JP7270192B2 - A method for acquiring data for diagnosing the presence or absence of cell cycle progression from the G2 phase to the M phase in kidney-derived cells in a subject, and its use - Google Patents

Description

The present invention relates to the progression of the cell cycle from _{the G2} phase to the M phase of kidney-derived cells in a subject (in other words, cell cycle arrest at the _G2 /M phase). The present invention relates to a method of acquiring data for diagnosing the presence or absence thereof, and its utilization.

Kidney injury is a disease with a high mortality rate and poor prognosis, especially severe acute kidney injury (AKI) with a mortality rate of over 50%.

Diagnosis of kidney injury (eg, acute kidney injury) is conventionally based on changes in serum creatinine levels and/or changes in urine output. However, since serum creatinine is a biomarker that changes in the amount 2-3 days after the onset of renal damage, it may be difficult to start treatment for renal damage if a diagnosis of renal damage is made based on changes in serum creatinine levels. delayed, resulting in a poor prognosis.

Therefore, L-FABP (L-type fatty acid binding protein), NGAL (neutrophil gelatinase associated lipocalin), TIMP-2 (tissue inhibitor of metalloproteinase-2), which are biomarkers whose amount changes early after renal damage occurs. ) and IGFBP7 (insulin-like growth factor-binding protein 7) are used for diagnosis of renal injury (for example, acute renal injury) (see Non-Patent Documents 1 and 2). TIMP-2 and IGFBP7 are biomarkers that can detect the presence _or absence of cell cycle progression from G1 phase to S phase in kidney-derived cells.

By the way, in recent years, the transition from acute kidney injury to chronic kidney disease requires the arrest of cell cycle progression from the _G2 phase to the M phase in renal epithelial cells and the transition from the _G2 phase to the M phase in renal epithelial cells. It has been suggested that it is related to fibrosis of renal tubulointerstitium that accompanies cessation of cycle progression (see Non-Patent Document 3).

HUI-MIAO JIA et al., “Prognostic value of cell cycle arrest biomarkers in patients at high risk for acute kidney injury: A systematic review and meta-analysis” Nephrology 22 (2017) p831-837 Anitha Vijayan, MD et al., “Clinical Use of Urine Biomarker [TIMP-2]×[IGFBP7] for acute Kidney Injury Risk Assessment”, Am J Kidney Dis. 2016 July, 68(1), p19-28 Li Yang et al., “Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury” nature medicine, Vol.16, No.5, May 2010, p535-543

However, in Non-Patent Document 3, when detecting the presence or absence of cell cycle progression from _G2 phase to M phase in kidney-derived cells (e.g., renal tubular cells), Observing the stained image of histone H3 phosphorylated in the tissue, or (ii) staining the nuclear DNA of cells cultured in vitro with Propidium Iodide, and subjecting the cells to FACS (Fluorescence activated cell sorting ) to observe the cell cycle. In other words, conventionally, there has been no technique for simply and noninvasively detecting the presence or absence of cell cycle progression from the _G2 phase to the M phase in kidney-derived cells (eg, renal tubular cells).

Therefore, one aspect of the present invention is to simply and non-invasively detect the presence or absence of cell cycle progression from G2 phase to M phase in kidney-derived cells ( _e.g. , renal tubular cells) using a clinical urine sample. The purpose is to provide technology to

In order to solve the above problems, according to one aspect of the present invention, a method for acquiring data for diagnosing the presence or absence of cell cycle progression from the _G2 phase to the M phase of kidney-derived cells in a subject includes: The method is characterized by including a step of detecting thioredoxin in urine collected from a specimen.

In order to solve the above problems, according to one aspect of the present invention, a kit for diagnosing the presence or absence of cell cycle progression from the _G2 phase to the M phase in kidney-derived cells in a subject is collected from the subject It is characterized by comprising a member for detecting thioredoxin in excreted urine.

To solve the above problems, according to one aspect of the present invention, a method for obtaining data for predicting recovery or chronicity of renal damage in a subject detects thioredoxin in urine collected from the subject. It is characterized by including steps.

According to one aspect of the present invention, in the data acquisition method for predicting recovery or chronicity of renal injury in a subject, the chronicization is cell cycle arrest from _G2 phase to M phase in kidney-derived cells Chronification through fibrosis of the renal tubulointerstitium associated with renal tubulointerstitium.

To solve the above problems, according to one aspect of the present invention, there is provided a kit for predicting recovery or chronicity of renal injury in a subject, comprising a member for detecting thioredoxin in urine collected from the subject It is characterized by having

According to one aspect of the present invention, in the kit for predicting recovery or chronification of renal injury in a subject, the chronification is performed in urine following cell cycle arrest from _G2 phase to M phase in kidney-derived cells. It may be chronic through fibrosis of the tubulointerstitium.

According to one aspect of the present invention, it is possible to diagnose the presence or absence of cell cycle progression from the _G2 phase to the M phase of kidney-derived cells in a subject.

According to one aspect of the present invention, recovery or chronicity of renal damage in a subject can be predicted.

FIG. 2 is a diagram showing the correlation between the amount of thioredoxin in urine and the cell cycle of kidney-derived cells in Examples of the present invention. FIG. 2 shows the effect of thioredoxin reductase inhibitors on cyclin B expression in Examples of the present invention. FIG. 2 shows the effect of thioredoxin reductase inhibitors on Cdc25C phosphorylation in Examples of the present invention. FIG. 2 shows the effect of thioredoxin reductase inhibitors on cyclin D expression in Examples of the present invention. FIG. 2 shows the effects of thioredoxin reductase inhibitors on the cell cycle in Examples of the present invention. (a) and (b) show the effect of inhibition of thioredoxin expression on the expression of cyclin B and phosphorylated histone H3 in an example of the present invention.

One embodiment of the present invention is described below, but the present invention is not limited thereto. The present invention is not limited to the configurations described below, and can be modified in various ways within the scope of the claims, and the technical means disclosed in different embodiments and examples. Embodiments and examples obtained by appropriate combinations are also included in the technical scope of the present invention. Also, all of the documents mentioned in this specification are incorporated herein by reference. In this specification, when "A to B" is described with respect to a numerical range, the description means "A or more and B or less".

[1. A method for obtaining data for diagnosing the presence or absence of cell cycle progression from the _G2 phase to the M phase in kidney-derived cells in a subject, and obtaining data for predicting recovery or chronic renal injury in a subject Acquisition method〕
The method of acquiring data for diagnosing the presence or absence of cell cycle progression from the _G2 phase to the M phase of kidney-derived cells in a subject according to the present embodiment includes the step of detecting thioredoxin in urine collected from the subject. including.

When a large amount of thioredoxin is contained in the urine of a subject, progression of the cell cycle from the _G2 phase to the M phase in the kidney-derived cells (e.g., renal tubular cells) of the subject is considered to be arrested. can judge. For example, when the amount B of thioredoxin present in the urine of a subject is greater than the amount A of thioredoxin present in the urine of a healthy subject or in the urine of a patient suffering from chronic kidney disease, It can be determined that cell cycle progression from the _G2 phase to the M phase has stopped in the subject's kidney-derived cells (eg, renal tubular cells). The ratio between the amount A and the amount B is not particularly limited, and may be, for example, "B>A", "B≧2×A", or "B≧5×A". "B≧10×A", "B≧50×A", or "B≧100×A".

On the other hand, when the urine of a subject does not contain thioredoxin or contains a small amount of thioredoxin, kidney-derived cells (e.g., tubular cells) of the subject undergo cell cycle transition from the _G2 phase to the M phase. It can be determined that progression is accelerated. For example, when the amount B of thioredoxin present in the urine of a subject is less than the amount A of thioredoxin present in the urine of a healthy subject or in the urine of a patient suffering from chronic kidney disease, It can be determined that cell cycle progression from the _G2 phase to the M phase is accelerated in kidney-derived cells (eg, renal tubular cells) of the subject. The amount ratio between the amount A and the amount B is not particularly limited, and may be, for example, "B<A", "B≦0.5×A", or "B≦0.2× A", may be "B ≤ 0.1 x A", may be "B ≤ 0.5 x A", or may be "B ≤ 0.01 x A" good too.

A method for obtaining data for predicting recovery or chronicity of renal damage in a subject according to the present embodiment includes a step of detecting thioredoxin in urine collected from the subject.

Non-Patent Document 3 states that the transition from acute kidney injury to chronic kidney disease involves arrest of cell cycle progression from G2 _phase to M phase in renal epithelial cells and transition from _G2 phase to M phase in renal epithelial cells. It has been suggested that fibrosis of the renal tubulointerstitium with cell cycle progression arrest to Based on the findings and the examples of the present application, not only the arrest of the cell cycle progression from the _G2 phase to the M phase in the subject, but also the chronic renal injury accompanied by fibrosis of the tubulointerstitium can be predicted. can do. Therefore, the chronification may be chronification via tubular interstitial fibrosis associated with cell cycle arrest from the _G2 phase to the M phase in kidney-derived cells. Further, the data acquisition method of the present embodiment may be a data acquisition method for predicting fibrosis of renal tubulointerstitium in a subject.

As described above, when a large amount of thioredoxin is contained in the urine of a subject, cell cycle progression from the _G2 phase to the M phase occurs in kidney-derived cells (e.g., renal tubular cells) of the subject. It can be determined that it has stopped. In this case, it can be determined that the renal disorder that has developed in the subject may become chronic in the future (or is highly likely to become chronic).

On the other hand, when the urine of a subject does not contain thioredoxin or contains a small amount of thioredoxin, kidney-derived cells (e.g., tubular cells) of the subject undergo cell cycle transition from the _G2 phase to the M phase. It can be determined that progression is accelerated. In this case, it can be determined that there is a possibility (or a high possibility of recovery) of the renal disorder that has developed in the subject in the future.

Examples of the kidney-derived cells include non-human mammals (e.g., mammals such as dogs, cats, sheep, goats, mice, rats, rabbits, cows, horses, and pigs), and human kidney-derived cells. can be done.

Examples of the kidney-derived cells include renal tubular cells, proximal renal tubular cells, distal renal tubular cells, and collecting duct cells.

Thioredoxin includes oxidized thioredoxin and reduced thioredoxin, and thioredoxin released from renal tubular cells (renal tubular cells) is oxidized thioredoxin.

In this embodiment, thioredoxin can be detected using a known method. Examples of such methods include Chemiluminescent Enzyme Immunoassay (CLEIA), Chemiluminescent Immunoassay (CLIA), ELISA (Enzyme-Linked Immuno Sorbent Assay), and Western blotting. be able to. From the viewpoint of detecting thioredoxin rapidly and with high sensitivity, the method is preferably a chemiluminescent enzyme immunoassay method or a chemiluminescence immunoassay method.

An example of a method for detecting thioredoxin is described below, but the present invention is not limited to this method.

The detection method includes (I) a pretreatment step of diluting a sample (specifically, urine) with a buffer solution and maintaining the pH of the sample within a desired range (eg, pH 6 to pH 9); ) detecting thioredoxin contained in the pretreated sample. Thioredoxin is preferably detected using an immunological reaction, and in particular, a chemiluminescence immunoassay method or a chemiluminescence enzyme immunoassay method can be suitably used.

The specimen used in the pretreatment step may be used as it is collected from the subject, or diluted with a desired solvent (e.g., water or buffer). may be used.

In the pretreatment step, the pH of the sample diluted with the buffer solution is maintained preferably in the range of 6-9, more preferably in the range of 6.5-8. With the above configuration, thioredoxin can be detected more accurately. In particular, urine can be of varying pH. Therefore, when using urine as a sample, it is preferable to adjust the pH in the pretreatment step.

For adjustment of pH, alkaline agents (e.g., sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, sodium carbonate aqueous solution, sodium hydrogen carbonate aqueous solution, glycine-NaOH buffer, tricine-NaOH buffer, phosphate buffer, borate buffer solution, Tris-HCl buffer) can be used.

In the pretreatment step, the sample is diluted with a buffer solution. Thereby, the salt concentration of the specimen can be adjusted to a concentration suitable for detecting thioredoxin, and as a result, thioredoxin can be detected more accurately.

As the buffer solution, for example, a buffer solution mixed with animal serum can be used. With this configuration, thioredoxin can be detected more accurately by stabilizing the antibody-antigen reaction. Examples of the buffer solution mixed with animal serum include PioCube FluAB sample pretreatment solution manufactured by Toyobo Co., Ltd.

The mixing ratio of the specimen and the buffer solution is not particularly limited, but for example, when the volume or mass of the specimen is "1", the volume or mass of the specimen is mixed with a buffer solution of "5 to 10". preferably. With this configuration, thioredoxin can be detected more accurately.

The step of detecting thioredoxin can be performed by CLEIA method, CLIA method, ELISA method, Western blotting method, or the like. From the viewpoint of rapidly detecting thioredoxin, it is preferable to detect thioredoxin according to the CLEIA method. Incidentally, the CLEIA method is known from Japanese Patent Application Laid-Open No. 2001-235471, etc., and this document is incorporated herein by reference.

More specifically, the step of detecting thioredoxin can also be performed as follows.

In one aspect, detecting thioredoxin may comprise the steps of:
(a) contacting the specimen treated in the pretreatment step with a first antibody that specifically binds to thioredoxin contained in the specimen and to which a ligand is bound, and thioredoxin and the first the step of forming a complex with the antibody of (first immune reaction),
(b) dropping the complex onto a porous filter bound to a capture agent of the ligand to allow the ligand to bind to the capture agent;
(c) dripping a labeled second antibody that specifically binds to the thioredoxin onto the porous filter to allow the second antibody to bind to the complex (second immune reaction);
(d) washing the porous filter; and
(e) detecting activity (eg, fluorescence) of the label bound to the porous filter;

In another embodiment, detecting thioredoxin may comprise the steps of:
(a) the sample treated in the pretreatment step, the first antibody that specifically binds to thioredoxin contained in the sample and is bound to the ligand, and the portion different from the first antibody contacting a second antibody that specifically binds to the thioredoxin and is labeled to form a complex of the thioredoxin, the first antibody, and the second antibody;
(b) dropping the complex onto a porous filter bound to a capture agent of the ligand to allow the ligand to bind to the capture agent;
(c) washing the porous filter; and
(d) detecting activity (eg, fluorescence) of the label bound to the porous filter;

Examples of the first antibody include an antibody labeled with biotin. Examples of porous filters include glass fiber filters. The second antibody can include an antibody labeled with an enzyme (eg, fluorescent protein, Horse Radish Peroxidase (HRP), or Alkaline Phosphatase (AP)). An example of an apparatus that can be used for the CLEIA method is POCube (registered trademark), a compact chemiluminescence immunoassay analyzer manufactured by Toyobo Co., Ltd.

[2. A kit for diagnosing the presence or absence of cell cycle progression from _G2 phase to M phase in kidney-derived cells in a subject, and a kit for predicting recovery or chronic renal injury in a subject]
The kit for diagnosing the presence or absence of cell cycle progression from the _G2 phase to the M phase in kidney-derived cells in a subject of the present embodiment comprises a member for detecting thioredoxin in urine collected from a subject. I have.

A kit for predicting recovery or chronicity of renal injury in a subject of the present embodiment comprises a member for detecting thioredoxin in urine collected from the subject.

The member can be, for example, a thioredoxin binding protein or an anti-thioredoxin antibody.

Thioredoxin-binding proteins include well-known thioredoxin-binding proteins (eg, Thioredoxin-interacting protein (TXNIP), Thioredoxin binding protein 2 (TBP-2), Vitamin D3 up-regulated protein 1 (VDUP-1), Apoptosis signal-regulating kinase 1 (ASK1) and Mitogen-activated protein kinase kinase kinase 5 (MAP3K5)) can be used.

Anti-thioredoxin antibodies may be polyclonal antibodies or monoclonal antibodies. Also, the antibody of said antibody can be any type of immunoglobulin. Specifically, it may be IgG, IgM, IgA, IgD or IgE. Alternatively, the antibody may be a Fab fragment, an F(ab') ₂ fragment or an Fc fragment.

The above antibodies can be obtained by various known methods (e.g., HarLow et al., "Antibodies: A laboratory manual, Cold Spring Harbor Laboratory, New York (1988)", Iwasaki et al., "Monoclonal Antibody Hybridoma and ELISA, Kodansha (1991)"). ). Alternatively, the antibody may be a commercially available antibody.

Monoclonal antibodies can be obtained by methods well known in the art, such as the hybridoma method (Kohler, G. and Milstein, C., Nature 256, 495-497 (1975)), the trioma method, the human B-cell hybridoma method (Kozbor, Immunology Today). 4, 72 (1983)) and the EBV-hybridoma method (see, eg, Monoclonal Antibodies and Cancer Therapy, Alan R Liss, Inc., 77-96 (1985))).

Fab, F(ab') ₂ and Fc fragments are produced by cleaving antibodies with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') ₂ fragments). , can be made.

A ligand or a label may be bound to the member. With this configuration, thioredoxin can be detected more easily and more accurately.

The kit of the present embodiment may be provided with a component other than a member for detecting thioredoxin (for example, a washing solution and/or a porous filter to which a ligand-capturing agent is bound).

The chronification may be chronification via tubular interstitial fibrosis associated with cell cycle arrest from the _G2 phase to the M phase in kidney-derived cells. Further, the kit of the present embodiment may be a kit for predicting fibrosis of renal tubulointerstitium in a subject.

[3. others〕
The present invention can also be configured as follows.

One aspect of the present invention includes the step of detecting thioredoxin in urine (e.g., urine collected from a subject), from _G2 phase to M phase of kidney-derived cells (e.g., tubular cells) in a subject. is a method for diagnosing the presence or absence of cell cycle progression.

One aspect of the present invention is a method for predicting recovery or chronicity of renal injury in a subject, comprising the step of detecting thioredoxin in urine (in urine collected from the subject).

One aspect of the present invention is a method of cell cycle arrest from the _G2 phase to the M phase comprising the step of administering an inhibitor of thioredoxin reductase.

<1. Thioredoxin in urine as an indicator of G2 _- to-M cell cycle arrest in acute kidney injury.
Urinary thioredoxin 1 (TRX) was detected by ELISA and immunohistochemical techniques using kidneys and urine collected from mice model of acute renal injury caused by renal ischemia reperfusion injury. ) and the cell cycle. The ELISA method and the immunohistochemical method followed known methods.

As shown in the upper part of FIG. 1, from the ELISA test, thioredoxin 1 decreased in the kidney (specifically, renal tubular cells) in acute kidney injury model mice, and accompanying this, thioredoxin 1 in the urine Thioredoxin 1 was found to increase. Specifically, the amount of thioredoxin 1 in urine peaked 12 hours after inducing acute kidney injury. Thereafter, the amount of thioredoxin 1 in urine decreased gradually.

As shown in the lower part of FIG. 1, in the kidney, 12 hours after acute kidney injury, renal tubular cells expressing phosphorylated histone H3, which is a cell cycle arrest marker in the G ₂ /M phase, were regenerated. numbers have peaked. Subsequently, the number of renal tubular cells expressing phosphorylated histone H3, a cell cycle arrest marker at the G ₂ /M phase, gradually decreased as the amount of thioredoxin 1 in urine decreased ( For example, see test results 72 hours after inducing acute kidney injury).

From this test, acute kidney injury causes a decrease or loss of thioredoxin 1 in the kidney (specifically, renal tubular cells), which causes redox failure in the kidneys (specifically, renal tubular cells), and urinary An increase in thioredoxin was found to be accompanied by an increase in the number of cells in the kidney that were arrested in cell cycle progression from the _G2 phase to the M phase. In other words, this test revealed that thioredoxin in urine serves as a biomarker for diagnosing the presence or absence of cell cycle progression from the _G2 phase to the M phase of kidney-derived cells.

<2. Effects of thioredoxin reductase inhibitors on cyclin B expression>
Mouse proximal tubular cells (mProx cells) or human renal epithelial cells (HEK293 cells) were cultured on culture plates in known media. When the cells cover about 50% of the bottom of the culture plate, thioredoxin reductase inhibitor (2,4-Dinitrochlorobenzene: DNCB) is added to the medium to a final concentration of 1 μM, 5 μM, or 10 μM. bottom. In this test, DNCB was dissolved in DMSO (dimethylsulfoxide) so that the concentration of DNCB was 30 μM, and 1/1000 of the volume of the medium was added to the medium. bottom. On the other hand, in the vehicle (control), 1/1000 of the amount of DMSO in the medium was added to the medium.

8 hours or 24 hours after adding DNCB or DMSO to the medium, remove the medium in the culture plate, add 2 mM dithiothreitol, 1 mM NaVO ₃ , Complete protease inhibitor (Roche Applied Science ) was added in RIPA (radioimmune precipitation assay) buffer. Then, whole cell lysate in which cells were dissolved in RIPA buffer was recovered.

The protein concentration of the collected whole cell lysate was measured by the Lowry method, the Bradford method, or the BCA method. Whole cell lysate corresponding to 20 μg of protein was subjected to SDS-PAGE gel electrophoresis to separate proteins based on size.

Cyclin B1 and β-actin were then detected by Western blotting.

Specifically, the SDS-PAGE gel after electrophoresis was brought into close contact with the nitrocellulose membrane, a voltage was applied to the proteins in the SDS-PAGE gel, and the proteins in the SDS-PAGE gel were transferred to the nitrocellulose membrane. .

The protein-transferred nitrocellulose membrane was soaked in a solution containing anti-cyclin B1 antibody and anti-β-actin antibody.

After washing the nitrocellulose membrane with 0.05% TBS-T, the nitrocellulose membrane was immersed in a solution containing a secondary antibody labeled with HRP (horseradish peroxidase).

After washing the nitrocellulose membrane with 0.05% TBS-T, cyclin B1 and β-actin were detected by chemiluminescence.

Furthermore, a value was calculated by dividing the density of the band corresponding to cyclin B1 by the density of the band corresponding to β-actin.

The test results are shown in FIG. As is clear from FIG. 2, the thioredoxin reductase inhibitor markedly decreased the expression level of cyclin B, which is required for cell cycle progression from the _G2 phase to the M phase. More specifically, the final concentration of the thioredoxin reductase inhibitor was 10 μM, and the expression level of cyclin B in a test in which the whole cell lysate was recovered 24 hours after the addition of DNCB to the medium was ) was down to 1% of the cyclin B expression level.

<3. Effect of thioredoxin reductase inhibitor on phosphorylation of Cdc25C>
Mouse proximal tubular cells (mProx cells) or human renal epithelial cells (HEK293 cells) were cultured on culture plates in known media. When the cells covered approximately 50% of the bottom of the culture plate, thioredoxin reductase inhibitor (DNCB) was added to the medium to a final concentration of 1 μM, 5 μM, or 10 μM. In this test, a solution of DNCB in DMSO was prepared so that the concentration of DNCB was 30 μM as the stock solution of DNCB, and 1/1000 of the volume of the medium was added to the medium. On the other hand, in the vehicle (control), 1/1000 of the amount of DMSO in the medium was added to the medium.

24 hours after the addition of DNCB or DMSO to the medium, the medium in the culture plate was removed, and 2 mM dithiothreitol, 1 mM NaVO ₃ , Complete protease inhibitor (Roche Applied Science) was added to the cells on the culture plate and RIPA was added. Buffer was added. Then, whole cell lysate in which cells were dissolved in RIPA buffer was recovered.

The protein concentration of the collected whole cell lysate was measured by the Lowry method, the Bradford method, or the BCA method. Whole cell lysate corresponding to 20 μg of protein was subjected to SDS-PAGE gel electrophoresis to separate proteins based on size.

Cdc25C was then detected by Western blotting.

Specifically, the SDS-PAGE gel after electrophoresis was brought into close contact with the nitrocellulose membrane, a voltage was applied to the proteins in the SDS-PAGE gel, and the proteins in the SDS-PAGE gel were transferred to the nitrocellulose membrane. .

The nitrocellulose membrane with transferred proteins was soaked in a solution containing anti-Cdc25C antibody.

After washing the nitrocellulose membrane with 0.05% TBS-T, the nitrocellulose membrane was immersed in a solution containing an HRP-labeled secondary antibody.

Next, after washing the nitrocellulose membrane with 0.05% TBS-T, Cdc25C (more specifically, two types of phosphorylated Cdc25C) were detected based on the chemiluminescence method.

Furthermore, the density of the band corresponding to highly phosphorylated Cdc25C (Hyper-P in FIG. 3) was changed to that of the band corresponding to low phosphorylated Cdc25C (Hypo-P in FIG. 3). The value was calculated by dividing by the

The test results are shown in FIG. As is clear from FIG. 3, the thioredoxin reductase inhibitor markedly reduced the expression level of highly phosphorylated Cdc25C, which is required for cell cycle progression from the _G2 phase to the M phase.

<4. Effects of thioredoxin reductase inhibitors on cyclin D expression>
Mouse proximal tubular cells (mProx cells) or human renal epithelial cells (HEK293 cells) were cultured on culture plates in known media. When the cells covered approximately 50% of the bottom of the culture plate, thioredoxin reductase inhibitor (DNCB) was added to the medium to a final concentration of 1 μM, 5 μM, or 10 μM. In this test, a solution of DNCB in DMSO was prepared so that the concentration of DNCB was 30 μM as the stock solution of DNCB, and 1/1000 of the volume of the medium was added to the medium. On the other hand, in the vehicle (control), 1/1000 of the amount of DMSO in the medium was added to the medium.

8 hours or 24 hours after adding DNCB or DMSO to the medium, remove the medium in the culture plate, add 2 mM dithiothreitol, 1 mM NaVO ₃ , Complete protease inhibitor ( RIPA buffer supplemented with Roche Applied Science) was added. Next, whole cell lysate in which human embryonic kidney cells were dissolved in RIPA buffer was recovered.

The protein concentration of the collected whole cell lysate was measured by the Lowry method, the Bradford method, or the BCA method. Whole cell lysate corresponding to 20 μg of protein was subjected to SDS-PAGE gel electrophoresis to separate proteins based on size.

Cyclin D1 and β-actin were then detected by Western blotting.

Specifically, the SDS-PAGE gel after electrophoresis was brought into close contact with the nitrocellulose membrane, a voltage was applied to the proteins in the SDS-PAGE gel, and the proteins in the SDS-PAGE gel were transferred to the nitrocellulose membrane. .

The protein-transferred nitrocellulose membrane was soaked in a solution containing anti-cyclin D1 antibody and anti-β-actin antibody.

After washing the nitrocellulose membrane with 0.05% TBS-T, the nitrocellulose membrane was immersed in a solution containing an HRP-labeled secondary antibody.

After washing the nitrocellulose membrane with 0.05% TBS-T, cyclin D1 and β-actin were detected by chemiluminescence.

The test results are shown in FIG. As is clear from FIG. 4, the thioredoxin reductase inhibitor did not affect the expression level of cyclin D, which is unrelated to cell cycle progression from the _G2 phase to the M phase. Cyclin D is involved in cell cycle arrest at the _G1 phase.

<5. Effects of thioredoxin reductase inhibitors on the cell cycle>
Mouse proximal tubular cells (mProx cells) were cultured on culture plates in a known medium. When mouse proximal tubular cells covered approximately 50% of the bottom of the culture plate, thioredoxin reductase inhibitor (DNCB) was added to the medium to a final concentration of 30 μM. In this test, a solution of DNCB in DMSO was prepared so that the concentration of DNCB was 30 μM as the stock solution of DNCB, and 1/1000 of the volume of the medium was added to the medium. On the other hand, in the vehicle (control), 1/1000 of the amount of DMSO in the medium was added to the medium.

Twenty-four hours after adding DNCB or DMSO to the medium, remove the medium in the culture plate and collect the mouse proximal tubular cells by adding a trypsin solution to the mouse proximal tubular cells on the culture plate. bottom.

The trypsin solution containing mouse proximal tubular cells was transferred to a 1.5 mL tube. A pipette was used to dissociate the mouse proximal tubule cells into individual cells, since clumps of multiple cells made it impossible to detect individual cells by the flow cytometer.

The trypsin solution containing mouse proximal tubular cells was centrifuged at 600 xg for 5 minutes. The supernatant was discarded, 1 mL of PBS (phosphate buffered saline) was added to the mouse proximal tubular cells, and the mouse proximal tubular cells were suspended in the PBS.

PBS containing mouse proximal tubular cells was centrifuged at 600 xg for 5 minutes. The supernatant was discarded, 150 μL of PBS was added to the mouse proximal tubular cells, and the mouse proximal tubular cells were suspended in the PBS.

While gently stirring the PBS containing mouse proximal tubular cells, 350 μL of 100% ethanol was slowly added to the PBS to bring the final concentration of ethanol to 70%. Additionally, 500 μL of 70% ethanol was added.

70% ethanol containing mouse proximal tubular cells was allowed to stand overnight at −20° C., thereby fixing the mouse proximal tubular cells with ethanol.

Mouse proximal tubular cells in 70% ethanol were centrifuged at 3,000 xg for 2 minutes. The supernatant was discarded, 1 mL of PBS containing 0.01% BSA (bovine serum albumin) was added to the mouse proximal tubular cells, and the mouse proximal tubular cells were suspended in the PBS. . PBS containing mouse proximal tubular cells was centrifuged at 3,000×g for 2 minutes. The supernatant was discarded, 1 mL of PBS containing 0.01% BSA was added to the mouse proximal tubular cells, and the mouse proximal tubular cells were suspended in the PBS. PBS containing mouse proximal tubular cells was centrifuged at 3,000×g for 2 minutes. The supernatant was discarded, 0.5 mL of PBS containing 0.01% BSA was added to the mouse proximal tubular cells, and the mouse proximal tubular cells were suspended in the PBS.

0.5 mL PBS containing 0.01% BSA supplemented with Propidium Iodide (final concentration 20 μg/mL) and RNase (final concentration 20 μg/mL) to PBS containing mouse proximal tubular cells In addition, mouse proximal tubular cells were dissociated into individual cells using a pipette. This stained DNA in the nuclei of mouse proximal tubular cells.

The fluorescence intensity of the stained mouse proximal tubular cells was measured using a flow cytometer, and the phase of the cell cycle of each mouse proximal tubular cell was examined according to a known method. rice field.

The test results are shown in FIG. As evident from FIG. 5, the thioredoxin reductase inhibitor increased the number of cells arrested in cell cycle progression from the _G2 phase to the M phase.

Thioredoxin exists intracellularly as oxidized thioredoxin or reduced thioredoxin. At steady state, oxidized thioredoxin is reduced to reduced thioredoxin by thioredoxin reductase and recycled.

When cells are given oxidative stress that exceeds the reducing ability of thioredoxin reductase or an inhibitor of thioredoxin reductase, oxidized thioredoxin becomes excessive in the cell, and the oxidized thioredoxin is released outside the cell. When the phenomenon occurs in kidney-derived cells (e.g., renal tubular cells) in vivo, oxidized thioredoxin in kidney-derived cells is released into the urine (references: Kasuno et al., AJP Renal Physiol, 2014, Vol.307, No.12, p1342-51).

Inhibition of thioredoxin reductase activity also arrests cell cycle progression from _G2 to M phase of kidney-derived cells (see Examples above).

These facts indicate that the release of thioredoxin in kidney-derived cells into urine and the arrest of cell cycle progression from _G2 phase to M phase of kidney-derived cells occur simultaneously. In the present invention, utilizing the new findings, by detecting thioredoxin in urine collected from a subject, the transition of kidney-derived cells (e.g., tubular cells) in the subject from the _G2 phase to the M phase It makes it possible to diagnose the presence or absence of cell cycle progression.

Furthermore, it has been suggested that the transition from acute kidney injury to chronic kidney disease is associated with arrest of cell cycle progression from G2 _phase to M phase in renal epithelial cells (see Non-Patent Document 3). ). Therefore, it can be said that recovery or chronicity of renal damage in a subject can be predicted by detecting thioredoxin in urine collected from the subject.

<6. Effect of Inhibition of Thioredoxin 1 Expression on Cyclin B and Phosphorylated Histone H3 Expression>
Mouse proximal tubular cells (mProx cells) or human renal epithelial cells (HEK293 cells) were cultured on culture plates in known media. When the cells cover approximately 50% of the bottom surface of the culture plate, siRNA that inhibits the expression of TRX1 (TRX siRNA: TRX small interfering RNA, MISSION (registered trademark) esiRNA human TXN manufactured by Sigma-Aldrich), or siRNA (Negative siRNA: Negative small interfering RNA, Sigma-Aldrich) that does not inhibit the expression of TRX1 so that the final concentration is 12 pmol / mL MISSION® siRNA Universal Negative Control #1, Product No. SIC001 from Co., Ltd.) was added to the medium.

Introduction of TRX siRNA and Negative siRNA into cells was performed using a Thermofisher Lipofectamine RNAiMAX Transfection Reagent kit according to the protocol attached to the kit.

72 hours after the introduction of TRX siRNA or Negative siRNA into the cells, the medium in the culture plate was removed, and 2 mM dithiothreitol, 1 mM NaVO ₃ and Complete protease inhibitor (Roche Applied Science) were added to the cells on the culture plate. RIPA (radioimmune precipitation assay) buffer was added. Then, whole cell lysate in which cells were dissolved in RIPA buffer was recovered.

The protein concentration of the collected whole cell lysate was measured by the Lowry method, the Bradford method, or the BCA method. Whole cell lysate corresponding to 20 μg of protein was subjected to SDS-PAGE gel electrophoresis to separate proteins based on size.

Thioredoxin 1, cyclin B1, phosphorylated histone H3, and β-actin were then detected by Western blotting.

Specifically, the SDS-PAGE gel after electrophoresis was brought into close contact with the nitrocellulose membrane, a voltage was applied to the proteins in the SDS-PAGE gel, and the proteins in the SDS-PAGE gel were transferred to the nitrocellulose membrane. .

The protein-transferred nitrocellulose membrane was soaked in a solution containing anti-thioredoxin 1 antibody, anti-cyclin B1 antibody, anti-histone H3 antibody, and anti-β-actin antibody.

After washing the nitrocellulose membrane with 0.05% TBS-T, the nitrocellulose membrane was immersed in a solution containing a secondary antibody labeled with HRP (horseradish peroxidase).

After washing the nitrocellulose membrane with 0.05% TBS-T, thioredoxin 1, cyclin B1, phosphorylated histone H3, and β-actin were detected based on the chemiluminescence method.

Further, values were calculated by dividing the density of the bands corresponding to thioredoxin 1, cyclin B1, and phosphorylated histone H3 by the density of the band corresponding to β-actin.

The test results are shown in FIGS. 6(a) and 6(b). As is clear from Figures 6(a) and 6(b), inhibition of thioredoxin 1 expression reduced the expression level of cyclin B, which is required for cell cycle progression from the _G2 phase to the M phase. Moreover, inhibition of thioredoxin 1 expression increased the expression level of phosphorylated histone H3, a cell cycle arrest marker at the G ₂ /M phase. The degree of decrease in the expression level of cyclin B and increase in the expression level of phosphorylated histone H3 depended on the amount of TRX siRNA used in the test.

One aspect of the present invention can be widely used in fields related to the cell cycle and renal disorders.

Claims (4)

A method for obtaining data for diagnosing the presence or absence of cell cycle progression from _G2 phase to M phase in kidney-derived cells in a subject, comprising the step of detecting oxidized thioredoxin in urine collected from the subject. A kit for diagnosing the presence or absence of cell cycle progression from _G2 phase to M phase in kidney-derived cells in a subject, comprising a member for detecting oxidized thioredoxin in urine collected from the subject. A method for obtaining data for predicting recovery or chronicity of renal damage in a subject, comprising the step of detecting oxidized thioredoxin in urine collected from the subject ,
A method for acquiring data , wherein the chronification is chronification mediated by tubular interstitial fibrosis associated with cell cycle arrest from the G2 phase to the M phase in kidney-derived cells _. A kit for predicting recovery or chronicity of renal injury in a subject, comprising a member for detecting oxidized thioredoxin in urine collected from the subject ,
The kit , wherein the chronification is chronification mediated by tubular interstitial fibrosis associated with cell cycle arrest from the G2 phase to the M phase in kidney- _derived cells.

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