Kidney protection useful agent

Abstract

[Problem] The purpose of the present invention is to further develop various useful agents having 3, 5-dihydroxy-4-methoxybenzyl alcohol (3, 5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient, which have been obtained by the inventor, and to provide a compound having 3, 5-dihydroxy-4-methoxybenzyl alcohol (3, 5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient. The present invention relates to a variety of useful agents, such as useful kidney-protecting agents having a kidney-protecting effect, which are useful kidney-protecting agents (1, 3, 5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient. [Solution] The present invention is characterized by having, as an active ingredient, 3, 5-dihydroxy-4-methoxybenzyl alcohol (3, 5-dihydroxy-4-methoxybenzyl alcohol), and by having a kidney protection effect.

Description

Technical Field

[0001] The present invention relates to a useful agent for protecting the kidney, and in particular to a useful agent for protecting the kidney, which contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and has a protective effect on the kidney. Background Art

[0002] The oxidative stress state refers to a state in which the balance between the oxidative and anti-oxidative functions in cells is disrupted, and an example thereof includes a state in which excessive reactive oxygen species (ROS: reactive oxygen species) accumulate in cells.

[0003] Moreover, oxidative stress is the cause of various diseases such as aging and kidney disease in humans.

[0004] Here, the mitochondria existing in cells and in cells are described. Mitochondria are organelles of eukaryotic organisms, are composed of a double biological membrane, have their own DNA (mitochondrial DNA = mtDNA), and can divide and proliferate.

[0005] In addition to participating in the synthesis of ATP (a nucleotide formed by the combination of adenosine and three molecules of phosphate, an important substance that plays a mediating role in the storage, supply, and transportation of energy in living organisms. It is adenosine triphosphate that releases energy as it hydrolyzes ADP (adenosine diphosphate)), mtDNA is also known as the site of oxygen respiration (aerobic respiration).

[0006] In addition, it also plays an important role in cell apoptosis. mtDNA and its gene products are also partially located on the cell surface, and their mutations are specifically eliminated by the natural immune system.

[0007] In the human body, there are hundreds or thousands of mitochondria in metabolically active cells such as the liver, kidneys, muscles, and brain, accounting for about 40% of the cytoplasm. On average, there are 300 to 400 mitochondria in each cell, accounting for 10% of the body weight.

[0008] In mitochondria, high-energy electrons and oxygen molecules are used to synthesize ATP (a nucleotide formed by combining adenosine with three molecules of phosphate, an important substance that plays a mediating role in the storage, supply, and transportation of energy in living organisms. It is adenosine triphosphate that releases energy by hydrolyzing ADP (adenosine diphosphate)). However, when ATP is produced, the above-mentioned excess reactive oxygen species (ROS) are produced as a byproduct.

[0009] Furthermore, under the hyperactive state of oxidative stress, the function of mitochondria is particularly reduced, and excessive reactive oxygen species (ROS) are generated. The reactive oxygen species (ROS) further promote the generation of lipid peroxides through free radical chains, falling into a vicious cycle. Therefore, in order to maintain the balance between the generation and removal of ROS, it is considered important to maintain the function of mitochondria.

[0010] In addition, there are particularly many mitochondria in the proximal tubule cells of the kidney (Bhargava P and Schnellmann RG, Nat Rev Nephrol, 13: 629-646, 2017). The reason is believed to be that a large amount of ATP is required when reabsorbing nutrients, etc. Therefore, it can be considered that mitochondria play an important role in the proximal tubule cells of the kidney.

[0011] Oysters are bivalves belonging to the family Mytilidae, and their habitat is not only in Japan but also throughout East Asia. Oysters contain a large amount of minerals such as calcium and zinc in addition to glycogen and protein, making them a high-nutrition food.

[0012] The present inventors have explored and studied physiologically active substances from oysters, and through the search for substances with antioxidant activity from oyster extracts, have discovered the epoch-making antioxidant substance 3,5-dihydroxy-4-methoxybenzyl alcohol (hereinafter referred to as DHMBA), and have invented many useful agents using this substance.

[0013] So far, the free radical scavenging ability of DHMBA and the activation of the Keap1-Nrf2 pathway and the induction of the expression of the accompanying antioxidant gene group (HO-1 and NQO1, etc.) have been confirmed (Fuda H, Watanabe M, et al., Food Chem, 176: 226-33, 2015; Joko S, Watanabe M, et al., J Funct Foods, 35: 245-255, 2017).

[0014] The former is called direct antioxidant capacity, and the latter is called indirect antioxidant capacity, but DHMBA has been confirmed to be a substance that has both functions. Moreover, studies have shown that the cytotoxicity of DHMBA is lower than that of existing antioxidants. However, it has not been confirmed whether DHMBA is useful for kidney protection. Therefore, the present invention focuses on mitochondria, uses human kidney proximal tubule cells HK-2, and verifies and confirms the useful effects of DHMBA on various protections of mitochondria. Furthermore, according to the verification, it is confirmed that the present invention has useful effects such as kidney protection.

[0015] Prior art literature

[0016] Patent Literature

[0017] Patent Document 1: Japanese Patent Application Publication No. 2017-132753 Summary of the invention

[0018] Problem that the invention aims to solve

[0019] The object of the present invention is to further develop the inventions of various useful agents containing 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient already obtained by the inventors, and to provide various useful agents such as a useful agent for kidney protection having a kidney-protective effect containing 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient.

[0020] Solutions for solving problems

[0021] The present invention is characterized in that it is a useful agent for kidney protection, which uses 3,5-dihydroxy-4-methoxybenzyl alcohol as an effective ingredient and has a kidney protection effect;

[0022] or

[0023] The invention is characterized in that it is a promoter for reducing ROS in mitochondria of human renal proximal tubular cells (HK-2) under oxidative stress stimulation, with 3,5-dihydroxy-4-methoxybenzylalcohol as an effective ingredient, and has the effect of reducing ROS in mitochondria of human renal proximal tubular cells (HK-2) under oxidative stress stimulation;

[0024] or

[0025] The invention is characterized in that it is a cytotoxicity inhibitor of human renal proximal tubular cells (HK-2) and a cell survival rate enhancer of human renal proximal tubular cells (HK-2), and uses 3,5-dihydroxy-4-methoxybenzyl alcohol as an effective ingredient, has a cytotoxicity inhibitory effect on human renal proximal tubular cells (HK-2), and has a cell survival rate enhancing effect on human renal proximal tubular cells (HK-2);

[0026] or

[0027] The invention is characterized in that it is an inhibitor of the increase of reactive oxygen species accumulated in the cells of human renal proximal tubular cells (HK-2), and contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, and has an inhibitory effect on the increase of reactive oxygen species accumulated in the cells of human renal proximal tubular cells (HK-2);

[0028] or

[0029] The invention is characterized in that it is an inhibitor of the increase of reactive oxygen species accumulated in the mitochondria of human renal proximal tubular cells (HK-2), and contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, and has an inhibitory effect on the increase of reactive oxygen species accumulated in the mitochondria of human renal proximal tubular cells (HK-2);

[0030] or

[0031] The invention is characterized in that it is a respiratory activator of mitochondria of human kidney proximal tubular cells (HK-2), with 3,5-dihydroxy-4-methoxybenzyl alcohol as an effective ingredient, and has the function of activating the respiration of mitochondria of human kidney proximal tubular cells (HK-2);

[0032] or

[0033] The invention is characterized in that it is a promoter for increasing the number of mitochondria in human renal proximal tubular cells (HK-2), using 3,5-dihydroxy-4-methoxybenzyl alcohol as an effective ingredient, and has the effect of increasing the number of mitochondria in human renal proximal tubular cells (HK-2);

[0034] or

[0035] The invention is characterized in that it is a mitochondrial fragmentation inhibitor and mitochondrial biosynthesis pathway activator in human renal proximal tubular cells (HK-2), with 3,5-dihydroxy-4-methoxybenzyl alcohol as an effective ingredient, and has the function of inhibiting the fragmentation of mitochondria in human renal proximal tubular cells (HK-2) and activating the mitochondrial biosynthesis pathway;

[0036] or

[0037] It is an enhancer for the basal respiration, maximal respiration and ATP production of mitochondria in human renal proximal tubular cells (HK-2), characterized in that it uses 3,5-dihydroxy-4-methoxybenzylalcohol as an active ingredient and has the effect of enhancing the basal respiration, maximal respiration and ATP production of mitochondria in human renal proximal tubular cells (HK-2).

[0038] Effects of the Invention

[0039] According to the present invention, the following excellent effects are achieved: not only can various useful agents containing 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient be provided, but also various useful agents such as a useful agent for kidney protection having a kidney-protecting effect containing 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is an explanatory diagram for explaining the strategy for exploring the effect of improving mitochondrial function.

[0041] Figure 2 This is an explanatory diagram for explaining the protective effect of DHMBA on cells under oxidative stress. HK-2 cells were cultured with 100 μM BSO and 1 to 500 μM DHMBA, and then cell protection tests were performed based on cytotoxicity (A) and cell viability (B). Mean ± SD (n = 5 to 6).

[0042] Figure 3 This is an explanatory diagram for explaining the effect of DHMBA on reducing intracellular ROS under oxidative stress. HK-2 cells were cultured with 100 μM BSO and 250 and 500 μM DHMBA, and then intracellular ROS fluorescence images (A) and fluorescence intensity (B) were measured by DCFDA staining. Mean ± SD (n = 6).

[0043] Figure 4 This is an explanatory diagram for explaining the effect of DHMBA on reducing ROS in mitochondria under oxidative stress. HK-2 cells were cultured with 100 μM BSO and 250 and 500 μM DHMBA, and then the fluorescence image (A) and fluorescence intensity (B) of ROS in mitochondria were measured by DHR123 staining. Mean ± SD (n = 6).

[0044] Figure 5This is an explanatory diagram for explaining the changes in the expression levels of genes related to cell and mitochondrial damage under oxidative stress stimulation by DHMBA. HK-2 cells were cultured with 100 μM BSO and 250 and 500 μM DHMBA, and the gene expression levels were measured by qPCR. Mean ± SD (n = 4).

[0045] Figure 6 This is an explanatory diagram for explaining the changes in the expression level of genes related to mitochondrial biogenesis of DHMBA under oxidative stress stimulation. HK-2 cells were cultured with 100 μM BSO and 250 and 500 μM DHMBA, and the expression level of genes related to mitochondrial biogenesis was measured by qPCR. Mean ± SD (n = 4).

[0046] Figure 7 This is an illustration of the effect of DHMBA on mitochondrial morphology and the changes in the expression of genes related to fusion and fission under oxidative stress. After culturing HK-2 cells with 100 μM BSO, 250 and 500 μM DHMBA, mitochondrial morphology was observed by MitoTracker (registered trademark) Green FM fluorescence staining (A), and the expression of genes related to mitochondrial fusion and fission was measured by qPCR (B). Mean ± SD (n = 4).

[0047] Figure 8 This is an illustration of the effect of DHMBA on mitochondrial oxygen consumption rate. After culturing HK-2 cells with 100 μM BSO and 500 μM DHMBA for 24 hours, the oxygen consumption rate was measured using an extracellular flux analyzer (A). The main index for evaluating mitochondrial function can be calculated by the change in oxygen consumption rate (B). Mean ± SD (n = 3-4).

[0048] Fig. 9 This is an illustration of the effect of DHMBA on mitochondrial function. After culturing HK-2 cells with 100 μM BSO and 500 μM DHMBA for 24 hours, the oxygen consumption rate was measured using an extracellular flux analyzer, and the main indicators for mitochondrial function evaluation (basal respiration, ATP production, maximum respiration, spare respiratory capacity, proton leak, mitochondrial-independent oxygen consumption) were calculated. Mean ± SD (n = 3-4). DETAILED DESCRIPTION

[0049] Hereinafter, the present invention will be described based on one embodiment shown in the drawings.

[0050] Example

[0051] First, various tests were conducted to verify whether DHMBA is useful in kidney protection and the like, and the contents of the tests will be described.

[0052] "Materials and experimental methods for each verification experiment"

[0053] [Cell culture was performed]

[0054] Human renal proximal tubular cells (HK-2) were subcultured in Dulbecco's Modified Eagle's Medium (DMEM, Nacalai Tesque) containing 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator.

[0055] [Cytotoxicity and viability assays were performed]

[0056] To confirm the cytoprotective effect of DHMBA on oxidative stress-induced human renal proximal tubular cells HK-2, the cytotoxicity and viability of the cells were evaluated using the oxidative stress inducer buthionine sulfoximine (BSO).

[0057] Specifically, HK-2 cells were seeded into 96-well plates at 3.0×10 3 cells / well and cultured for 24 hours.

[0058] Next, 150 μL / well was added to make the final concentration of BSO 100 μM and the final concentration of DHMBA 1 - 500 μM. After culturing for 48 hours, 50 μL of the culture medium supernatant was transferred to a new 96-well plate, and a mixture (45:1) of the catalyst solution and the dye solution of the LDH cytotoxicity detection kit (Takara Bio Inc.) was added at 50 μL / well. After standing at room temperature for 30 minutes, the absorbance at 490 nm was measured using a microplate reader (Wallac 1420 ARVO Mx plate reader, PerkinElmer) to determine the cytotoxicity. In addition, 10 μL / well of the CCK-8 reagent (Dojindo Laboratories) was added to the plate with the cultured cells, and after culturing at 37°C for 2 hours, the absorbance at 450 nm was measured using a microplate reader (Wallac 1420 ARVO Mx plate reader) to evaluate the cell viability. The cytotoxicity and viability of the cells were calculated based on the absorbance values relative to the control group (control, 0 μg / mL) (n = 5 - 6 for each group).

[0059] [Measurement of intracellular ROS production was performed]

[0060] To evaluate the changes in intracellular ROS induced by DHMBA under oxidative stress stimulation, the production of intracellular ROS was measured.

[0061] HK-2 cells were seeded into a black 96-well plate at a density of 3.0 × 10 3 cells / well and cultured for 24 hours. Then, add 100 μL / well to make the final concentration of BSO 100 μM, and the final concentration of DHMBA 250 and 500 μM. After 24 hours, remove the supernatant and add 100 μL / well of DCFDA reagent (Sigma) diluted to 25 μM with serum-free DMEM, and culture at 37°C for 40 minutes. After further washing with PBS, add 100 μL / well of PBS, and measure the fluorescence at an excitation wavelength of 485 nm and a fluorescence wavelength of 535 nm using a microplate reader (Wallac1420ARVO Mx plate reader) (n=6 for each group), and observe and take pictures using a fluorescence microscope BZ-9000 (Keyence).

[0062] [Measurement of ROS production in mitochondria]

[0063] In order to verify the effect of DHMBA on the generation of ROS in mitochondria promoted by oxidative stress stimulation, ROS in mitochondria were observed using DHR123 and the fluorescence intensity was measured.

[0064] HK-2 cells were seeded into a black 24-well plate at 2.0×104 cells / well and cultured for 24 hours. Then, 500 μL / well was added to make the final concentration of BSO 100 μM and the final concentration of DHMBA 250 and 500 μM. After 24 hours, the supernatant was removed, and 250 μL / well of DHR123 reagent (Fuji Fiberchem Wako Pure Pharmaceutical) diluted to 5 μM with serum-free DMEM was added, and cultured at 37°C for 40 minutes.

[0065] After further washing with PBS, 500 μL of PBS was added, and the cells were observed and photographed using a fluorescence microscope BZ-9000 (Keyence). Then, the cells were peeled off from the bottom with a scraper and fully pipetted, and then transferred to a black 96-well plate at 200 μL / well, and the fluorescence was measured at an excitation wavelength of 505 nm and a fluorescence wavelength of 534 nm using a microplate reader (Wallac 1420 ARVO Mx plate reader) (n = 6 for each group).

[0066] [Measurement of gene expression levels related to cell and mitochondrial damage]

[0067] Under oxidative stress stimulation, ENDOG present in mitochondria transfers to the nucleus, playing a role in fragmenting nuclear DNA. In addition, when nuclear DNA is damaged, BIK increases and interacts with mitochondrial intrinsic factors to induce apoptosis, a type of cell death (Li LY, et al., Nature, 412: 95-99, 2001; Kutuk O, et al., PLoS One, 12: e0182809, 2017).

[0068] Therefore, the present invention verifies the effect of DHMBA on the cell death markers BIK and ENDOG which are increased under oxidative stress stimulation.

[0069] HK-2 cells were seeded into 6-well plates at a density of 5.0 × 10 4 cells / well and cultured for 24 hours. Next, add 2 mL / well to a final concentration of 100 μM BSO and 250 and 500 μM DHMBA. After 24 hours, use the NucleoSpin (registered trademark) RNA kit (MACHEREY-NAGEL) to recover the extracted total RNA. The concentration of the purified RNA was quantified by Nanodrop (Invitrogen). According to the protocol of ReverTra Ace (registered trademark) qPCR RT Master Mixwith gDNA Remover (TOYOBO), cDNA was synthesized from 1.0 μg of total RNA. Gene expression levels were measured according to the protocol of THUNDERBIRD (registered trademark) SYBR (registered trademark) qPCR Mix (TOYOBO). Use CFX Connect TM Real-time PCR analysis system (BioRad) was used for measurement. -(ΔΔCT) Method for analysis.

[0070] The gene expression level of the control group was set to 1 for comparison. The graph is represented by mean ± SD (n = 4 per group). Each expression level was corrected using the expression level of β-actin, a housekeeping gene. The primer sequences used are shown in Table 1.

[0071] [Table 1]

[0072] Table 1. Primer sequences of genes related to cell and mitochondrial damage

[0073]

[0074] F: forward primer; R: reverse primer

[0075] [Measurement of gene expression levels related to mitochondrial biogenesis]

[0076] It has been reported that mitochondrial biogenesis is regulated by activation of the PPARα / PGC1α / NRF1 / TFAM pathway (Uittenbogaard M and Chiaramello A, Current pharmaceutical design, 20:5574-5933, 2014).

[0077] The expression levels of genes related to mitochondrial biogenesis were measured in the same manner as in the previous item. The control group was set as 1 for comparison. The graphs are represented by mean ± SD (n = 3 per group). Each expression level was corrected by the expression level of β-actin. The primer sequences used are shown in Table 2.

[0078] [Table 2]

[0079] Table 2. Primer sequences for genes related to mitochondrial biogenesis

[0080]

[0081] F: forward primer; R: reverse primer

[0082] [Observation of mitochondrial morphology]

[0083] Excessive ROS induces mitochondrial fission, causing mitochondrial fragmentation and functional impairment (Zorov D, et al., Cells, 8: 175, 2019). In order to observe whether DHMBA affects mitochondrial morphological changes under oxidative stress stimulation, double staining of mitochondria and nuclei was performed using MitoTracker (registered trademark) Green FM (ThermoFisher Scientific) that responds to the membrane potential difference of mitochondria and Hoechst33342 (Dojindo Chemical Research Institute Co., Ltd.) that binds to nuclear DNA.

[0084] HK-2 cells were seeded into 35 mm culture dishes at a density of 5.0 × 10 4 Cells / dish were cultured for 24 hours. Then, 2 mL / well was added to make the final concentration of BSO 100 μM and the final concentration of DHMBA 250 and 500 μM. After 24 hours, 2 mL / dish of a mixed solution of 0.1 μM MitoTracker (registered trademark) Green FM and 5 μg / mL Hoechst33342 was added, cultured at 37°C for 20 minutes, washed with PBS, and 1 mL of PBS was added. Fluorescence images were obtained at 100 times using a fluorescence microscope BZ-9000 (Keyence).

[0085] [Measurement of gene expression levels related to mitochondrial fusion and fission]

[0086] The expression levels of genes related to mitochondrial fusion (OPA1) and fission (DRP1 and FIS1) were measured in the same manner as in the previous item. The control group was set as 1 for comparison. The graphs are represented by mean ± SD (n = 4 per group). Each expression level was corrected by the expression level of β-actin. The primer sequences used are shown in Table 3.

[0087] [Table 3]

[0088] Table 3. Primer sequences of genes related to mitochondrial fusion and fission

[0089]

[0090] F: forward primer; R: reverse primer

[0091] [Measurement of mitochondrial respiratory capacity]

[0092] The extracellular flux analyzer XFp (Agilent Technologies) was used to investigate the improving effect of DHMBA on the respiratory capacity of mitochondria under oxidative stress.

[0093] HK-2 cells were cultured at 1.5×10 4 Cells / well were inoculated into XFp cell culture microplates (Agilent Technologies) and cultured for 24 hours. Then, 200 μL / well was added to make the final concentration of BSO 100 μM and the final concentration of DHMBA 250 and 500 μM. After 24 hours of culture, it was replaced with running medium (XF DMEM Medium, pH 7.4, Agilent Technologies) containing 20mM GlutaMAX (Gibco), 10mM pyruvate (Gibco), and glucose (10mM), and cultured for 1 hour at 37°C in a CO2-free incubator. The calibration plate that had been hydrated for 24 hours was placed on the extracellular flux analyzer XFp for calibration. After the calibration, the calibration plate and cell plate were replaced to measure the oxygen consumption rate (oxygen consumption rat: OCR). First, the basal respiratory rate was measured, and then oligomycin (final concentration: 2 μM) was added to inhibit the production of ATP. Next, FCCP (final concentration: 1 μM) was added to measure the maximum respiratory rate. Finally, rotenone / antimycin A (final concentration: 0.25 μM) was added to measure the mitochondrial-independent respiration rate. In all measurements, each was measured three times.

[0094] [Statistical processing performed]

[0095] The obtained data were expressed as mean ± standard deviation (SD) and analyzed using JMP Pro16 (SAS Institute Inc.) Dunnett's test was used for multiple comparisons between groups, and p < 0.05 was set as the statistically significant level.

[0096] [Results and Investigation]

[0097] [About cytotoxicity and survival rate]

[0098] When 100 μM BSO was added to HK-2 cells (100 μM BSO + 0 μM DHMBA group), strong cytotoxicity was observed ( Figure 2 A) and low cell viability ( Figure 2 B). Next, by the combined treatment of 100 μM BSO and DHMBA, 3.9 μM or more DHMBA inhibited the cytotoxicity ( Figure 2 A), an increase in cell survival was observed ( Figure 2 B).

[0099] This result is considered to indicate that cell death was induced by BSO stimulation but was inhibited by DHMBA.

[0100] [About the production of intracellular ROS]

[0101] An increase in intracellular ROS was observed in cells supplemented with BSO, and in the co-treatment with DHMBA, the production of ROS was reduced to the same extent as in the control group ( Figure 3 A). In addition, from the measured Figure 3 The same result was obtained for the fluorescence intensity value of B. Therefore, the study showed that although intracellular ROS accumulated due to the stimulation of BSO, DHMBA inhibited the accumulation.

[0102] Here, Figure 3 The graphs show the effect of DHMBA in reducing intracellular ROS under oxidative stress stimulation. After HK-2 cells were cultured with 100 μM BSO and 250 and 500 μM DHMBA, the fluorescence image (A) and fluorescence intensity (B) of intracellular ROS were measured by DCFDA staining.

[0103] [About the production of ROS in mitochondria]

[0104] like Figure 4 As shown in A, the accumulation of ROS was also observed in the mitochondria by adding BSO, and the accumulation of ROS in the mitochondria was inhibited in the group with simultaneous addition of DHMBA. Figure 4The same results were obtained for the fluorescence intensity values ​​of B. The results show that ROS accumulate in mitochondria due to the stimulation of BSO, but DHMBA inhibits the increase.

[0105] Here, Figure 4 The graph shows the effect of DHMBA on reducing ROS in mitochondria under oxidative stress. After HK-2 cells were cultured with 100 μM BSO and 250 and 500 μM DHMBA, the fluorescence image (A) and fluorescence intensity (B) of ROS in mitochondria were measured by DHR123 staining. Mean±SD (n=6).

[0106] [About the expression level of genes related to cell and mitochondrial damage]

[0107] like Figure 5 As shown, the gene expression levels of BIK and ENDOG increased under oxidative stress stimulation, but were reduced to the same level as the control group by adding DHMBA. From these results, it can be considered that cell death was caused by BSO stimulation, but cell damage was reduced and cell death was inhibited by adding DHMBA.

[0108] Here, Figure 5 This is a graph showing changes in the expression levels of genes related to cell and mitochondrial damage under oxidative stress stimulation of DHMBA, and is a graph obtained by measuring gene expression levels by qPCR after culturing HK-2 cells with 100 μM BSO and 250 and 500 μM DHMBA. Mean±SD (n=4).

[0109] [About the expression level of genes related to mitochondrial biogenesis]

[0110] like Figure 6 As shown, due to oxidative stress, the gene expression of PPARα, which is related to mitochondrial biogenesis, increased, while the expression of PGC1α gene decreased. In addition, by adding DHMBA, the expression of PGC1α and TFAM increased. This result suggests that the mitochondrial biogenesis pathway was activated by adding DHMBA, and the number of mitochondria may increase.

[0111] Here, Figure 6 This is a graph showing changes in the expression level of genes related to mitochondrial biogenesis of DHMBA under oxidative stress stimulation. HK-2 cells were cultured with 100 μM BSO and 250 and 500 μM DHMBA, and the expression levels of genes related to mitochondrial biogenesis were measured by qPCR. Mean±SD (n=4).

[0112] [Observation of mitochondrial morphology and determination of gene expression levels related to fusion and fission]

[0113] like Figure 7 As shown in A, in the control group, mitochondria were observed to be long and network-like. On the other hand, under oxidative stress stimulation, mitochondrial fragmentation was induced, and the combined treatment with DHMBA reduced the fragmented mitochondria, and a mitochondrial morphology similar to that of the control group was observed. From this result, it can be inferred that the increase in ROS in cells and mitochondria caused by the addition of BSO caused mitochondrial fragmentation, but DHMBA suppressed the fragmentation.

[0114] In addition, the expression levels of genes OPA1, which is related to mitochondrial fusion, and DRP1 and FIS1, which are related to fission, were investigated. Under oxidative stress conditions, the expression level of OPA1 was suppressed, while the expression level of FIS1 increased ( Figure 7 B) It is believed that changes in these genes induce mitochondrial fragmentation, which is related to Figure 7 The morphological observation results of mitochondria were consistent with those of A. In addition, since the addition of DHMBA reduced the expression level of FIS1, it can be considered that DHMBA has an inhibitory effect on mitochondrial fragmentation.

[0115] Here, Figure 7 The figure shows the effect of DHMBA on mitochondrial morphology and the changes in the expression of genes related to fusion and fission under oxidative stress stimulation. After HK-2 cells were cultured with 100 μM BSO, 250 and 500 μM DHMBA, the mitochondrial morphology was observed by MitoTracker (registered trademark) Green FM fluorescence staining (A), and the expression of genes related to mitochondrial fusion and fission was measured by qPCR (B). Mean ± SD (n = 4).

[0116] [About the respiratory capacity of mitochondria]

[0117] Since mitochondria play an important role in energy production, this study evaluated the effect of DHMBA on mitochondrial respiratory capacity. By adding inhibitors to the mitochondrial electron transport system, the oxygen consumption rate (OCR) was measured over time. Figure 8 A. In addition, the changes in oxygen consumption rate caused by various inhibitors can be shown as Figure 8 B is shown. Furthermore, Fig. 9 Indicates changes in various mitochondrial functions. No impairment of mitochondrial function was observed after 24-hour addition of BSO, but basal and maximum respiration of mitochondria and ATP production increased after addition of DHMBA. This suggests that DHMBA is expected to have the effect of improving these mitochondrial functions.

[0118] Here, Figure 8is a graph showing the effect of DHMBA on mitochondrial oxygen consumption rate. After HK-2 cells were cultured with 100 μM BSO and 500 μM DHMBA for 24 hours, the oxygen consumption rate was measured using an extracellular flux analyzer ( Figure 8 A). By changing the oxygen consumption rate, the main indicators for evaluating mitochondrial function can be calculated ( Figure 8 B). Mean±SD (n=3-4).

[0119] also, Fig. 9 This is a graph showing the effect of DHMBA on mitochondrial function. After culturing HK-2 cells with 100 μM BSO and 500 μM DHMBA for 24 hours, the oxygen consumption rate was measured using an extracellular flux analyzer, and the main indicators for mitochondrial function evaluation (basal respiration, ATP production, maximum respiration, spare respiratory capacity, proton leak, mitochondrial-independent oxygen consumption) were calculated. Mean ± SD (n = 3-4).

[0120] [in conclusion]

[0121] Under oxidative stress induced by the addition of BSO, the amount of ROS in cells and mitochondria increased, inducing cell death, while the addition of DHMBA reduced the production of ROS, showing a cytoprotective effect.

[0122] In addition, it is believed that the addition of DHMBA can inhibit mitochondrial fragmentation and activate the mitochondrial biogenesis pathway, thereby protecting mitochondria from oxidative stress damage.

[0123] In addition, the results of mitochondrial respiratory capacity measurement showed that mitochondrial basal respiration, ATP production, and maximum respiration increased under oxidative stress. The above results show that DHMBA has a protective effect on cells and mitochondria and promotes mitochondrial function in human renal proximal tubular HK-2 cells under oxidative stress.

[0124] [About the manufacturing method of the present invention]

[0125] A method for producing a kidney-protective agent having a kidney-protective effect and the like containing the above-mentioned 3,5-dihydroxy-4-methoxybenzylalcohol as an active ingredient will be described.

[0126] 3,5-dihydroxy-4-methoxybenzyl alcohol can be obtained from oysters as described above, and the inventors have obtained many patents regarding methods for efficiently extracting and producing 3,5-dihydroxy-4-methoxybenzyl alcohol from the oysters. In addition, 3,5-dihydroxy-4-methoxybenzyl alcohol can also be produced by synthesis, and the inventors have also obtained a patent regarding the synthesis method.

[0127] In addition, regarding 3,5-dihydroxy-4-methoxybenzyl alcohol, it is presumed in the present invention that 3,5-dihydroxy-4-methoxybenzyl alcohol has many protective effects on various human internal organs, and experiments were conducted to verify this and the invention was made.

[0128] Therefore, the useful agent of the present invention is produced after performing these verifications.

Claims

1. A useful agent for protecting kidneys, It is characterized in that It contains 3,5-dihydroxy-4-methoxybenzyl alcohol as its active ingredient and has a kidney-protective effect.

2. A promoter for reducing ROS in mitochondria of human renal proximal tubular cells (HK-2) under oxidative stress, It is characterized in that The active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol, which has the effect of reducing ROS in mitochondria of human renal proximal tubular cells (HK-2) under oxidative stress.

3. A cytotoxicity inhibitor of human renal proximal tubular cells (HK-2) and a cell survival rate enhancer of human renal proximal tubular cells (HK-2), It is characterized in that The active ingredient of the invention is 3,5-dihydroxy-4-methoxybenzyl alcohol, which has an inhibitory effect on cytotoxicity of human renal proximal tubular cells (HK-2) and an effect of increasing the cell survival rate of human renal proximal tubular cells (HK-2).

4. An inhibitor of the increase of reactive oxygen species accumulated in the cells of human renal proximal tubule cells (HK-2), It is characterized in that The active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol, which has an inhibitory effect on the increase of reactive oxygen species accumulated in human renal proximal tubular cells (HK-2).

5. An inhibitor of the increase of reactive oxygen species accumulated in the mitochondria of human renal proximal tubular cells (HK-2), It is characterized in that The active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol, which has an inhibitory effect on the increase of reactive oxygen species accumulated in the mitochondria of human renal proximal tubular cells (HK-2).

6. A respiratory activator for mitochondrial cells of human kidney proximal tubule cells (HK-2), It is characterized in that The active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol, which has the effect of activating the respiration of mitochondria in human renal proximal tubular cells (HK-2).

7. An agent for increasing the number of mitochondria in human renal proximal tubular cells (HK-2), It is characterized in that The active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol, which has the effect of increasing the number of mitochondria in human renal proximal tubular cells (HK-2).

8. A mitochondrial fragmentation inhibitor and mitochondrial biosynthesis pathway activator in human renal proximal tubule cells (HK-2), It is characterized in that The invention contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, has the effect of inhibiting the fragmentation of mitochondria in human renal proximal tubular cells (HK-2) and activating the biosynthetic pathway of the mitochondria.

9. An enhancer for enhancing mitochondrial basal respiration, maximal respiration and ATP production in mitochondria of human renal proximal tubular cells (HK-2), It is characterized in that The active ingredient is 3,5-dihydroxy-4-methoxybenzylalcohol, which has the effect of improving the mitochondrial basal respiration, maximum respiration and ATP production of mitochondria in human renal proximal tubular cells (HK-2).

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