Method for producing a cytotoxicity inhibitor for human renal proximal tubular cells (HK2)

Abstract

To further develop the inventions of useful agents including 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, which the present inventors have already acquired, and to provide useful agents, such as a nephroprotective useful agent having a nephroprotective useful effect, including 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient.SOLUTION: The present invention is characterized by having a nephroprotective useful effect and including 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an agent useful for renal protection, and particularly to an agent useful for renal protection having a renal protection - useful action with 3,5 - dihydroxy - 4 - methoxybenzyl alcohol as an active ingredient.

Background Art

[0002] The oxidative stress state means a state in which the balance between oxidation and antioxidant functions in cells is disrupted. For example, a state in which excessive reactive oxygen species (ROS) accumulate in cells can be cited. And the oxidative stress state causes various diseases such as human aging and kidney diseases.

[0003] Here, regarding cells and mitochondria existing in cells, mitochondria are organelles of eukaryotic cells, consisting of a double biological membrane, having unique DNA (mitochondrial DNA = mtDNA), and dividing and proliferating.

[0004] The mtDNA is involved in life phenomena other than the synthesis of ATP (a nucleotide in which three phosphates are bound to adenosine, an important substance that mediates the storage, supply, and transport of energy in the living body. It is adenosine triphosphate that releases energy upon hydrolysis to ADP (adenosine diphosphate)), and is also known as the site of oxygen respiration (aerobic respiration).

[0005] It also plays an important role in cell apoptosis. Some of the mtDNA and its gene products are also localized on the cell surface, and their mutations are specifically eliminated by the innate immune system.

[0006] In humans, hundreds or thousands of mitochondria are found in metabolically active cells such as those in the liver, kidneys, muscles, and brain, making up about 40% of the cytoplasm. On average, there are 300-400 mitochondria in a single cell, and they account for 10% of body weight throughout the entire body.

[0007] Mitochondria utilize high-energy electrons and oxygen molecules to synthesize ATP (a nucleotide consisting of adenosine bonded to three phosphate groups, a vital substance that mediates the storage, supply, and transport of energy in living organisms; adenosine triphosphate, which releases energy upon hydrolysis to ADP (adenosine diphosphate)). However, the production of ATP also generates the aforementioned excess reactive oxygen species (ROS) as a byproduct.

[0008] Under conditions of increased oxidative stress, mitochondrial function, in particular, declines, leading to the production of excessive reactive oxygen species (ROS). These ROS then promote the formation of lipid peroxides through a radical chain reaction, creating a vicious cycle. Therefore, maintaining mitochondrial function is considered important to maintain a balance between ROS production and removal.

[0009] Furthermore, mitochondria are particularly abundant in the proximal tubular cells of the kidney (Bhargava P and Schnellmann RG, Nat Rev Nephrol, 13: 629-646, 2017). This is thought to be because a large amount of ATP is required when reabsorbing nutrients. Therefore, mitochondria are considered to play an important role in the proximal tubular cells of the kidney.

[0010] The Pacific oyster (Cornus flavimanus) is a bivalve mollusk belonging to the family Ostreidae in the order Ostreida, and its habitat extends throughout East Asia, including Japan. Pacific oysters are a highly nutritious food source, containing large amounts of glycogen, protein, and minerals such as calcium and zinc.

[0011] The inventor of this case conducted research and exploration of physiologically active substances from Pacific oysters, and as a result of searching for substances with antioxidant properties from oyster extracts, discovered the groundbreaking antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (hereinafter referred to as DHMBA), and invented many useful agents using it.

[0012] To date, DHMBA has been shown to have radical scavenging capabilities and activate the Keap1-Nrf2 pathway, leading to the induction of antioxidant gene expression (such as HO-1 and NQO1) (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).

[0013] The former is called direct antioxidant capacity, and the latter is called indirect antioxidant capacity, but it has been demonstrated that DHMBA is a substance that possesses both functions. Furthermore, it has been revealed that DHMBA has lower cytotoxicity than existing antioxidants. However, it had not been confirmed to date whether DHMBA is useful for renal protection. Therefore, in this invention, we focused on mitochondria and verified and confirmed the useful protective effects of DHMBA on mitochondria using human renal proximal tubular cells HK-2. Furthermore, from the above verification, it was confirmed that this invention has useful effects such as renal protection. [Prior art documents] [Patent Documents]

[0014] [Patent Document 1] Japanese Patent Publication No. 2017-132753 [Overview of the project] [Problems that the invention aims to solve]

[0015] The present invention further develops the invention of each useful agent having 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, which the present inventor has already obtained, and aims to provide each useful agent such as a renal protective agent having a renal protective useful action with 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient.

Means for Solving the Problems

[0016] The present invention Human renal proximal tubular cells (HK-2) were stimulated with oxidative stress by adding 100 μM of an oxidative stress inducer (BSO) to human renal proximal tubular cells (HK-2). To the human renal proximal tubular cells (HK-2) that have been subjected to the oxidative stress stimulation described above, 3,5-dihydroxy-4-methoxybenzyl alcohol was added at a concentration of 3.9 μM or higher. The absorbance of human renal proximal tubular cells (HK-2) after addition was measured to determine cytotoxicity, and the measured value was obtained. Based on the obtained measurement values, we obtained verification results showing that the cytotoxicity of human renal proximal tubular cells (HK-2) can be suppressed by the aforementioned additive. The obtained verification results were obtained. manufactured a cytotoxicity inhibitor of human renal proximal tubular cells (HK-2) with 3,5-dihydroxy-4-methoxybenzyl alcohol at a concentration of 3.9 μM or more Use characterized by this or Human renal proximal tubular cells (HK-2) were stimulated with oxidative stress by adding 100 μM of an oxidative stress inducer (BSO) to human renal proximal tubular cells (HK-2). To the human renal proximal tubular cells (HK-2) that have been subjected to the oxidative stress stimulation described above, 3,5-dihydroxy-4-methoxybenzyl alcohol was added at a concentration of 3.9 μM or higher. The fluorescence intensity of human renal proximal tubular cells (HK-2) after addition was measured to obtain the measurement value. From the obtained measurements, it was found that the addition of the above-mentioned additive affected human renal proximal tubular cells (HK-2). The cell survival rate Obtain increasing verification results, The obtained verification results were obtained. manufactured a cell survival rate increasing agent of human renal proximal tubular cells (HK-2) with 3,5-dihydroxy-4-methoxybenzyl alcohol at a concentration of 3.9 μM or more Use characterized by this or Human renal proximal tubular cells (HK-2) were stimulated with oxidative stress by adding 100 μM of an oxidative stress inducer (BSO) to human renal proximal tubular cells (HK-2). To the human renal proximal tubular cells (HK-2) that have been subjected to the oxidative stress stimulation described above, 3,5-dihydroxy-4-methoxybenzyl alcohol is added at a concentration of 250 μM or 500 μM. ​​ Regarding human renal proximal tubular cells (HK-2) after addition: DHR123 staining We obtained intracellular ROS fluorescence images and fluorescence intensity measurements using this method. From the obtained measurements, the addition of the above-mentioned additive to human renal proximal tubular cells (HK-2) accumulated in mitochondria We obtained verification results that reduce the increase in ROS. The obtained verification results were obtained. The aforementioned 3,5-dihydroxy-4-methoxybenzyl alcohol Use We have manufactured an agent that suppresses the increase of ROS accumulated in the mitochondria of human renal proximal tubular cells (HK-2). It is characterized by the following: or Human renal proximal tubular cells (HK-2) were stimulated with oxidative stress by adding 100 μM of an oxidative stress inducer (BSO) to human renal proximal tubular cells (HK-2). To the human renal proximal tubular cells (HK-2) that have been subjected to the oxidative stress stimulation described above, 3,5-dihydroxy-4-methoxybenzyl alcohol is added at a concentration of 250 μM or 500 μM. After addition, basal and maximal respiration of mitochondria in human renal proximal tubular cells (HK-2) were measured and values ​​were obtained. Based on the obtained measurements, the addition Activates mitochondrial respiration in human renal proximal tubular cells (HK-2). Obtain the verification results, The obtained verification results were obtained. The aforementioned 3,5-dihydroxy-4-methoxybenzyl alcohol Use We manufactured a respiratory activator for mitochondria in human renal proximal tubular cells (HK-2). It is characterized by the following: or Human renal proximal tubular cells (HK-2) were stimulated with oxidative stress by adding 100 μM of an oxidative stress inducer (BSO) to human renal proximal tubular cells (HK-2). To the human renal proximal tubular cells (HK-2) that have been subjected to the oxidative stress stimulation described above, 3,5-dihydroxy-4-methoxybenzyl alcohol is added at a concentration of 250 μM or 500 μM. After adding the substance, human renal proximal tubular cells (HK-2) were subjected to qPCR to measure the expression levels of mitochondrial biosynthesis-related genes, and measurements indicating activation of the mitochondrial biosynthesis pathway were obtained. Based on the obtained measurement values, we obtained verification results showing that the aforementioned additive increases the number of mitochondria in human renal proximal tubular cells (HK-2). The obtained verification results were obtained. The aforementioned 3,5-dihydroxy-4-methoxybenzyl alcohol Use We manufactured an agent that promotes the increase of mitochondrial numbers in human renal proximal tubular cells (HK-2). It is characterized by the following: or Human renal proximal tubular cells (HK-2) were stimulated with oxidative stress by adding 100 μM of an oxidative stress inducer (BSO) to human renal proximal tubular cells (HK-2). To the human renal proximal tubular cells (HK-2) that have been subjected to the oxidative stress stimulation described above, 3,5-dihydroxy-4-methoxybenzyl alcohol is added at a concentration of 250 μM or 500 μM. After the addition of the substance, human renal proximal tubular cells (HK-2) were subjected to observation of mitochondrial morphology by Green FM fluorescence staining, and measurement of mitochondrial fusion and fission-related gene expression levels by qPCR. Based on the observation and measurement results, the addition of the substance was observed to be effective. Mitochondrial fragmentation of human renal proximal tubular cells (HK-2) Observational and verification results were obtained to suppress the suppression of Observation results and verification results were obtained. The aforementioned 3,5-dihydroxy-4-methoxybenzyl alcohol Use We have manufactured a human renal proximal tubular cell (HK-2) mitochondrial fragmentation inhibitor that has an inhibitory effect on mitochondrial fragmentation of human renal proximal tubular cells (HK-2). It is characterized by the following: or Human renal proximal tubular cells (HK-2) were stimulated with oxidative stress by adding 100 μM of an oxidative stress inducer (BSO) to human renal proximal tubular cells (HK-2). To the human renal proximal tubular cells (HK-2) that have been subjected to the oxidative stress stimulation described above, 3,5-dihydroxy-4-methoxybenzyl alcohol is added at a concentration of 250 μM or 500 μM. The ATP production values ​​of mitochondria in human renal proximal tubular cells (HK-2) after addition were measured to obtain the values. From the obtained measurement values, the addition ATP production in mitochondria of human renal proximal tubular cells (HK-2) We obtained verification results that increased it, The obtained verification results were obtained. The aforementioned 3,5-dihydroxy-4-methoxybenzyl alcohol Use Mitochondria of human renal proximal tubular cells (HK-2) An agent that increases ATP production Manufactured, It is characterized by the following: [Effects of the Invention]

[0017] The present invention not only provides various useful agents containing 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, but also offers the excellent effect of providing various useful agents, such as a renal protective agent having a renal protective effect, also containing 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient. [Brief explanation of the drawing]

[0018] [Figure 1] This is an explanatory diagram illustrating strategies for exploring the effects of improving mitochondrial function. [Figure 2] This diagram illustrates the cytoprotective effect of DHMBA under oxidative stress. HK-2 cells were cultured with 100 μM BSO and 1-500 μM DHMBA, followed by cytoprotection tests using cytotoxicity (A) and cell viability (B). Mean ± SD (n = 5-6). [Figure 3] This diagram illustrates 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 intracellular ROS fluorescence images (A) and fluorescence intensity (B) were measured by DCFDA staining. Mean ± SD (n = 6). [Figure 4] This diagram illustrates the effect of DHMBA on reducing mitochondrial ROS under oxidative stress. HK-2 cells were cultured with 100 μM BSO and 250 and 500 μM DHMBA, and then mitochondrial ROS fluorescence images (A) and fluorescence intensity (B) were measured by DHR123 staining. Mean ± SD (n = 6). [Figure 5] This diagram illustrates the changes in cellular and mitochondrial dysfunction-related gene expression levels in response to DHMBA under oxidative stress. HK-2 cells were cultured with 100 μM BSO and 250 and 500 μM DHMBA, after which gene expression levels were measured by qPCR. Mean ± SD (n = 4). [Figure 6] This diagram illustrates the changes in mitochondrial biosynthesis-related gene expression levels in DHMBA under oxidative stress. HK-2 cells were cultured with 100 μM BSO and 250 and 500 μM DHMBA, after which mitochondrial biosynthesis-related gene expression levels were measured by qPCR. Mean ± SD (n = 4). [Figure 7]This diagram illustrates the effects of DHMBA on mitochondrial morphology and changes in the expression levels of fusion and fission-related genes under oxidative stress. HK-2 cells were cultured with 100 μM BSO and 250 and 500 μM DHMBA. Mitochondrial morphology was observed using MitoTracker® Green FM fluorescence staining (A), and mitochondrial fusion and fission-related gene expression levels were measured by qPCR (B). Mean ± SD (n = 4). [Figure 8] This diagram illustrates the effect of DHMBA on mitochondrial oxygen consumption rate. HK-2 cells were cultured with 100 μM BSO and 500 μM DHMBA for 24 hours, after which oxygen consumption rate was measured using an extracellular flux analyzer (A). Key indicators for mitochondrial function can be calculated from the changes in oxygen consumption rate (B). Mean ± SD (n = 3-4). [Figure 9] This diagram illustrates the effects of DHMBA on mitochondrial function. HK-2 cells were cultured with 100 μM BSO and 500 μM DHMBA for 24 hours. Oxygen consumption rates were then measured using an extracellular flux analyzer, and key indicators of mitochondrial function (basal respiration, ATP production, maximal respiration, respiratory reserve, proton leak, and mitochondrial-independent oxygen consumption) were calculated. Mean ± SD (n = 3-4). [Modes for carrying out the invention]

[0019] The present invention will be described below based on an embodiment shown in the figure. [Examples]

[0020] First, we conducted various studies to verify whether DHMBA could demonstrate usefulness in areas such as renal protection, and we will explain the details of those studies.

[0021] "Materials and experimental methods for each verification experiment" (Cell culture was performed) 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-streptmycin at 37°C in a 5% CO2 incubator.

[0022] (Cytotoxicity and survival rate tests were conducted.) To confirm the protective effect of DHMBA on human renal proximal tubular cells (HK-2 cells) against oxidative stress, cell toxicity and viability were evaluated using the oxidative stress inducer buthionine sulfoximine (BSO). Specifically, HK-2 cells are placed in a 96-well plate in a 3.0 × 10⁶ arrangement. 3 Seeds were seeded to a cell / well ratio and cultured for 24 hours.

[0023] Next, 150 μL / well of BSO was added to a final concentration of 100 μM and DHMBA to a final concentration of 1-500 μM. After 48 hours of incubation, 50 μL of the supernatant was taken and transferred to a new 96-well plate, and 50 μL / well of a mixture of catalyst solution and dye solution (45:1) from the LDH Cytotoxicity Detection Kit (Takara Bio Inc.) was added. After standing at room temperature for 30 minutes, the absorbance at 490 nm was measured using a plate reader (Wallac 1420 ARVO Mx plate reader, PerkinElmer) to measure cytotoxicity. In addition, 10 μL / well of CCK-8 reagent (Dojin Chemical Laboratories Co., Ltd.) was added to the plate in which the cells had been cultured, and after incubation at 37°C for 2 hours, the absorbance at 450 nm was measured using a plate reader (Wallac 1420 ARVO Mx plate reader) to evaluate cell viability. Cell toxicity and viability were calculated from absorbance values ​​relative to the control (0 μg / mL) (n = 5-6 for each group).

[0024] (Intracellular ROS production was measured.) To evaluate the changes in intracellular ROS induced by DHMBA under oxidative stress stimulation, the amount of intracellular ROS produced was measured. HK-2 cells were placed in a black 96-well plate in a 3.0 × 10⁶ arrangement. 3 Cells were seeded to a concentration of cells / well and cultured for 24 hours. Next, 100 μL / well of BSO was added to a final concentration of 100 μM, and 100 μL / well of DHMBA to final concentrations of 250 μM and 500 μM. After 24 hours, the supernatant was removed, and 100 μL / well of DCFDA reagent (Sigma), diluted to 25 μM in serum-free DMEM, was added, and the cells were cultured at 37°C for 40 minutes. After further washing with PBS, 100 μL / well of PBS was added, and fluorescence was measured at excitation wavelength 485 nm and fluorescence wavelength 535 nm using a plate reader (Wallac 1420 ARVO Mx plate reader) (n = 6 for each group). The fluorescence was observed and photographed using a fluorescence microscope BZ-9000 (Keyence).

[0025] (Mitochondrial ROS production levels were measured.) To investigate the effect of DHMBA on mitochondrial ROS production, which is promoted by oxidative stress stimulation, mitochondrial ROS were observed and fluorescence intensity was measured using DHR123.

[0026] HK-2 cells are placed in a black 24-well plate in a 2.0 × 10⁶ arrangement. 4 Cells were seeded to a density of cells / well and cultured for 24 hours. Next, 500 μL / well of BSO was added to a final concentration of 100 μM, and 500 μM of DHMBA was added to final concentrations of 250 μM and 500 μM. After 24 hours, the supernatant was removed, and 250 μL / well of DHR123 reagent (Fujifilm Wako Pure Chemical Industries) diluted to 5 μM in serum-free DMEM was added, and the cells were cultured at 37°C for 40 minutes.

[0027] 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). Subsequently, the cells were detached from the bottom with a scraper and thoroughly pipetted. Then, 200 μL / well was transferred to a black 96-well plate, and fluorescence was measured at an excitation wavelength of 505 nm and an emission wavelength of 534 nm using a plate reader (Wallac 1420 ARVO Mx plate reader) (n = 6 for each group).

[0028] (We measured the expression levels of genes related to cellular and mitochondrial dysfunction.) Under oxidative stress stimulation, ENDOG, present in mitochondria, translocates to the nucleus and acts on nuclear DNA fragmentation. Furthermore, when nuclear DNA is damaged, BIK increases, interacting 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).

[0029] Therefore, in this invention, we investigated the effects of DHMBA on the cell death markers BIK and ENDOG, which increase under oxidative stress stimulation. 5.0 × 10⁶ HK-2 cells in a 6-well plate 4Cells were seeded to a density of cells / well and cultured for 24 hours. Next, 2 mL / well of BSO was added to a final concentration of 100 μM, and 2 mL / well of DHMBA to final concentrations of 250 μM and 500 μM. After 24 hours, total RNA was extracted and collected using the NucleoSpin® RNA Kit (MACHEREY-NAGEL). The concentration of purified RNA was quantified using Nanodrop (Invitrogen). cDNA was synthesized from 1.0 μg of total RNA according to the ReverTra Ace® qPCR RT Master Mix with gDNA Remover (TOYOBO) protocol. Gene expression levels were measured according to the THUNDERBIRD® SYBR® qPCR Mix (TOYOBO) protocol. CFX Connect TM The measurement was performed using a real-time PCR analysis system (BioRad), and 2 -(ΔΔCT) The analysis was performed using the specified method.

[0030] The gene expression levels of the control group were set to 1 for comparison. The figures are shown as mean ± SD (n = 4 for each group). Each expression level was corrected for the expression level of the housekeeping gene β-actin. The sequences of the primers used are shown in Table 1.

[0031] [Table 1]

[0032] (We measured the expression levels of genes related to mitochondrial biosynthesis.) Mitochondrial biosynthesis has been reported to be regulated via the activation of the PPARα / PGC1α / NRF1 / TFAM pathway (Uittenbogaard M and Chiaramello A, Current pharmaceutical design, 20: 5574-5933, 2014). The expression levels of genes related to mitochondrial biosynthesis were measured in the same manner as described in the previous section. A control group was set as 1 for comparison. Figures are shown as mean ± SD (n = 3 for each group). Each expression level was corrected for the expression level of β-actin. The sequences of the primers used are shown in Table 2.

[0033] [Table 2]

[0034] (We performed morphological observations of mitochondria.) Excessive production of ROS induces mitochondrial fission, leading to mitochondrial fragmentation and dysfunction (Zorov D, et al., Cells, 8: 175, 2019). To observe whether DHMBA affects mitochondrial morphological changes under oxidative stress stimulation, double staining of mitochondria and the nucleus was performed using MitoTracker® Green FM (Thermo Fisher Scientific), which responds to the mitochondrial membrane potential difference, and Hoechst33342 (Dojin Chemical Laboratories Co., Ltd.), which binds to nuclear DNA.

[0035] HK-2 cells were placed in a 35 mm dish in a 5.0 × 10⁶ area. 4 Cells were seeded to a concentration of 100 μM per dish and cultured for 24 hours. Next, 2 mL / well of BSO and 2 mL / well of DHMBA were added to final concentrations of 250 μM and 500 μM respectively. 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, and the cells were cultured at 37°C for 20 minutes. After washing with PBS, 1 mL of PBS was added, and fluorescence images were obtained at 100x magnification using a fluorescence microscope BZ-9000 (Keyence).

[0036] (Mitochondrial fusion and fission-related gene expression levels were measured.) The expression levels of genes related to mitochondrial fusion (OPA1) and fission (DRP1 and FIS1) were measured in the same manner as described above. The control group was set to 1 for comparison. Figures are shown as mean ± SD (n = 4 for each group). Each expression level was corrected for β-actin expression. The sequences of the primers used are shown in Table 3.

[0037] [Table 3]

[0038] (We measured the respiratory capacity of mitochondria.) We investigated the effect of DHMBA on improving mitochondrial respiratory capacity under oxidative stress using the extracellular flux analyzer XFp (Agilent Technologies).

[0039] HK-2 cells were placed in an XFp Cell Culture Miniplate (Agilent Technologies) in a 1.5 × 10⁶ layer. 4Cells were seeded at a rate of cells / well and cultured for 24 hours. Next, 200 μL / well of BSO was added to a final concentration of 100 μM, and 200 μL / well of DHMBA to final concentrations of 250 μM and 500 μM. After 24 hours of culture, the medium was changed to running medium (XF DMEM Medium, pH 7.4, Agilent Technologies) containing 20 mM GlutaMAX (Gibco), 10 mM Pyruvate (Gibco), and Glucose (10 mM), and the cells were cultured for 1 hour in an incubator at 37°C without CO2 control. Calibration plates, which had been hydrated for 24 hours, were placed in an extracellular flux analyzer XFp and calibration was performed. After calibration, the calibration plate and cell plate were exchanged, and the oxygen consumption rate (OCR) was measured. The basal respiratory rate was measured first, followed by the addition of oligomycin (final concentration: 2 μM) to inhibit ATP production. Next, FCCP (final concentration: 1 μM) was added, and the maximum respiratory rate was measured. Finally, rotenone / antimycin A (final concentration: 0.25 μM) was added, and the mitochondrial-independent respiration rate was measured. All measurements were performed three times.

[0040] (Statistical processing was performed) The obtained data were expressed as mean ± standard deviation (SD) and analyzed using JMP Pro 16 (SAS Institute Inc.). Dunnett's test was used for multiple comparisons between groups, with p < 0.05 being the statistical significance level.

[0041] (Results and Discussion) (Regarding cytotoxicity and survival rate) When HK-2 cells were treated with 100 μM BSO (100 μM BSO + 0 μM DHMBA group), strong cytotoxicity (Figure 2A) and low cell viability (Figure 2B) were observed. Next, co-treatment with 100 μM BSO and DHMBA showed that DHMBA at concentrations of 3.9 μM or higher suppressed cytotoxicity (Figure 2A) and increased cell viability (Figure 2B). These results suggest that BSO stimulation induced cell death, but DHMBA suppressed it.

[0042] (Regarding intracellular ROS production) An increase in intracellular ROS was observed in cells treated with BSO, and co-treatment with DHMBA reduced ROS production to a level similar to that of the control group (Figure 3A). The same result was obtained from the fluorescence intensity measurements in Figure 3B. Therefore, it was revealed that BSO stimulation led to intracellular ROS accumulation, but DHMBA suppressed this accumulation.

[0043] Figure 3 shows the effect of DHMBA on reducing intracellular ROS under oxidative stress stimulation. HK-2 cells were cultured with 100 μM BSO and 250 and 500 μM DHMBA, and intracellular ROS fluorescence images (A) and fluorescence intensity (B) were measured by DCFDA staining.

[0044] (Regarding the amount of ROS produced in mitochondria) As shown in Figure 4A, ROS accumulation was observed in mitochondria upon addition of BSO, and suppression of mitochondrial ROS accumulation was observed in the group simultaneously treated with DHMBA. The same results were obtained from the fluorescence intensity measurements in Figure 4B. From these results, it became clear that BSO stimulation led to mitochondrial ROS accumulation, but DHMBA suppressed this increase.

[0045] Figure 4 shows the effect of DHMBA on reducing mitochondrial ROS under oxidative stress stimulation. HK-2 cells were cultured with 100 μM BSO and 250 and 500 μM DHMBA, and then mitochondrial ROS fluorescence images (A) and fluorescence intensity (B) were measured by DHR123 staining. Mean ± SD (n = 6).

[0046] (Regarding the expression levels of genes related to cellular and mitochondrial dysfunction) As shown in Figure 5, the gene expression levels of BIK and ENDOG increased under oxidative stress stimulation, but decreased to the same level as the control group upon addition of DHMBA. These results suggest that cell death occurred due to BSO stimulation, but cell damage was reduced and cell death was suppressed by the addition of DHMBA.

[0047] Figure 5 shows the changes in cellular and mitochondrial dysfunction-related gene expression levels in response to DHMBA under oxidative stress stimulation. HK-2 cells were cultured with 100 μM BSO and 250 and 500 μM DHMBA, after which gene expression levels were measured by qPCR. Mean ± SD (n = 4).

[0048] (Regarding the expression levels of genes related to mitochondrial biosynthesis) As shown in Figure 6, oxidative stress increased the gene expression level of PPARα, which is involved in mitochondrial biosynthesis, but decreased the gene expression level of PGC1α. Furthermore, the addition of DHMBA increased the expression levels of both PGC1α and TFAM. These results suggest that DHMBA addition may have activated the mitochondrial biosynthesis pathway and increased the number of mitochondria.

[0049] Figure 6 shows the changes in mitochondrial biosynthesis-related gene expression levels in DHMBA under oxidative stress stimulation. HK-2 cells were cultured with 100 μM BSO and 250 and 500 μM DHMBA, and then mitochondrial biosynthesis-related gene expression levels were measured by qPCR. Mean ± SD (n = 4).

[0050] (Regarding mitochondrial morphological observation and measurement of gene expression levels related to fusion and fission) As shown in Figure 7A, in the control group, mitochondria were observed to be long and linear, forming a network. On the other hand, under oxidative stress stimulation, mitochondrial fragmentation was induced, and co-treatment with DHMBA reduced the number of fragmented mitochondria, resulting in a mitochondrial morphology similar to that of the control group. From these results, it was inferred that the increase in ROS within cells and mitochondria due to BSO addition caused mitochondrial fragmentation, but that this fragmentation was suppressed by DHMBA.

[0051] Furthermore, we investigated the expression levels of the OPA1 gene, which is related to mitochondrial fusion, and the DRP1 and FIS1 genes, which are related to fission. Under oxidative stress conditions, OPA1 expression was suppressed and FIS1 expression was increased (Figure 7B). It is thought that these changes in genes induced mitochondrial fragmentation, which is consistent with the results of mitochondrial morphological observations in Figure 7A. In addition, the addition of DHMBA reduced FIS1 expression, suggesting that DHMBA had an inhibitory effect on mitochondrial fragmentation.

[0052] Figure 7 shows the effects of DHMBA on mitochondrial morphology and changes in the expression levels of fusion and fission-related genes under oxidative stress stimulation. HK-2 cells were cultured with 100 μM BSO and 250 and 500 μM DHMBA. Mitochondrial morphology was observed by MitoTracker (registered trademark) Green FM fluorescence staining (A), and mitochondrial fusion and fission-related gene expression levels were measured by qPCR (B). Mean ± SD (n = 4).

[0053] (Regarding mitochondrial respiratory capacity) Since mitochondria play a crucial role in energy production, this study evaluated the effects of DHMBA on mitochondrial respiratory capacity. Figure 8A shows the results of measuring the oxygen consumption rate (OCR) over time with the addition of inhibitors to the mitochondrial electron transport chain. Furthermore, the changes in oxygen consumption rate due to various inhibitors can be interpreted as shown in Figure 8B. Figure 9 further shows the changes in various mitochondrial functions. While no impairment of mitochondrial function was observed with 24-hour BSO addition, DHMBA addition increased basal and maximal mitochondrial respiration and ATP production. This suggests that DHMBA can be expected to enhance these mitochondrial functions.

[0054] Figure 8 shows the effect of DHMBA on mitochondrial oxygen consumption rate. HK-2 cells were cultured with 100 μM BSO and 500 μM DHMBA for 24 hours, after which the oxygen consumption rate was measured using an extracellular flux analyzer (Figure 8A). Key indicators for evaluating mitochondrial function can be calculated from the changes in oxygen consumption rate (Figure 8B). Mean ± SD (n = 3-4).

[0055] Furthermore, Figure 9 shows the effect of DHMBA on mitochondrial function. HK-2 cells were cultured in 100 μM BSO and 500 μM DHMBA for 24 hours. Oxygen consumption rates were then measured using an extracellular flux analyzer, and key indicators of mitochondrial function (basal respiration, ATP production, maximal respiration, respiratory reserve, proton leak, and mitochondrial-independent oxygen consumption) were calculated. Mean ± SD (n = 3-4).

[0056] (Conclusion) Under oxidative stress conditions induced by BSO addition, the amount of ROS in cells and mitochondria increased, inducing cell death. However, the addition of DHMBA reduced ROS production, demonstrating a cytoprotective effect. Furthermore, it is thought that the addition of DHMBA suppressed mitochondrial fragmentation, activated the mitochondrial biosynthesis pathway, and protected mitochondria from oxidative stress damage. Furthermore, measurements of mitochondrial respiratory capacity showed that basal mitochondrial respiration, ATP production, and maximal respiration increased under oxidative stress conditions. These results indicate that DHMBA has protective effects on cells and mitochondria, as well as promoting mitochondrial function, in human renal proximal tubular cells HK-2 under oxidative stress stimulation.

[0057] (Regarding the manufacturing method of the present invention) This paper describes a method for producing a renal protective agent having a renal protective effect, which contains the aforementioned 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient.

[0058] As mentioned above, 3,5-dihydroxy-4-methoxybenzyl alcohol can be obtained from Pacific oysters, and the inventors of this case have already obtained numerous patents for methods of efficiently extracting and producing 3,5-dihydroxy-4-methoxybenzyl alcohol from Pacific oysters. Furthermore, 3,5-dihydroxy-4-methoxybenzyl alcohol can also be produced by synthesis, and the inventors of this case have already obtained patents for such synthesis methods.

[0059] Furthermore, in this invention, we surmised that 3,5-dihydroxy-4-methoxybenzyl alcohol has many protective benefits for various human organs, and we conducted experiments to verify this, leading to the invention. Therefore, the useful agent of this invention is manufactured after these verifications have been carried out.

Claims

1. To stimulate human renal proximal tubular cells (HK-2) with oxidative stress, 100 μM of an oxidative stress inducer (BSO) is added, along with 3,5-dihydroxy-4-methoxybenzyl alcohol at a concentration of 3.9 μM or higher. The absorbance of human renal proximal tubular cells (HK-2) after addition was measured to determine cytotoxicity, and the measured value was obtained. Based on the obtained measurement values, we obtained verification results showing that the cytotoxicity of human renal proximal tubular cells (HK-2) can be suppressed by the aforementioned additive. Using 3,5-dihydroxy-4-methoxybenzyl alcohol at a concentration of 3.9 μM or higher, for which verification results were obtained, a cytotoxicity inhibitor for human renal proximal tubular cells (HK-2) was manufactured. A method for producing a cytotoxicity inhibitor for human renal proximal tubular cells (HK-2), characterized by the following:

2. To stimulate human renal proximal tubular cells (HK-2) with oxidative stress, 100 μM of an oxidative stress inducer (BSO) is added, along with 3,5-dihydroxy-4-methoxybenzyl alcohol at a concentration of 3.9 μM or higher. The absorbance of human renal proximal tubular cells (HK-2) after addition was measured to obtain the measurement value. From the obtained measurements, we obtained verification results showing that the addition of the above-mentioned substance increases the cell viability of human renal proximal tubular cells (HK-2). Using 3,5-dihydroxy-4-methoxybenzyl alcohol at a concentration of 3.9 μM or higher, for which verification results were obtained, a cell viability enhancer for human renal proximal tubular cells (HK-2) was manufactured. A method for producing a cell viability increasing agent for human renal proximal tubular cells (HK-2), characterized by the following:

3. Adding 100 μM of an oxidative stress inducer (BSO) to human renal proximal tubular cells (HK-2) to stimulate oxidative stress, and adding 3,5-dihydroxy-4-methoxybenzyl alcohol at a concentration of 250 μM or 500 μM, For human renal proximal tubular cells (HK-2) after addition, mitochondrial ROS fluorescence images and fluorescence intensity measurements were obtained using DHR123 staining. Based on the obtained measurements, we obtained verification results showing that the addition of the above-mentioned substance reduces the increase in ROS accumulated in the mitochondria of human renal proximal tubular cells (HK-2). Using the 3,5-dihydroxy-4-methoxybenzyl alcohol for which verification results were obtained, an inhibitor of the increase in ROS accumulated in the mitochondria of human renal proximal tubular cells (HK-2) was manufactured. A method for producing an agent that inhibits the increase of ROS accumulated in the mitochondria of human renal proximal tubular cells (HK-2), characterized by the above.

4. To stimulate human renal proximal tubular cells (HK-2) with oxidative stress, 100 μM of an oxidative stress inducer (BSO) is added, along with 3,5-dihydroxy-4-methoxybenzyl alcohol at a concentration of 500 μM. After addition, basal and maximal respiration of mitochondria in human renal proximal tubular cells (HK-2) were measured and values ​​were obtained. Based on the obtained measurements, we obtained verification results showing that the aforementioned additive activates mitochondrial respiration in human renal proximal tubular cells (HK-2). Using the 3,5-dihydroxy-4-methoxybenzyl alcohol for which verification results were obtained, a respiratory activator for human renal proximal tubular cells (HK-2) mitochondria was manufactured. A method for producing a respiratory activator for mitochondria of human renal proximal tubular cells (HK-2), characterized by the following:

5. Adding 100 μM of an oxidative stress inducer (BSO) to human renal proximal tubular cells (HK-2) to stimulate oxidative stress, and adding 3,5-dihydroxy-4-methoxybenzyl alcohol at a concentration of 250 μM or 500 μM, After adding the substance, human renal proximal tubular cells (HK-2) were subjected to qPCR to measure the expression levels of mitochondrial biosynthesis-related genes, and measurements indicating activation of the mitochondrial biosynthesis pathway were obtained. Based on the obtained measurement values, we obtained verification results showing that the aforementioned additive increases the number of mitochondria in human renal proximal tubular cells (HK-2). Using the 3,5-dihydroxy-4-methoxybenzyl alcohol for which verification results were obtained, an agent that promotes the increase of mitochondrial number in human renal proximal tubular cells (HK-2) was manufactured. A method for producing an agent that promotes the increase of mitochondrial numbers in human renal proximal tubular cells (HK-2), characterized by the above.

6. Adding 100 μM of an oxidative stress inducer (BSO) to human renal proximal tubular cells (HK-2) to stimulate oxidative stress, and adding 3,5-dihydroxy-4-methoxybenzyl alcohol at a concentration of 250 μM or 500 μM, After adding the substance, human renal proximal tubular cells (HK-2) were subjected to observation of mitochondrial morphology by Green FM fluorescence staining, and measurement of mitochondrial fusion and fission-related gene expression levels by qPCR. Based on the observation and measurement results, observation and verification results were obtained showing that the addition suppresses mitochondrial fragmentation in human renal proximal tubular cells (HK-2). Using the 3,5-dihydroxy-4-methoxybenzyl alcohol for which the observed and verified results were obtained, a human renal proximal tubular cell (HK-2) mitochondrial fragmentation inhibitor was manufactured that has an inhibitory effect on mitochondrial fragmentation of human renal proximal tubular cells (HK-2). A method for producing an inhibitor of mitochondrial fragmentation of human renal proximal tubular cells (HK-2), characterized by the above.

7. To stimulate human renal proximal tubular cells (HK-2) with oxidative stress, 100 μM of an oxidative stress inducer (BSO) is added, along with 3,5-dihydroxy-4-methoxybenzyl alcohol at a concentration of 500 μM. The ATP production values ​​of mitochondria in human renal proximal tubular cells (HK-2) after addition were measured to obtain the values. From the obtained measurements, we obtained verification results showing that the addition of the above-mentioned substance increases ATP production in the mitochondria of human renal proximal tubular cells (HK-2). Using the 3,5-dihydroxy-4-methoxybenzyl alcohol for which verification results were obtained, an agent for increasing ATP production in the mitochondria of human renal proximal tubular cells (HK-2) was manufactured. A method for producing an agent that increases ATP production in the mitochondria of human renal proximal tubular cells (HK-2), characterized by the above.

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