JP2024034246A - Method of testing for idiopathic nephrotic syndrome - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a test method that enables efficacy evaluation of steroid treatment on an idiopathic nephrotic syndrome patient without relying on the steroid treatment.

SOLUTION: A test method disclosed herein comprises: bringing a specimen of a subject with idiopathic nephrotic syndrome into contact with a sample containing cells, basement membranes, and integrin appearing at interfaces between the cells and the basement membrane; measuring the activation degree of the integrin caused by the contact with the specimen; and evaluating efficacy of steroid treatment for the subject on the basis of a result of the measurement.

SELECTED DRAWING: Figure 1

COPYRIGHT: (C)2024,JPO&INPIT

Description

The present disclosure relates to a method of testing for idiopathic nephrotic syndrome.

Idiopathic nephrotic syndrome is a kidney disease common in children that causes large amounts of protein to leak from the urine and a decrease in protein in the blood. Idiopathic nephrotic syndrome is divided into steroid-sensitive nephrotic syndrome (SSNS), in which proteinuria disappears with steroid treatment, and steroid-resistant nephrotic syndrome (steroid-resistant nephrotic syndrome, in which proteinuria does not disappear even with steroid treatment). resistant nephrotic syndrome (SRNS) (for example, see Non-Patent Document 1).

Clin Exp Nephrol (2017) 21:651–657

Currently, it is possible to determine whether idiopathic nephrotic syndrome is SSNS or SRNS by uniformly administering steroid therapy at the maximum dose for 4 to 6 weeks, and then determining whether or not there is a response to steroid therapy. It is being done. For this reason, there are problems such as a delay in starting highly effective treatment for patients with SRNS.
In view of the above circumstances, an object of the present invention is to provide a testing method that can evaluate the effectiveness of steroid treatment for patients with idiopathic nephrotic syndrome without relying on steroid treatment.

Specific means for achieving the above object include the following embodiments.
<1> Contacting a specimen of a subject suffering from idiopathic nephrotic syndrome with a sample containing cells, a basement membrane, and an integrin expressed at the interface between the cells and the basement membrane;
Measuring the degree of activation of the integrin due to contact with the specimen;
A testing method comprising the step of evaluating the effectiveness of steroid treatment for the subject based on the results of the measurement.
<2> The test method according to <1>, wherein the cells are human glomerular epithelial cells.
<3> The testing method according to <1> or <2>, wherein the integrin includes αvβ3 integrin.
<4> The inspection method according to any one of <1> to <3>, wherein the measurement is performed using a total internal reflection illumination fluorescence microscope.
<5> The test method according to any one of <1> to <4>, wherein the measurement is performed by an immunological method.
<6> The test method according to any one of <1> to <5>, wherein the specimen is serum, plasma, or urine.

According to the present disclosure, a test method is provided that can evaluate the effectiveness of steroid treatment for patients with idiopathic nephrotic syndrome without relying on steroid treatment.

It is a graph showing the degree of activation of αvβ3 integrin measured in Examples. It is a graph showing the degree of activation of αvβ3 integrin measured in Examples. 3 is a graph showing an ROC curve created from the results shown in FIG. 2.

Embodiments of the present disclosure will be described below. These descriptions and examples are illustrative of the invention and are not intended to limit the scope of the invention.
In this specification, a numerical range indicated using "-" indicates a range that includes the numerical values written before and after "-" as the minimum and maximum values, respectively.

The testing method of the present disclosure includes:
A step of contacting a specimen of a subject suffering from idiopathic nephrotic syndrome with a sample containing cells, a basement membrane, and an integrin expressed at the interface between the cells and the basement membrane (hereinafter also referred to as the first step);
a step of measuring the degree of activation of the integrin due to contact with the specimen (hereinafter also referred to as second step);
The method includes a step (hereinafter also referred to as a third step) of evaluating the effectiveness of steroid treatment for the subject based on the results of the measurement.

As a result of studies conducted by the present inventors, integrin is activated when a sample from a subject suffering from idiopathic nephrotic syndrome is brought into contact with a sample containing cells, basement membrane, and integrin expressed at the interface between the cells and basement membrane. It was found that the degree of integrin activation is related to the attributes of the subject.
Specifically, the degree of integrin activation in a sample that has been in contact with a sample from a subject who is SRNS is significantly higher than the degree of activation of integrin in a sample that has been in contact with a sample from a subject who is SSNS. Understood.

That is, the effectiveness of steroid treatment for a subject can be evaluated based on the measurement results of the degree of integrin activation in a sample that has been brought into contact with a sample from the subject.
For this reason, for example, it is possible to evaluate the effectiveness of steroid treatment for a subject without undergoing long-term steroid treatment, and for subjects whose effectiveness of steroid treatment is evaluated to be low, treatment other than steroid treatment can be performed. You can start quickly.

(1st step)
In the first step, a specimen from a subject suffering from idiopathic nephrotic syndrome is brought into contact with a sample containing cells, a basement membrane, and an integrin expressed at the interface between the cells and the basement membrane.

The cells contained in the sample are not particularly limited as long as they express integrin at the interface with the basement membrane.
Cells that express integrin at the interface with the basement membrane include human glomerular epithelial cells. Human glomerular epithelial cells (also referred to as podocytes) are cells that together with endothelial cells and basement membranes as an extracellular matrix form the blood vessel walls of capillaries that constitute the glomeruli in the kidney. Human glomerular epithelial cells play the role of preventing proteins contained in blood in capillaries from leaking into urine during ultrafiltration performed in the glomerulus.

The type of integrin contained in the sample is not particularly limited, and can be selected depending on the type of cells contained in the sample. For example, when the cells contained in the sample are human glomerular epithelial cells, the integrin expressed at the interface between the cells and the basement membrane includes αvβ3 integrin.

The form of the sample used in the first step is not particularly limited as long as it can come into contact with the test subject's specimen.
From the viewpoint of measurement accuracy and workability of the degree of integrin activation at the interface between cells and basement membrane, it is preferable that the sample be placed on the substrate, and the sample should be placed on the substrate having a planar region. More preferably, it is arranged in a plane area. The thickness of the basement membrane is not particularly limited, but is usually about 50 nm to 150 nm.

The material of the base material on which the sample is placed is not particularly limited, and can be selected depending on the method for measuring the degree of integrin activation at the interface between cells and basement membrane. For example, when light is transmitted through a base material during measurement, the material of the base material can be selected from materials with light transmittance such as glass.

The method for preparing the sample used in the first step is not particularly limited, and can be carried out by any conventional method. The cells contained in the sample may be established cell lines or normal cells.

The specimen of the subject that is brought into contact with the sample can be selected from, for example, serum, plasma, urine, and the like.

The time period for which the test subject's specimen is brought into contact with the sample in the first step is not particularly limited.
From the viewpoint of sufficiently activating integrin in the sample, the above-mentioned time is preferably 5 minutes or more, more preferably 10 minutes or more, and even more preferably 20 minutes or more.
From the viewpoint of maintaining good cell conditions, the above-mentioned time is preferably 24 hours or less, more preferably 12 hours or less, and even more preferably 3 hours or less.
The sample after contact with the specimen may be fixed using formalin or the like.

(Second process)
In the second step, the degree of integrin activation due to contact with the specimen is measured.
In the present disclosure, "activation of integrin" refers to a change in the structure of integrin that allows it to bind to a ligand. "Degree of integrin activation" refers to the ratio of activated integrin (integrin in a state that exhibits ligand binding ability) to all integrins (including active and inactive types) expressed in a sample. means.

From the viewpoint of measurement accuracy, it is preferable to measure the degree of integrin activation by an immunological method. In the present disclosure, an immunological method refers to a method of detecting a signal emitted by a labeling substance using an antigen-antibody reaction.
For example, the second step may include a step of adding an antibody whose antigen is activated integrin to the sample after contacting with the specimen. In this case, the labeling substance may be bound to an antibody whose antigen is activated integrin, or it may be bound to another substance such as a secondary antibody.

Specifically, immunological methods include fluorescence immunoassay (FIA), which uses a fluorescent substance as a labeling substance, bioluminescent immunoassay (BLIA), which uses a bioluminescent substance as a labeling substance, and electrochemistry as a labeling substance. Electrochemiluminescence immunoassay (ECLIA) that uses a luminescent substance, enzyme immunoassay (EIA) that uses an enzyme as a labeling substance, radioimmunoassay (RIA) that uses a radioactive isotope as a labeling substance, Examples include chemiluminescent immunoassay (CIA) that uses chemiluminescent substances. Among these, fluorescence immunoassay (FIA) is preferred from the viewpoints of stability, versatility, and safety.

From the viewpoint of measurement accuracy, it is preferable to measure the degree of integrin activation using a total internal reflection fluorescence microscope (TIRFM).

In measurements using TIRFM, signals are detected using evanescent light, which is light that slightly leaks out in the direction opposite to the direction of reflection when light is totally reflected at an interface between materials with different refractive indexes.
For example, when light is irradiated from the opposite side of the base material to the side where the sample is placed, and when the irradiated light is totally reflected at the interface between the base material and the sample, the evanescent light that seeps out to the sample side excites the labeled substance. Use it as light.
Since the reach depth of evanescent light is extremely shallow, it selectively detects active integrin signals that exist near the interface between cells and the basement membrane, which is the measurement target area of the sample, and detects active integrin signals that exist outside the sample measurement target area. It is possible to exclude signals from type integrins (for example, those that are taken up into cells during the process of metabolism or recycling and are not immediately involved in cell adhesion) from the detection target.

(Third step)
In the third step, the effectiveness of the steroid treatment for the subject is evaluated based on the results of the measurements performed in the second step.
As described above, the degree of integrin activation in a sample that has been contacted with a specimen from a subject who is SRNS is significantly higher than the degree of activation of integrin in a sample that has been contacted with a specimen from a subject who is SSNS.
Therefore, for example, if the degree of integrin activation measured in the second step exceeds a predetermined level, the idiopathic nephrotic syndrome that the subject is suffering from is steroid resistant, and the effectiveness of steroid treatment is low. It can be determined that

EXAMPLES Hereinafter, the present invention will be explained in detail with reference to Examples, but the present invention is not limited to these Examples.

(1) Sample preparation and integrin activation treatment A sample in which a healthy human glomerular epithelial cell line (ciPods) is differentiated in a TIRFM dish, and the differentiated cells are arranged on the dish surface via the basement membrane. was created. Next, the samples were divided into the following seven types and treated to activate αvβ3 integrin (30 minutes), and the treated samples were fixed with formalin. A primary antibody (AP5) against activated αvβ3 integrin was added to each of the fixed samples to cause an antigen-antibody reaction. Since AP5 is an antibody produced in a mouse, an antigen-antibody reaction was generated using a fluorescently labeled anti-mouse IgG antibody as a secondary antibody, and active αvβ3 integrin was fluorescently labeled. The patient sera used for sample processing were n=1 each for SRNS and SSNS, and the same patient's sera at the time of relapse and time of remission were used.

Non: Negative control (no treatment)
HC: Negative control (treated with serum from a healthy person)
Mn: positive control (treated with divalent cation (Mn))
SRNS RL: Treated with relapse serum from SRNS patients SRNS RM: Treated with remission serum from SRNS patients SSNS RL: Treated with relapse serum from SSNS patients SSNS RM: Treated with remission serum from SSNS patients

(2) Measurement of the degree of activation of αvβ3 integrin Using TIRFM, excitation light of a fluorescent label is irradiated from the opposite side of the dish to the side where the sample is placed, and the interface between the human glomerular epithelial cells of the sample and the basement membrane is The signal intensity of the antibody bound to activated αvβ3 integrin in the vicinity was measured. In the measurement, a 60x objective lens was used, and 30 fields of view were photographed for each sample. After correcting the background noise in each field of view, the signal intensity of the antibody bound to activated αvβ3 integrin was divided by the number of cells in the same field of view to calculate the signal intensity per cell for each field of view. The calculated median signal intensity per cell was taken as the signal intensity of each sample.

(3) Results The measurement results of signal intensity are shown in Figure 1. Each plot in FIG. 1 shows the signal intensity of activated αvβ3 integrin per cell, and the larger the value, the greater the degree of activation of αvβ3 integrin.

As shown in Figure 1, when comparing the results of treatment with relapse serum (SRNS RL) and remission serum (SRNS RM) from SRNS patients, the signal intensity when treated with remission serum was similar to that of the negative control. The signal intensity when treated with relapse serum was significantly higher than that of the negative control.
On the other hand, when comparing the results of treatment with relapse serum (MCNS RL) and remission serum (MCNS RM) from SSNS patients, the signal intensity in both cases is comparable to that of the negative control.
From the above results, it can be seen that activation of αvβ3 integrin is induced when a sample is treated with a specimen from a patient who has developed idiopathic nephrotic syndrome and whose idiopathic nephrotic syndrome is steroid-resistant.

(4) Tests and results using samples from multiple patients The same experiments as above were conducted using the recurrence sera of existing cohort samples, the SRNS group (n = 9) and the SSNS group (n = 14). . The signal intensity of each sample was converted into a ratio to the signal intensity of a negative control (Non) performed in the same experiment and used for statistical analysis. The analysis results of signal intensity are shown in FIG. 2.
As shown in FIG. 2, the signal intensity when the sample was treated with the relapse serum of the SRNS patient was significantly higher than the signal intensity when the sample was treated with the relapse serum of the SSNS patient. In other words, the degree of αvβ3 integrin activation when the sample was treated with the relapse serum of an SRNS patient was significantly higher than the degree of αvβ3 integrin activation when the sample was treated with the relapse serum of an SSNS patient. I understand.

FIG. 3 shows an ROC curve created from the experimental results shown in FIG. 2. The AUC of the ROC curve shown in FIG. 3 is 0.8889, and the cutoff value determined by Youden index is 0.166 (sensitivity 78%, specificity 93%), and the test accuracy is judged to be good.

Claims (6)

contacting a specimen of a subject suffering from idiopathic nephrotic syndrome with a sample containing cells, a basement membrane, and an integrin expressed at the interface between the cells and the basement membrane;
Measuring the degree of activation of the integrin due to contact with the specimen;
A testing method comprising the step of evaluating the effectiveness of steroid treatment for the subject based on the results of the measurement. The testing method according to claim 1, wherein the cells are human glomerular epithelial cells. The testing method according to claim 1, wherein the integrin includes αvβ3 integrin. The inspection method according to claim 1, wherein the measurement is performed using a total internal reflection illumination fluorescence microscope. The test method according to claim 1, wherein the measurement is performed by an immunological method. The test method according to claim 1, wherein the specimen is serum, plasma, or urine.