity and diagnosing method using thereof - Google Patents

Abstract

The present invention relates to a biomarker composition for diagnosing susceptibility to nephrotoxicity induced by drugs and a method for predicting nephrotoxicity susceptibility using the same. More specifically, AKR1C1 (Aldo-Keto Reductase Family 1 Member C1) is used as an active ingredient. Provided is a diagnostic biomarker composition containing nephrotoxicity sensitivity.
As it was confirmed that the expression level of AKR1C1 was reduced in the liver tissue of subjects whose nephrotoxicity was induced by drugs such as cisplatin before drug administration compared to the control group, the expression level of AKR1C1 was found to be reduced in the kidney tissues of subjects before cisplatin administration. It can be provided as a diagnostic biomarker for nephrotoxicity susceptibility to predict toxicity susceptibility in advance, and using this, nephrotoxicity that may be induced by drug administration can be effectively predicted in advance before drug administration.

Description

Biomarker composition for diagnosing nephrotoxicity sensitivity and prediction method using the same {BIOMARKER COMPOSITION FOR DIAGNOSING SUSCEPTIBILITY TO NEPHROTOXICITY AND DIAGNOSING METHOD USING THEREOF}

The present invention relates to a biomarker composition for diagnosing susceptibility to nephrotoxicity induced by drugs and a method for predicting nephrotoxicity susceptibility using the same.

The kidney is a very important organ that contributes to maintaining body homeostasis by excreting exogenous toxic substances and their resulting toxic metabolites out of the body through the process of absorption, detoxification, and excretion. Therefore, the kidneys are always exposed to toxic substances and are prone to toxicity due to drugs or oxidative stress (Ichimura et al., 2008; Rached et al., 2008; Sieber et al., 2009; Ferguson et al., 2008 ). Kidney toxicity is a major bottleneck in the new drug development process, accounting for more than 20% of organ toxicity in experimental animals in non-clinical toxicity tests (Kohli et al., 2000; Naughton et al., 2008; Nagai and Takano, 2010). . Because the kidneys function normally when mildly damaged, it is difficult to observe functional changes before significant damage occurs. Currently, clinical toxicological evaluation of kidney function is conducted by measuring blood urea nitrogen (BUN) or serum creatinine (sCr), but these methods have low sensitivity and require 70-80% of renal epithelial cells to be damaged for detection. (Rached et al., 2008; Kirtane et al., 2005). Additionally, enzymes released into urine from damaged kidney cells (γ-glutamyl transferase (γ-GT), N-acetyl-β-D-glucosaminidase (NAG)) do not exist stably in urine and are quickly decomposed (Ferguson et al. , 2008; Han et al., 2008). Diagnosis of nephrotoxicity using existing indicator substances that utilize proteins present in blood or urine is difficult to diagnose at an early stage, so discovery of more sensitive and accurate biomarkers for nephrotoxicity is required (Aicher et al ., 1998,; Devarajan et al., 2008). Currently, several methods are being developed to confirm nephrotoxicity. In particular, attempts were made to determine whether nephrotoxicity was present, focusing on the expression of genes.

Meanwhile, Cisplatin, which has been reported as a new nephrotoxic drug, is a first-generation anticancer drug mainly used to treat testicular cancer, bladder cancer, ovarian cancer, and prostate cancer, and has been reported to be toxic to both cancer cells and normal cells. Cisplatin damages DNA due to oxidative stress such as superoxide, which is produced when metabolized by CYP2E1 in the liver, and inhibits ATP production by interfering with mitochondrial electron transfer enzymes #1 and #4. In addition, a decrease in the mitochondrial membrane potential reduces the intake of calcium ions and causes an abnormality in antioxidant function, causing toxicity.

In addition, since cisplatin directly binds to guanine in the intra and interstrands of DNA and interferes with transcription, thereby inducing apoptosis, research is needed on methods to predict in advance whether one may exhibit drug sensitivity before taking cisplatin.

Republic of Korea Patent No. 10-1498481 (published on March 27, 2014)

The purpose of the present invention is to provide a biomarker composition that can predict an individual's sensitivity to nephrotoxicity to a drug, and by using this to predict nephrotoxicity that may be induced by drug administration before drug administration, We aim to provide a method to prevent toxicity in advance.

In order to achieve the above object, the present invention provides a diagnostic biomarker composition for nephrotoxicity sensitivity containing AKR1C1 (Aldo-Keto Reductase Family 1 Member C1) as an active ingredient.

The present invention provides a kit for diagnosing nephrotoxicity sensitivity, which includes as an active ingredient an agent capable of measuring the expression level of AKR1C1.

In addition, the present invention includes the steps of collecting biological samples isolated from an individual before drug administration; Confirming the expression level of AKR1C1 in the collected samples; And it provides a method for predicting nephrotoxicity susceptibility including; comparing the AKR1C1 expression level with the control group to confirm the level of decrease.

According to the present invention, as it was confirmed that the expression level of AKR1C1 in the kidney tissue of subjects whose nephrotoxicity was induced by drugs such as cisplatin before drug administration was reduced compared to the control group, AKR1C1 was expressed in the kidney tissues of subjects before the administration of cisplatin. It can be provided as a diagnostic biomarker for nephrotoxicity susceptibility to predict in advance the susceptibility to nephrotoxicity caused by cisplatin.

Using the biomarker composition, nephrotoxicity that may be induced by drug administration can be effectively predicted in advance before drug administration.

Figure 1 shows the results of comparing the expression levels of Akr1c1 mRNA of the present invention.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention expected that among the genes in kidney tissue that show innate expression differences in each individual, there would be genes highly related to the sensitivity to nephrotoxicity caused by drugs, and tested experimental animals that showed nephrotoxicity after cisplatin administration and cisplatin administration. As a result of confirming the gene expression pattern in the liver tissue before drug administration in experimental animals that showed insensitive nephrotoxicity even after treatment, the expression level of Akr1c1 in the kidney tissue before cisplatin administration in subjects that showed nephrotoxicity was the expression level in subjects that showed insensitive nephrotoxicity. As it was confirmed that it was lower, the present invention was completed.

The present invention provides a biomarker composition for diagnosing nephrotoxicity sensitivity containing AKR1C1 (Aldo-Keto Reductase Family 1 Member C1) as an active ingredient.

In this specification, “AKR1C1 (Aldo-Keto Reductase Family 1 Member C1; NM_001033697.1)” exists in chromosome 10p14-15 region and belongs to the aldehyde ketone reductase superfamily. The AKR family acts as a catalyst to convert aldehydes and ketones into alcohols. In addition, AKR1C1 increases drug resistance for cancer treatment and is also involved in antioxidant activity.

The expression level of AKR1C1 may be checked in kidney tissue before drug administration.

The nephrotoxicity may be induced by a drug, and preferably may be induced by cisplatin.

In this specification, "biomarker" is an indicator that can detect changes in the body, and is a substance that can confirm the normal or pathological state of a living organism and whether there is a change in this, and includes polypeptides, nucleic acids, lipids, glycolipids, and glycoproteins. , organic biomolecules such as sugars (monosaccharides, disaccharides, oligosaccharides, etc.), and can be used to diagnose susceptibility to nephrotoxicity due to drugs as in the present invention.

The present invention provides a kit for diagnosing nephrotoxicity sensitivity, which includes as an active ingredient an agent capable of measuring the expression level of AKR1C1.

Agents capable of measuring the expression level of AKR1C1 may be primers or probes that specifically bind to the AKR1C1 gene, antibodies, peptides, aptamers, or compounds that specifically bind to the AKR1C1 protein, but are limited thereto. no.

As used herein, a "primer" is a nucleic acid sequence with a short free 3' hydroxyl group, which is capable of forming base pairs with a complementary template and serves as a starting point for copying the template strand. refers to a short nucleic acid sequence. Primers can initiate DNA synthesis in the presence of four different nucleotide triphosphates and reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature.

The primer is a primer specific for the gene and can be either sense or antisense nucleic acid, typically having a 7 to 50 nucleotide sequence, and as long as it does not change the basic nature of the primer that serves as the starting point for DNA synthesis, additional features can be mixed. PCR conditions and lengths of sense and antisense primers can be appropriately selected according to techniques known in the art.

As used herein, “probe” refers to a nucleic acid fragment such as RNA or DNA that can specifically bind to mRNA, ranging from a few bases to hundreds of bases in length, and is labeled so that it can bind to a specific mRNA. You can check the presence or absence of and expression level. Probes may be formed in the form of oligonucleotide probes, single strand DNA probes, double strand DNA probes, RNA probes, etc. Appropriate probes and hybridization conditions can be appropriately selected according to techniques known in the art.

As used herein, “antibody” is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. In the present invention, the antibody refers to an antibody that specifically binds to the protein, and the antibody can be prepared according to conventional methods in the art. The types of antibodies include polyclonal antibodies or monoclonal antibodies, and may include all immunoglobulin antibodies. The antibody is meant to be intact, having two full-length light chains and two full-length heavy chains. Additionally, the antibodies may also include special antibodies such as humanized antibodies.

As used herein, “peptide” has the advantage of high binding affinity to the target substance and does not undergo denaturation even during heat/chemical treatment. Additionally, because the molecule size is small, it can be used as a fusion protein by attaching it to another protein. Specifically, it can be used by attaching it to a polymer protein chain, so it can be used as a diagnostic kit and drug delivery material.

As used herein, “aptamer” refers to a special type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure and is capable of binding to a target molecule with high affinity and specificity. It refers to a type of polynucleotide composed of . As described above, aptamers can specifically bind to antigenic substances in the same way as antibodies, but are composed of polynucleotides that are more stable than proteins, have a simpler structure, and are easier to synthesize, so they can be used as a replacement for antibodies. You can.

The kit may be a primer kit, DNA chip kit, or protein chip kit, but is not limited thereto. The kit may further include an antibody that selectively recognizes a marker for diagnosing nephrotoxicity susceptibility, as well as one or more other component compositions, solutions, or devices suitable for the analysis method.

Preferably, a microarray for predicting nephrotoxicity induced by cisplatin that integrates oligonucleotides or complementary strand molecules containing all or part of the AKR1C1 gene sequence that showed a change in expression level in the liver tissue of the subject before drug administration. Chips may be provided. The microarray chip compares the expression of the AKR1C1 gene in liver tissue before drug administration, enabling more meaningful and accurate comparison and analysis of nephrotoxicity that may be induced by cisplatin administration.

The present invention includes the steps of collecting biological samples isolated from an individual before drug administration; Confirming the expression level of AKR1C1 in the collected samples; And it provides a method for predicting nephrotoxicity susceptibility including; comparing the AKR1C1 expression level with the control group to confirm the level of decrease.

The type of the subject is not limited as long as it is an animal that can cause nephrotoxicity, but may specifically be a mammal, for example, a human (Homo sapiens).

The biological sample is isolated from an individual or control group before drug administration, and may be selected from the group consisting of urine, blood, cells, and tissues in which the expression level of the protein or gene thereof is different from that of the normal control group, and more preferably, liver. It may be an organization, but is not limited thereto.

The expression level of the gene was determined by next generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), and RNase. It may be measured using one or more methods selected from the group consisting of RNase protection assay (RPA), microarray, and northern blotting, but is not limited thereto.

In addition, the expression or activity level of the protein can be measured using Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, or octerony. (Ouchterlony) One or more methods selected from the group consisting of immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, and flow cytometry (FACS). It can be measured through, but is not limited to.

If the expression level of AKR1C1 is lower than that of the control group, it can be predicted that the sensitivity to nephrotoxicity due to the drug is high.

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Example 1> Identification of biomarkers for predicting nephrotoxicity sensitivity

SD rats (7 weeks old), 10 males and 10 females, were divided into 4 groups: male control (male control; hereinafter referred to as M-Con), female control (hereinafter referred to as F-Con), Each group was randomly divided into a male cisplatin treated group (male cisplatin; hereinafter referred to as M-CIS) and a female cisplatin treated group (female cisplatin; hereinafter referred to as F-CIS). Cisplatin was dissolved in physiological saline at a concentration of 25 mg/kg and administered intraperitoneally as a single dose to the cisplatin treatment group, and physiological saline was administered to the control group. An autopsy was performed 48 hours after administration, and through serum analysis (BUN, Creatinine) and histopathological analysis, it was confirmed that nephrotoxicity was more severe in males than in females due to cisplatin administration. Afterwards, RNA was extracted from kidney tissue and RNA seq (Illumina Novaseq6000) was performed to compare and analyze gene expression patterns in the four groups. (Fold change 2 or more, p value 0.05 or less).

As a result, as shown in Figure 1, the expression level of AKR1C1 mRNA was about 2.02 times higher in the F-Con group than in the M-con group. In the cisplatin treatment group, the expression of AKR1C1 mRNA decreased in both genders. However, the expression level of AKR1C1 mRNA was approximately 2.39 times higher in the F-CIS group than in the M-CIS group. The tendency for higher AKR1C1 mRNA expression in females compared to males was maintained even by cisplatin administration. As a result, it was determined that the higher expression pattern of AKR1C1 mRNA in females in the cisplatin administration group and control group may be the cause of alleviation of cisplatin-induced nephrotoxicity.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. do. That is, the actual scope of the present invention is defined by the appended claims and their equivalents.

Claims (9)

A biomarker composition for diagnosing susceptibility to nephrotoxicity induced by cisplatin, containing AKR1C1 (Aldo-Keto Reductase Family 1 Member C1) as an active ingredient. delete delete According to claim 1,
The AKR1C1 is,
A biomarker composition characterized in that the expression level is confirmed in kidney tissue before drug administration. A kit for diagnosing susceptibility to nephrotoxicity induced by cisplatin, comprising as an active ingredient an agent capable of measuring the expression level of AKR1C1. According to claim 5,
An agent capable of measuring the expression level of AKR1C1,
A kit for diagnosing nephrotoxicity sensitivity, comprising a primer or probe that specifically binds to the AKR1C1 gene, an antibody, peptide, aptamer, or compound that specifically binds to the AKR1C1 protein. Collecting biological samples isolated from the subject before drug administration;
Confirming the expression level of AKR1C1 in the collected samples; and
A method for predicting susceptibility to nephrotoxicity induced by cisplatin, including the step of confirming the level of reduction by comparing the AKR1C1 expression level with the control group. delete delete