Use of a DNA methyltransferase inhibitor for the preparation of a medicament for the treatment of polycystic kidney disease

Abstract

The application discloses application of DNA methyltransferase inhibitors in preparation of drugs for treating polycystic kidney disease. Preferably, the DNA methyltransferase inhibitor is decitabine or azacitidine, and the polycystic kidney disease is polycystic kidney disease caused or aggravated by mTOR activation. The application uses Tsc2 knockout mouse embryo fibroblasts MEF and Tsc2 kidney-specific knockout mice, finds that after treatment of the DNA methyltransferase inhibitor decitabine, cell proliferation is inhibited, polycystic kidney disease of the mice is relieved, and kidney function is partially recovered. The application provides a new and effective technical means for treatment of polycystic kidney disease.

Description

TECHNICAL FIELD

[0001] The present application relates to a new drug for treating polycystic kidney disease, in particular to the application of DNA methyltransferase inhibitor in treating polycystic kidney disease. The present application belongs to the technical field of medicine. BACKGROUND

[0002] Polycystic kidney disease is an important cause affecting the quality of life and life safety of patients, mainly manifested as bilateral kidney enlargement, kidney pain, hematuria and hypertension, etc. Ultimately, about 50% of patients develop renal failure before the age of 60. Current research suggests that cyst epithelial cell proliferation is an important factor in promoting the occurrence and development of polycystic kidney disease. Abnormalities in cAMP, mTOR, WNT, VEGF, EGFR, PI3K / Akt, Hippo, etc. are important mechanisms for causing abnormal proliferation of cyst epithelial cells.

[0003] mTOR is a highly conserved serine / threonine kinase, which exists in the form of mTORC1 and mTORC2 two complexes in the cell, regulates transcription and protein synthesis, and regulates cell proliferation, survival, apoptosis, autophagy, etc. The activity of mTORC1 is mainly negatively regulated by the TSC1 / 2 complex. Mutations in the TSC1 or TSC2 gene cause dysfunction of the protein complex encoded by the gene, leading to overactivation of mTORC1, which results in a kind of autosomal genetic disease, tuberous sclerosis complex (TSC). About 35-50% of TSC patients show polycystic kidney disease. In addition, polycystic kidney disease is commonly seen in clinical practice with mutations in PKD1 or PKD2 genes, and the pathogenesis is complex, while activation of the mTOR signaling pathway is considered to be one of the important pathogenesis mechanisms.

[0004] For a long time, the treatment of polycystic kidney disease has been mainly symptomatic treatment, which cannot effectively delay the progression of the disease, so the prognosis of most patients is poor. With the continuous deepening of research, specific drugs that inhibit cyst growth have been found, such as mTOR inhibitors, vasopressin II receptor antagonists, somatostatin analogues, etc. Among them, tolvaptan is found to be effective and approved for clinical application, but its serious side effects such as liver damage hinder its clinical application. Although mTOR inhibitors such as everolimus have been approved as the first-line treatment for TSC renal angiomyolipoma, their effectiveness for polycystic kidney disease has not been fully evaluated clinically. Moreover, the side effects of mTOR inhibitors, such as activation of the Akt pathway, also hinder their clinical application.

[0005] Therefore, it is urgent to find an effective drug for polycystic kidney disease, thereby providing new options for the treatment of patients. SUMMARY

[0006] The purpose of the present application is to provide a new effective drug for treating polycystic kidney disease.

[0007] In order to achieve the above-mentioned purpose, the application adopts the following technical means:

[0008] The application finds that, after Tsc2 knockout causes mTOR activation in Tsc2 knockout mouse embryonic fibroblasts (MEF), the expression of miR-142-3p is down-regulated, and the cell proliferation is inhibited after overexpression of miR-142-3p. In Tsc2 kidney-specific knockout mice, the kidney shows obvious polycystic kidney disease, and the expression level of miR-142-3p in the kidney is obviously lower than that of the control mice. MiR-142-3p can inhibit the proliferation of various cells, including tumor cells, vascular smooth muscle cells, immune cells and hematopoietic cells. There are various mechanisms for the regulation of the expression of miR-142-3p in cells, and DNA methylation is the main reason for the down-regulation of miR-142-3p. DNA methyltransferase inhibitors can inhibit DNA methylation by inhibiting DNA methyltransferase and reducing the methylation of CpG islands. In view of this, the application finds that, after the Tsc2 knockout MEF is treated with the DNA methyltransferase inhibitor decitabine, the expression level of miR-142-3p in the Tsc2 knockout MEF is obviously increased, and the cell proliferation is inhibited; the growth of cysts in Tsc2 kidney-specific knockout mice is inhibited, and the kidney function is partially restored.

[0009] Therefore, on the basis of the above-mentioned research, the application proposes the application of the DNA methyltransferase inhibitor in the preparation of a drug for treating polycystic kidney disease.

[0010] Preferably, the DNA methyltransferase inhibitor can inhibit the methylation of miR-142-3p.

[0011] Preferably, the DNA methyltransferase inhibitor is decitabine or azacitidine.

[0012] Preferably, the polycystic kidney disease is polycystic kidney disease caused or aggravated by mTOR activation.

[0013] Preferably, the treatment object is a mammal or a human.

[0014] Preferably, the drug is a tablet, a dispersible tablet, a tablet containing a tablet, an oral disintegrating tablet, a sustained-release tablet, a capsule, a soft capsule, a dripping pill, a granule, an injection, a powder injection or an aerosol.

[0015] Preferably, the DNA methyltransferase inhibitor is used as the only effective component or in combination with other drugs for treating polycystic kidney disease.

[0016] Compared with the prior art, the application has the following beneficial effects:

[0017] The present application uses Tsc2 knockout MEF cells and Tsc2 kidney-specific knockout mice, and finds that after treatment with DNA methyltransferase inhibitor decitabine, cell proliferation is inhibited, polycystic kidney disease in mice is alleviated, and kidney function is partially restored. The present application provides a new and effective technical means for the treatment of polycystic kidney disease. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The phosphorylation level of S6, a downstream molecule reflecting mTOR activation in MEF, and the expression level of miR-142-3p. ** P<0.01.

[0019] Figure 2 The effect of overexpression of miR-142-3p on Tsc2 - / - MEF proliferation. *** P<0.001.

[0020] Figure 3 The changes of kidney and miR-142-3p of Tsc2 kidney-specific knockout mice. ** P<0.01; *** P<0.001.

[0021] Figure 4 The effect of decitabine treatment on Tsc2 - / - MEF on miR-142-3p and cell activity. * P<0.05.

[0022] Figure 5 The changes of kidney morphology and function of Tsc2 kidney-specific knockout mice after decitabine treatment. A, general morphology of mice and kidneys; B, kidney body ratio; C, HE staining of kidney sections; D, PCNA immunohistochemistry; E, percentage of PCNA positive cells in cyst epithelial cells; F, concentration of blood BUN. ** P<0.01; *** P<0.001; **** P<0.0001.

[0023] Figure 6 The expression level of miR-142-3p in the kidney of Tsc2 kidney-specific knockout mice after decitabine treatment. * P<0.05. DETAILED DESCRIPTION

[0024] The application will be further described in connection with the specific embodiments. However, the application is not limited to the following embodiments. Those skilled in the art should understand that the details and forms of the technical solutions of the application can be modified or replaced without departing from the spirit and scope of the application, and such modifications and replacements fall within the protection scope of the application.

[0025] Example 1, effect of Tsc2 knockout on mTOR activity and expression level of miR-142-3p in MEF

[0026] 1. Experimental purpose

[0027] To study the effect of Tsc2 knockout in cells on mTOR activity and expression level of miR-142-3p

[0028] 2. Instruments and equipment

[0029] 2.1 CO2 cell incubator, THERMO FISHER

[0030] 2.2 Real-time quantitative PCR instrument, Roche

[0031] 2.3 Vertical single-sided double-person super-clean workbench, Suzhou Purification Equipment Factory

[0032] 2.4 Inverted biological microscope, CKX41, Olympus

[0033] 2.5 Electrophoresis tank and membrane transfer instrument, Beijing Liuyi

[0034] 2.6 Gel imaging instrument, BioRad

[0035] 3. Experimental materials and preparation

[0036] 3.1 Tsc2 + / + MEF and Tsc2 - / - MEF cells.

[0037] 3.2 DMEM cell culture medium (GIBCO): containing 10% inactivated newborn calf serum (GIBCO).

[0038] 3.3 0.25% trypsin solution, THERMO FISHER.

[0039] 3.4 Phosphate buffer solution (PBS): NaCl 8g, KCl 0.2g, Na2HPO41.15g, KH2PO40.2g, dissolved in 1L double distilled water, 121℃ high pressure sterilization for 20min, 4℃ storage.

[0040] 3.5 Trizol reagent, THERMO FISHER.

[0041] 3.6 Small RNA Reverse Transcription Kit and Small RNA Real-Time Quantitative PCR Kit, Applied biosystems

[0042] 3.7 Antibodies: Anti-TSC2 antibody (1 : 1000, Cell Signaling Technology); β-Actin antibody (1 : 20000, sigma); Anti-p-S6 antibody (5: 1000, Cell Signaling Technology); Anti-S6 antibody (1 : 1000, Cell Signaling Technology)

[0043] 3.8 Cell lysis buffer (full gold); precast gel (GenScript); PVDF membrane (Millipore); ECL luminescence solution (THERMO FISHER).

[0044] 4. Experimental methods

[0045] (1) RNA extraction

[0046] Discard the culture medium in the cell culture dish, first wash it with 1 ml of PBS, then add 1 ml of Trizol and react on the shaker for 30 minutes. Collect all the liquid in an RNase-free 1.5 ml EP tube. Add the corresponding volume of chloroform to the EP tube according to the ratio of 200 μl of chloroform per 1 ml of Trizol, mix well by inverting, and then stand at room temperature for 3 minutes. Centrifuge at 4C°, 12000 rpm for 15 minutes. The liquid separates into three layers. Use a pipette to suck the upper colorless aqueous phase into a new RNase-free 1.5 ml EP tube. Add an equal volume of isopropanol, and react on ice for 10 minutes. Centrifuge at 4C°, 12000 rpm for 15 minutes. RNA precipitate appears at the bottom of the EP tube. Carefully remove the supernatant. Wash with 75% ethanol prepared in advance by adding DEPC water, then centrifuge at 4C°, 12000 rpm for 5 minutes, discard the supernatant, and dry. Add an appropriate amount of RNase-free water to mix according to the size of the precipitate. Use a UV spectrophotometer to determine the concentration of RNA.

[0047] (2) Reverse transcription

[0048]

[0049]

[0050] The mass of the RNA sample in the reaction system is 1-10 ng

[0051] The reaction conditions are 16C° for 30 minutes, 42C° for 30 minutes, and 85C° for 5 minutes.

[0052] (3) Real-time quantitative PCR

[0053] The reaction system is as follows:

[0054]

[0055] The reaction conditions are 50C°, 2 minutes; 95C°, 10 minutes; 95C°, 15 seconds; 60C°, 60 seconds; and the reaction is performed for 40 cycles.

[0056] (4) Immunoblotting

[0057] The supernatant of the cells is discarded, and the cells are washed with PBS, and then 1 ml of PBS is added to collect the cell mass into a 1.5 ml EP tube, which is centrifuged at 5000 rpm for 5 minutes at 4C. The supernatant is discarded, and the cells are washed twice with cold PBS. 100 μl of lysis solution containing phosphatase / protease inhibitors is added to each cell mass, and the cells are lysed for 30 minutes on ice. After centrifugation at 15000 rpm for 10 minutes at 4C, the supernatant is taken.

[0058] SDS-PAGE electrophoresis: The precast gel is fixed in the electrophoresis tank, and the pre-prepared electrophoresis buffer is poured into the electrophoresis tank, and the air bubbles are chased out by tilting or gently shaking. The comb is pulled vertically, and the electrophoresis buffer is added to the tank. According to the protein loading amount of 40 μg, the sample volume is calculated, and the sample is carefully added, and then 1x loading buffer is added to each loading well to the same volume. Electrophoresis is performed at 100 volts for about 100 minutes, and when the bromophenol blue indicator runs to the bottom of the gel, the electrophoresis is terminated.

[0059] Membrane transfer: The filter paper and PVDF membrane are cut according to the size of the gel, the PVDF membrane is activated with methanol for 10 seconds, washed with distilled water for 3 minutes, and soaked in cold transfer solution for 3 minutes. The PVDF membrane, gel, and filter paper are soaked in the transfer buffer, and the PVDF membrane, gel, and filter paper are placed in the order of 6 layers of filter paper, PVDF membrane, gel, and 6 layers of filter paper from top to bottom, and the PVDF membrane is labeled, the edges are aligned, and the air bubbles between the layers are ensured to be free before assembling the transfer clamp. The transfer clamp is placed in the transfer tank filled with transfer solution, and the transfer conditions are set as constant current 300 mA and 2 hours for membrane transfer.

[0060] Blocking: After the membrane transfer is completed, the PVDF membrane is placed in 1x TBST (0.1M, pH 7.4) for 5 minutes, and then placed in a 3% BSA solution diluted in TBST, and incubated at room temperature for 1 hour.

[0061] Antibody incubation: After blocking is completed, the membrane is washed with TBST solution for 3 times x 10 minutes / time. The primary antibody is incubated overnight at 4C. The membrane is washed with TBST solution for 3 times x 10 minutes / time. Goat anti-rabbit / mouse secondary antibody (1:20000) conjugated with horseradish peroxidase (HRP-) is added, and incubated at room temperature for 1 hour. The membrane is washed with TBST solution for 3 times x 10 minutes / time.

[0062] Result collection: mix the ECL kit (Thermo) A and B solution in a ratio of 1:1, then incubate on the membrane surface, after 5 minutes at room temperature, use the protein gel imager to scan the PVDF membrane and obtain the picture.

[0063] 5、Experimental results

[0064] As shown in Figure 1 Tsc2 - / - The level of S6 phosphorylation, a downstream molecule reflecting mTOR activation, was increased in MEF cells, and the expression level of miR-142-3p was significantly lower than that of Tsc2 + / + MEF cells.

[0065] 6、Conclusion

[0066] Knocking out Tsc2 gene in cells can significantly activate mTOR and reduce the expression level of miR-142-3p.

[0067] Example two, the effect of miR-142-3p on the proliferation of Tsc2 knockout MEF

[0068] 1、Experimental purpose

[0069] Study the effect of miR-142-3p on the proliferation of Tsc2 - / - MEF

[0070] 2、Instruments and equipment

[0071] 2.1 CO2 cell incubator, THERMOFISHER

[0072] 2.2 Multifunctional enzyme marker, Synergy NEO, BioTek

[0073] 2.3 Vertical single-sided double-person super-clean workbench, Suzhou Purification Equipment Factory

[0074] 2.4 Inverted biological microscope, CKX41, Olympus

[0075] 3、Experimental materials and preparation

[0076] 3.1 Tsc2 - / - MEF cells

[0077] 3.2 DMEM cell culture medium (GIBCO): containing 10% inactivated newborn calf serum (GIBCO).

[0078] 3.30.25% trypsin solution, THERMOFISHER

[0079] 3.4 Phosphate buffer (PBS): NaCl 8 g, KCl 0.2 g, Na2HPO4 1.15 g, KH2PO4 0.2 g, dissolved in 1 L double distilled water, autoclaved at 121 °C for 20 min, stored at 4 °C.

[0080] 3.5 CCK8 solution, Dojindo

[0081] 3.6 Lipofectamine RNAiMAX, THERMOFISHER

[0082] 4. Experimental method

[0083] Cells in logarithmic growth phase were trypsinized and washed, then suspended in culture medium containing 10% fetal bovine serum, counted by trypan blue staining method, and the cell suspension density was adjusted to 1 x 10 5 Cells / ml. In a 6-well plate, 2 ml of cells were added to each well, and incubated at 37 °C, 5% CO2 in a cell incubator overnight. The control miRNA, miR-142-3p or miR-142-3p inhibitor was diluted to 100 μl OMEM, with a final concentration of 10 nM, and Lipofectamine RNAiMAX 5 μl was diluted to 100 μl OMEM, mixed and incubated for 5 min, then mixed with the miRNA and transfection reagent and incubated for 20 min, then added to the corresponding cell culture dish, and after 4 h of transfection, the culture medium was completely discarded and 2 ml of fresh culture medium was added. After 48 h, the cells were plated in a 96-well plate, 5000 cells per well, with three replicates. Untreated cells were used as a blank control. The 96-well plate was incubated at 37 °C, 5% CO2 in a cell incubator for 48 h. 10 μl of CCK-8 solution was added to each well, and incubation was continued in the incubator for 4 h. The optical density (OD) at 450 nm was measured and plotted.

[0084] 5. Experimental results

[0085] As shown in Figure 2 , overexpression of miR-142-3p reduced the activity of Tsc2 - / - MEF cells, while overexpression of miR-142-3p inhibitor enhanced the activity of Tsc2 - / - MEF cells.

[0086] 6. Conclusion

[0087] miR-142-3p inhibited the proliferation of Tsc2 - / - MEF cells.

[0088] Example Three, Changes in Kidney and miR-142-3p of Tsc2 Kidney-specific Knockout Mice

[0089] 1. Purpose of the experiment

[0090] Investigation of the changes of kidney and miR-142-3p in Tsc2 kidney-specific knockout mice

[0091] 2、Instrument and equipment

[0092] 2.1 Real-time quantitative PCR instrument, Roche

[0093] 2.2 Dissection tools

[0094] 3、Experimental materials and preparation

[0095] 3.1 Tsc2 kidney-specific knockout mice and control mice

[0096] 3.2 Trizol reagent, THERMOFISHER.

[0097] 3.3 Small RNA reverse transcription kit and small RNA real-time quantitative PCR kit, Applied biosystems

[0098] 4、Experimental method

[0099] 4.1 Observation of kidney phenotype:

[0100] Dissect the mice at 35 days after birth, take the kidney, take pictures, weigh, measure the body weight, and calculate the kidney body ratio.

[0101] 4.2 Detection of miR-142-3p expression level:

[0102] (1) RNA extraction

[0103] Prepare a clean mortar on ice, pour an appropriate amount of liquid nitrogen for quick pre-cooling, then quickly take out the low-temperature stored animal tissue block and put it into the mortar, so that the tissue immersed in liquid nitrogen becomes hard, use the grinding instrument to grind the hard tissue block into powder, immediately transfer the powder into a 1.5ml RNase-free EP tube, add 1ml Trizol. Shake for 30 minutes under room temperature, add 200 microliters of chloroform, react for 10 minutes, centrifuge at 4C°, 12000 rpm for 15 minutes, and the liquid is divided into three layers. Use a pipette to suck the upper colorless aqueous phase into a new RNase-free 1.5ml EP tube. Add an equal volume of isopropanol, react for 10 minutes on ice, centrifuge at 4C°, 12000 rpm for 15 minutes, and RNA precipitate appears at the bottom of the EP tube. Carefully remove the supernatant. Add 75% ethanol prepared in advance by DEPC water for washing, then centrifuge at 4C°, 12000 rpm for 5 minutes, discard the supernatant, and dry on paper. According to the size of the precipitate, add an appropriate amount of RNase-free water and mix well. Use a UV spectrophotometer to determine the concentration of RNA.

[0104] Reverse transcription and real-time quantitative PCR were performed as described in Example 1.

[0105] 5、Experimental results

[0106] As shown in Figure 1, the kidneys of Tsc2 kidney-specific knockout mice were significantly larger than those of control mice, the kidney-body ratio was significantly increased, and the expression level of miR-142-3p was significantly lower than that of control mice. Figure 3

[0107] 6、Conclusion

[0108] Knocking out the Tsc2 gene in mouse kidneys can cause polycystic kidney disease and significantly reduce the expression level of miR-142-3p.

[0109] Example 4, Tsc2 treated with decitabine - / - Effect of MEF on miR-142-3p and cell activity

[0110] 1、Experimental purpose

[0111] Study of Tsc2 treated with decitabine - / - Effect of MEF on miR-142-3p and cell activity

[0112] 2、Instruments and equipment

[0113] 2.1 CO2 cell incubator, THERMOFISHER

[0114] 2.2 Real-time quantitative PCR instrument, Roche

[0115] 2.3 Vertical single-sided double-person super-clean workbench, Suzhou Purification Equipment Factory

[0116] 2.4 Inverted biological microscope, CKX41, Olympus

[0117] 2.5 Multifunctional enzyme marker, SynergyNEO, BioTek

[0118] 3、Experimental materials and preparation

[0119] 3.1 Tsc2 + / + (WT) MEF and Tsc2 - / - MEF cells.

[0120] 3.2 DMEM cell culture medium (GIBCO): containing 10% inactivated newborn calf serum (GIBCO).

[0121] 3.3 0.25% trypsin solution (Trypsin): purchased from Invitrogen Company, stored at -20°C.

[0122] ​3.4 Phosphate buffer (PBS): NaCl 8 g, KCl 0.2 g, Na2HPO4 1.15 g, KH2PO4 0.2 g, dissolved in 1 L double distilled water, autoclaved at 121 °C for 20 min, stored at 4 °C.

[0123] 3.5 Trizol reagent, THERMOFISHER.

[0124] 3.6 Small RNA reverse transcription kit and small RNA real-time quantitative PCR kit, Applied biosystems

[0125] 3.7 Decitabine, Selleck

[0126] 4. Experimental method

[0127] 4.1 Detection of the expression level of miR-142-3p after decitabine treatment

[0128] 4x10 5 Tsc2 - / - MEF cells were plated in 6-well plates, 24 hours later, decitabine was added to make the final concentration 1, 2, 5 μM, PBS was used as blank control, after 24 hours of continuous culture, cell groups were collected, RNA was extracted, reverse transcription was performed, and real-time quantitative PCR detection was performed, as in Example One.

[0129] 4.2 CCK8 method for detecting cell activity after decitabine treatment

[0130] Two groups of 5000 cells were plated in 96-well plates, 24 hours later, decitabine was added to make the final concentration 0.5, 2, 5 μM, PBS was used as blank control, after 48 hours of continuous culture, 10 μl of CCK8 solution was added to each well, and incubation was continued for 4 hours, and the OD value at 450 nm was determined by enzyme marker. Taking the cell activity of the control cells as 100%, the cell activity of various treatments was calculated by OD value.

[0131] 5. Experimental results

[0132] As Figure 4 shown, the expression level of miR-142-3p in Tsc2 - / - MEF cells was significantly up-regulated after decitabine treatment, and cell proliferation was significantly inhibited.

[0133] 6. Conclusion

[0134] Decitabine treatment can significantly restore the decrease in the expression level of miR-142-3p caused by the knockout of Tsc2 gene in cells, and inhibit cell proliferation.

[0135] Example Five, Effect of decitabine treatment on mouse polycystic kidney disease and miR-142-3p expression level

[0136] 1. Objectives of the experiment

[0137] To study the effect of decitabine treatment on the expression level of miR-142-3p in mice with polycystic kidney disease

[0138] 2. Instruments and equipment

[0139] 2.1 Real-time quantitative PCR instrument, Roche

[0140] 2.2 Dissection tools

[0141] 2.3 Multifunctional enzyme marker, SynergyNEO, BioTek

[0142] 2.4 Digital slice scanner, 3DHISTECH

[0143] 3. Experimental materials and preparation

[0144] 3.1 Tsc2 kidney-specific knockout mice and control mice

[0145] 3.2 Trizol reagent, THERMOFISHER

[0146] 3.3 Small RNA reverse transcription kit and small RNA real-time quantitative PCR kit, Applied biosystems

[0147] 3.4 Decitabine, Selleck

[0148] 3.5 Urea nitrogen urease method detection kit, Nanjing Jiancheng

[0149] 3.6 Anti-PCNA antibody, goat anti-rabbit secondary antibody, Cell signaling

[0150] 4. Experimental methods

[0151] 4.1 Observation of kidney phenotype:

[0152] From 11 days after birth, Tsc2 kidney-specific knockout mice were injected intraperitoneally with decitabine at a dose of 0.02 mg / kg or 0.1 mg / kg, twice a day, for three times. After three injections, the mice were dissected at 20 days after birth, and the kidneys were taken out, photographed, weighed, and the kidney weight ratio was calculated. At the same time, Tsc2 kidney-specific knockout mice injected intraperitoneally with the same volume of normal saline were set as the control group.

[0153] 4.2 HE staining

[0154] The kidney was cut in half along the long axis and fixed in 4% paraformaldehyde for 48 hours at room temperature. The tissue was dehydrated in the following gradient: 50% ethanol for 1 hour, 70% ethanol for 1 hour, 80% ethanol for 1 hour, 90% ethanol for 40 minutes, 95% ethanol for 40 minutes, and 100% ethanol for 40 minutes twice. The tissue was cleared in xylene for 25 minutes twice and then embedded in paraffin. Sections were cut (5 mm) and placed in a 60 C° oven for 1 hour.

[0155] The sections were deparaffinated in the following gradient: xylene for 15 minutes twice, anhydrous ethanol, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, and water (3-4 minutes each). The sections were stained with hematoxylin for 9-15 minutes, washed with water, de-stained with hydrochloric acid alcohol for 2 seconds, washed with water, counterstained with ammonia for 2 seconds, washed with water, and stained with eosin for 1 minute. The sections were dehydrated in the following gradient: 90% ethanol, 95% ethanol, and anhydrous ethanol twice (3-4 minutes each). The sections were dried with a hair dryer, cleared in xylene, and mounted with resin. The staining was complete.

[0156] The sections were then scanned using a digital slide scanner to obtain high-definition images of the tissue.

[0157] 4.3 Immunohistochemistry

[0158] After the sections were deparaffinated, they were reacted with 3% hydrogen peroxide in the dark for 30 minutes. The sections were then subjected to antigen retrieval in a pressure cooker at 121 C° for 5 minutes using citrate buffer. After the sections were allowed to cool naturally, they were washed with PBS three times for 5 minutes each and then blocked with sheep serum working solution for 1 hour. After the blocking was complete, the sections were incubated with anti-PCNA antibody overnight at 4 C°. The next day, the sections were washed with PBS three times for 5 minutes each. After the sections were incubated with goat anti-rabbit secondary antibody at room temperature for 40 minutes, they were washed with PBS three times for 5 minutes each, incubated with SAB complex for 40 minutes to enhance antigen-antibody binding, washed with PBS three times for 5 minutes each, and then incubated with DAB to develop color. The sections were then washed with tap water, stained with hematoxylin for 10 seconds, and washed with water. The sections were dehydrated in a gradient of alcohol, cleared in xylene, mounted with resin, and stained. The percentage of PCNA-positive cells in the epithelial cells of the cysts in the two groups of mice was counted.

[0159] 4.4 Detection of blood urea nitrogen levels

[0160] The sample to be tested (7 μl) was placed in a centrifuge tube, and the prepared buffer enzyme solution (87.5 μl) was added. Blank and standard tubes were set up, and 7 μl of water and a standard solution were added, respectively. The mixture was mixed and placed in a 37 C° water bath for 10 minutes. Then, 350 μl of phenol color reagent and 350 μl of alkaline sodium hypochlorite were added to each tube, respectively, and the mixture was mixed and placed in a 37 C° water bath for 10 minutes. Then, the liquid in each tube was equally divided into three wells of a 96-well plate, and the absorbance OD value at 640 nm was measured using a microplate reader. Then, the following formula was used to obtain the results.

[0161]

[0162] The detection of 4.5 miR-142-3p expression levels was the same as in Example 3.

[0163] 5. Experimental Results

[0164] like Figure 5 and Figure 6 As shown, decitabine treatment of Tsc2 kidney-specific knockout mice resulted in significantly smaller kidneys, a significantly reduced kidney-to-body ratio, significantly smaller cysts observed at the tissue level, a significant decrease in PCNA-positive cells (a proliferation marker), and a significant decrease in blood urea nitrogen levels, an indicator of renal function. The expression level of miR-142-3p was significantly upregulated.

[0165] 6. Conclusion

[0166] Decitabine treatment can significantly inhibit the occurrence and development of polycystic kidney disease caused by Tsc2 gene knockout, significantly improve renal function, and significantly upregulate miR-142-3p expression level.

Claims

1. Use of a DNA methyltransferase inhibitor for the preparation of a medicament for the treatment of polycystic kidney disease resulting from a genetic deletion, wherein the DNA methyltransferase inhibitor is decitabine. Tsc2 polycystic kidney disease resulting from a genetic deletion, wherein the DNA methyltransferase inhibitor is decitabine.

2. Use according to claim 1, wherein The DNA methyltransferase inhibitor is capable of inhibiting methylation of miR-142-3p.

3. The use according to claim 1, wherein The subject of the treatment is a mammal or a human.

4. The use according to claim 1, wherein The medicine is a dispersible tablet, a tablet, an oral disintegrating tablet, a sustained-release tablet, a capsule, a dripping pill, a granule, an injection, a powder injection or an aerosol.

5. The use according to claim 1, wherein The DNA methyltransferase inhibitor as the sole active ingredient or in combination with other agents useful in the treatment of Tsc2 Additional agents for polycystic kidney disease resulting from genetic deletion.

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