Application of epirubicin in medicine for treating obstructive nephropathy or other chronic nephropathy

Abstract

The invention discloses an application of epirubicin in preparation of a medicine for treating obstructive nephropathy or other chronic nephropathy. The invention also discloses an application of epirubicin in preparation of a small-molecule inhibitor of B56 delta protein. The epirubicin disclosed by the invention has an obvious treatment effect on obstructive nephropathy renal tubule injury, and the epirubicin disclosed by the invention can be used for obviously relieving the renal tubule injury caused by lipid accumulation in tubule epithelial cells after single-side ureter ligation of a mouse. The epirubicin disclosed by the invention can obviously relieve lipid accumulation caused by activation of a fatty acid de novo synthesis pathway in a tubule epithelial cell after single-side ureter ligation of a mouse. In-vivo and in-vitro studies show that epirubicin can inhibit B56delta and promote the increase of the phosphorylation level of acetyl-CoA carboxylase 1 (ACC1), so that the de novo synthesis path of fatty acid is blocked, the metabolic reconstitution of renal tubular epithelial cells is resisted, and the renal tubular injury progress is relieved.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to the application of epirubicin in medicine for treating obstructive nephropathy or other chronic kidney diseases. Background Art

[0002] Obstructive nephropathy is a type of disease that causes damage to kidney function and structure due to urinary tract obstruction. Obstructive nephropathy remains an important cause of renal insufficiency and will eventually progress to end-stage renal failure (ESRD), requiring maintenance hemodialysis or kidney transplantation to maintain life, which not only seriously affects personal health and quality of life, but also poses a major challenge to the social economy.

[0003] Renal tubular injury is one of the main pathological manifestations of obstructive nephropathy. The degree of renal tubular injury closely affects the rate of renal function decline and long-term prognosis of obstructive nephropathy. Previous studies on the mechanism of obstructive nephropathy have shown that renal tubular cells undertake important physiological functions such as concentrating urine, maintaining acid-base, and water-electrolyte balance, all of which rely on a large amount of ATP. This energy is mainly provided by fatty acid oxidation (FAO) mediated by densely distributed mitochondria and peroxisomes in renal tubular epithelial cells to support their powerful reabsorption function. When urinary tract obstruction occurs, a large amount of lipid accumulation and fatty acid oxidation disorders occur in renal tubular epithelial cells. The energy generated cannot meet their own metabolic needs, and renal tubular epithelial cells undergo phenotypic changes, leading to renal tubular injury and decreased renal metabolic capacity, thereby causing a continuous decline in renal function.

[0004] Therefore, we need to actively study the metabolic remodeling of renal tubular epithelial cells and the regulatory mechanism of lipid metabolism under physiological and pathological conditions, find corresponding therapeutic therapies, promote FAO, increase intracellular ATP energy supply, and reduce tubular damage, which is of great significance for controlling the progression of obstructive nephropathy, improving renal function, and delaying chronic renal failure. However, the current treatment of obstructive nephropathy mainly focuses on relieving urinary tract obstruction (such as surgery, catheter drainage, etc.) and symptomatic treatment (such as the use of anti-inflammatory drugs and antioxidant drugs). However, these methods cannot reverse the kidney damage that has already occurred, especially the tubular damage and loss of renal function in the chronic stage. There is a lack of research on restoring the activity of fatty acid oxidation in the kidney. Therefore, it is urgent to develop better drug targets to treat renal tubular damage in patients with obstructive nephropathy and improve the prognosis and quality of life of patients.

[0005] PP2A is one of the serine / threonine protein phosphatases widely expressed in eukaryotic cells, accounting for about 0.3%-1% of the total cellular protein. The PP2A holoenzyme usually exists in the form of a trimer, consisting of a scaffolding A subunit and a catalytic C subunit as the core enzyme, and finally the regulatory B subunit and the core enzyme together form the PP2A holoenzyme. Studies have shown that PP2A participates in the metabolic remodeling of tubular epithelial cells in obstructive nephropathy, by dephosphorylating ACC1 (acetyl-CoA carboxylase 1), increasing lipid synthesis in tubular cells, inhibiting fatty acid oxidation, reducing the production of intracellular ATP, affecting the survival of tubular cells, and gradually declining renal function, accelerating the progression of ESRD.

[0006] One of the most important characteristics of regulatory subunit B is its diversity, including four families: B, B′, B″ and B″′. The core enzyme may produce more than 75 PP2A holoenzymes by combining with different subtypes of B subunits. It is precisely because of the rich variety of B subunits that, after binding to PP2Acα, PP2A has the substrate specificity and intracellular targeting of the holoenzyme, can accurately regulate various cellular functions, is widely distributed throughout the body and has diverse effects. Therefore, if the activity of the catalytic subunit of PP2A is inhibited to combat the progression of obstructive nephropathy, it may not only fail to achieve the expected therapeutic effect, but also produce serious toxic side effects. The regulatory subunit B has the substrate specificity of the holoenzyme, so if the activity of the holoenzyme is inhibited by specifically targeting the relevant regulatory subunits, the phosphorylation state of the specific substrate can be accurately regulated. Therefore, it is of great clinical significance to identify and study the molecular mechanism by which regulatory subunit B mediates PP2A targeting ACC1 to regulate lipid metabolism in renal tubular epithelial cells and its role in the progression of renal tubular damage, but there are very few studies in this area. The results showed that PP2A inhibits fatty acid synthesis by targeting dephosphorylation of Ser79 of ACC1 through the regulatory subunit B56δ. Since there are currently no inhibitors against B56δ, it is very important to screen its small molecule inhibitors and evaluate their role in the treatment of obstructive nephropathy.

[0007] Epirubicin (EPI) is an antibiotic anti-tumor and is an isomer of doxorubicin. It has anti-tumor effects by inhibiting topoisomerase. Epirubicin is a forkhead box protein p3 (Foxp3) inhibitor that can inhibit the activity of regulatory T cells and has a good therapeutic effect on a variety of transplanted tumors.

[0008] There are currently no reports on the use of epirubicin to improve renal tubular damage and treat obstructive nephropathy. Summary of the invention

[0009] Purpose of the invention: The technical problem to be solved by the present invention is to provide a new indication for epirubicin, namely, the use of epirubicin in a drug for treating obstructive nephropathy and renal tubular damage-related diseases.

[0010] The technical problem that the present invention needs to solve is to use epirubicin in the preparation of small molecule inhibitors of B56δ protein.

[0011] Technical solution: To achieve the above technical objectives, the present invention provides the use of epirubicin in the preparation of drugs for treating obstructive nephropathy or other chronic kidney diseases.

[0012] Wherein, the working concentration of epirubicin is 0.5-50 μM. Preferably, the working concentration of epirubicin is 5 μM.

[0013] Wherein, the obstructive nephropathy includes obstructive nephropathy renal tubular damage.

[0014] Wherein, the obstructive nephropathy renal tubular damage includes obstructive nephropathy caused by unilateral ureteral ligation.

[0015] The application includes reducing lipid accumulation in tubular epithelial cells caused by mild unilateral ureteral ligation.

[0016] Among them, the application includes reducing lipid accumulation caused by activation of the de novo fatty acid synthesis pathway in tubular epithelial cells after unilateral ureteral ligation.

[0017] Among them, the application includes inhibiting B56δ, reducing PP2A holoenzyme activity, and promoting increased ACC1 phosphorylation levels.

[0018] Wherein, the medicine includes a single or compound preparation.

[0019] Wherein, the dosage form of the drug includes injection, soft capsule, ointment, suppository or aerosol.

[0020] Wherein, the chronic kidney disease also includes IgA nephropathy, diabetic nephropathy, membranous nephropathy or chronic interstitial nephritis.

[0021] The present invention also includes the use of epirubicin in preparing small molecule inhibitors of B56δ protein.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following advantages: Epirubicin of the present invention has a significant therapeutic effect on renal tubular damage in obstructive nephropathy, and the Epirubicin of the present invention can significantly reduce renal tubular damage caused by lipid accumulation in the tubules after unilateral ureteral ligation in mice. Epirubicin of the present invention can significantly reduce lipid accumulation caused by activation of the de novo synthesis pathway of fatty acids in tubular epithelial cells after unilateral ureteral ligation in mice. The in vivo and in vitro studies of the present invention show that epirubicin can inhibit B56δ, promote the increase of ACC1 phosphorylation level, block lipid synthesis, counteract metabolic remodeling of tubular epithelial cells, and alleviate the progression of tubular damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The lipid accumulation in the kidneys of the control group mice (Vehicle) and the Epirubicin injection group mice before and after UUO surgery (CTL) and 10 days after UUO surgery. Figure 1 A is the oil red stained kidney tissue sections of mice in each group; Figure 1 B is the detection of TG content in kidney tissue of mice in each group; Figure 1 C shows glycogen staining (upper row) and Masson staining (lower row) of kidney tissue sections of mice in each group; Figure 1 D is the semi-quantitative statistics of the fibrosis area in Figure C.

[0024] Figure 2 This is the lipid accumulation in NRK-52E cells after treatment with Epirubicin. Figure 2 A is the detection of lipid droplets in cells using Bodipy dye (comparison between TGF-β-treated cells and TGF-β-treated + EPI-treated cells); Figure 2 B is for Figure 2 Semi-quantitative statistics of A; Figure 2 C is the quantitative detection of TG content in cells (untreated NRK-52E cells and NRK-52E cells treated with TGF-β) without EPI and after adding EPI treatment; Figure 2 D is Western blot detection of the protein level and phosphorylation level of ACC1 in cells (untreated NRK-52E cells and NRK-52E cells treated with TGF-β) after adding different concentrations of EPI treatment, Tubulin is the internal reference protein; Figure 2 E is the MTT assay for the toxic effect of Epirubicin on tubular epithelial cells; Figure 2 F: Western blot detection of the inhibitory effect of Epirubicin on B56δ.

[0025] Figure 3 To detect the expression of B56δ in renal tubular epithelial cells of chronic kidney disease. Figure 3 A is the collection of renal tissue biopsy specimens from clinical patients with IgA nephropathy, diabetic nephropathy (DN), chronic interstitial nephritis (CIN), membranous nephropathy (MN), etc. for immunohistochemical staining. Figure 3 B is immunohistochemical staining of mouse kidneys 10 days after UUO surgery. Figure 3 C: Western blot detection of B56δ protein expression in the kidneys of mice at different time points after UUO surgery. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0027] The method used in the embodiment of the present invention is briefly described as follows:

[0028] 1. Construction of unilateral ureteral obstruction (UUO) nephropathy model

[0029] CD-1 male mice aged 2 months and weighing 20-22 g were selected and anesthetized with 45 mg / kg pentobarbital intraperitoneally. The abdominal cavity was opened under sterile conditions, and the left ureter was bluntly separated. The ureter was permanently ligated with 3-0 surgical thread at the upper 1 / 3 and lower 1 / 3 of the ureter, and then the abdominal cavity was closed layer by layer. The mice were killed on the 10th day after surgery, and the renal tissue specimens on the surgical side and the contralateral side were collected.

[0030] 2. Collection of renal tissue specimens

[0031] After the mice were killed, the abdominal cavity was opened along the midline of the abdomen, the kidneys were collected and divided into three parts perpendicular to the long axis: one part was placed in 4% neutral formaldehyde, fixed at 4°C for 48 h and then embedded in paraffin for immunohistochemical staining with pathological stains; one part was embedded in OCT embedding medium and stored at -80°C for preparation of frozen sections and oil red staining; one part was used for immunoblotting detection and stored at -80°C.

[0032] 3. Oil red staining of kidney tissue

[0033] Routine OCT embedding, frozen section, 10μm; fix the sections in 4% paraformaldehyde for 30min, wash with water; soak in 60% isopropanol for 30min; stain in 60% oil red staining solution, stain in a 37℃ water bath overnight; differentiate in 60% isopropanol for a few seconds, wash with water; stain the cell nucleus with hematoxylin for 1-2min, wash with water; turn blue with ammonia for a few seconds; rinse with tap water, observe the cell nucleus dyed blue under a microscope; differentiate with 1% hydrochloric acid ethanol for a few seconds, wash with water; seal with glycerol and take pictures.

[0034] 4. Western blot

[0035] Tissue and cell sample processing: Use RIPA lysis buffer to grind tissue (about 100 mg) on ​​ice using a glass homogenizer or use a cell scraper to collect cells, then aspirate the homogenate into the corresponding EP tube, centrifuge at 16000 rpm × 30min, take the supernatant, take the protein homogenate supernatant and dilute it at a ratio of 1:50, and use the BCA protein detection kit to detect the protein concentration. The standard protein concentration is 1 mg / mL. Then use 4×SDS sample buffer (125mM Tris-HCl, 4% SDS, 20% glycerol, 100mM dithiothreitol, 0.2% bromophenol blue) and deionized water to adjust to the same concentration. Depending on the target protein, the tissue loading amount is 50-100μg / well, and the cell specimen is 10-20μg / well. RAPA lysis buffer formula: 1×PBS (pH 7.4), 1% NP-40, 0.1% sodium deoxycholate, 1μM sodium orthovanadate, 100nM PMSF, Protease Inhibitor Cocktail (1:100 dilution), Phosphatase Inhibitor Cocktail 2 (1:100 dilution), Phosphatase Inhibitor Cocktail 3 (1:100 dilution).

[0036] Western blotting procedure:

[0037] 1) Electrophoresis: Add the sample to the SDS-polyacrylamide gel loading well and use 80V voltage to separate protein molecules of different sizes;

[0038] 2) Transfer: Transfer the protein to a nitrocellulose membrane at 100 V in a transfer buffer (containing 48 mM Tris-HCl, 39 mM glycine, 0.037% SDS, 20% methanol) at 4°C. The transfer time is 60-75 min depending on the molecular weight of the target protein.

[0039] 3) Blocking: Incubate with TBST containing 2% BSA for 1 h at room temperature to block nonspecific areas on the membrane;

[0040] 4) Primary antibody incubation: dilute the primary antibody with TBST containing 2% BSA and then incubate at 4°C overnight;

[0041] 5) Wash the membrane: Wash the membrane three times with TBST at room temperature, 15 min each time;

[0042] 6) Secondary antibody incubation: dilute the secondary antibody with TBST containing 5% skim milk and incubate at room temperature for 1 h;

[0043] 7) Wash the membrane: Wash the membrane three times with TBST at room temperature, 15 min each time;

[0044] 8) Color development and scanning analysis: Use ECL solution to develop the protein signal, use X-ray light to show the target in a dark box, scan the image with a scanner, and finally use Image J software to analyze the relative content value of the protein signal.

[0045] 5. MTT colorimetric assay

[0046] In a 96-well plate, add 5 mg / L MTT (APExBIO) solution to the cell culture medium at 10 μL / well and incubate in a 37°C incubator for 2 hours in the dark. Remove the supernatant, add DMSO at 100 μL / well and place in a 37°C incubator for 5 minutes. Measure the OD value with an ELISA reader at a wavelength of 570 nm.

[0047] 6. BODIPY staining method

[0048] BODIPY493 / 503 2μg / μL, staining for 30min; discard the culture medium, fix with 4% paraformaldehyde for 30min, rinse with PBS 3 times, 5min each time; stain with DAPI for 1min, rinse quickly with PBS; seal the slides and observe under a fluorescence microscope.

[0049] 7. Triglyceride detection

[0050] Triglyceride assay kit was used to determine triglyceride content according to the instructions. The specific operation is as follows: ① Tissue sample: Accurately weigh the tissue weight, add 9 times the volume of homogenization medium at a ratio of weight (g): volume (mL) = 1:9, mechanically homogenize under ice-water bath conditions, centrifuge at 2500 rpm for 10 minutes, and take the supernatant for testing. ② Cell sample: Take out the prepared cell suspension, centrifuge at 1000 rpm for 10 minutes, discard the supernatant, and keep the cell pellet; wash with PBS 1 to 2 times, centrifuge at 1000 rpm for 10 minutes, discard the supernatant, and keep the cell pellet; add 0.2 to 0.3 mL of PBS for homogenization, ultrasonically disrupt or manually homogenize under ice-water bath conditions, and directly measure the prepared homogenate without centrifugation. The sample to be tested, the standard (2.68mmol / L) and pure water were added to a 96-well plate at 2.5μL per well, and then 250μL of working solution was added to each well, the plate was shaken to mix, incubated at 37°C for 10 minutes, and the wavelength was 500nm. The absorbance value of each well was measured by an enzyme reader. The triglyceride content was calculated according to the formula: (sample OD-blank OD) / (standard OD-blank OD)×standard concentration (2.68mmol / L) / test tissue sample homogenate protein concentration.

[0051] 8. Immunohistochemical staining of mouse kidney tissue sections

[0052] (1) Dewaxing: xylene No. I tank 20 min - xylene No. II tank 20 min - anhydrous ethanol No. I tank 5 min - anhydrous ethanol No. II tank 5 min - 95% ethanol 5 min - 80% ethanol 5 min - 60% ethanol 5 min - deionized water immersion 3 times, 4 minutes each time. Here, No. I and No. II tanks refer to different containers, but the contents are the same.

[0053] (2) Prepare 3% hydrogen peroxide: Immerse the slices in a solution of 3% hydrogen peroxide, cover with a lid, and soak in a sealed container at room temperature for 10 minutes to eliminate the activity of endogenous peroxidase. Then take out the slices and rinse them with deionized water three times, 3 minutes each time.

[0054] (3) Immerse the slices in a container containing alkaline antigen retrieval solution, cover the lid, boil in a microwave oven on high heat, then turn to low heat and continue boiling the retrieval solution for 25 minutes. Cool naturally in a fume hood. After cooling, wash with PBS buffer for 3 minutes each time, for a total of 3 washes.

[0055] (4) Blocking: Block with TBST + 2% BSA at room temperature for 1 h to prevent nonspecific binding of antibodies.

[0056] (5) Prepare anti-B56δ primary antibody (abcam, ab188323), remove excess liquid on the renal tissue sections, wipe the area around the sections with dry gauze, add primary antibody, incubate at room temperature for 3 h, and wash with PBS buffer after incubation, 3 min / time, for a total of 3 washes.

[0057] (6) Prepare secondary antibody (Quanhui International, provided in RQ7025 kit), remove excess liquid on the renal tissue sections, wipe the area around the sections with dry gauze, add biotin-labeled secondary antibody, incubate at room temperature for 1 hour, and wash with PBS buffer after incubation, 3 minutes each time, for a total of 3 washes.

[0058] (7) Prepare DAB colorimetric solution (provided in RQ7025 kit, Quanhui International), remove excess liquid from the renal tissue sections, wipe the area around the sections with dry gauze, add colorimetric solution, observe under a microscope, and then put into water to terminate the reaction.

[0059] (8) Imaging: The sections were observed and images were acquired using a Nikon Eclipse E600 fluorescence microscope, and the images were finally exported.

[0060] 9. PAS (Periodic Acid-Schiff) staining of mouse kidney tissue sections

[0061] (1) Dewaxing: xylene No. I tank 20 min - xylene No. II tank 20 min - anhydrous ethanol No. I tank 5 min - anhydrous ethanol No. II tank 5 min - 95% ethanol 5 min - 80% ethanol 5 min - 60% ethanol 5 min - deionized water immersion 3 times, 4 minutes each time. Here, No. I and No. II tanks refer to different containers, but the contents are the same.

[0062] (2) The slices were oxidized in 1% periodic acid for 10-15 min and then washed with water.

[0063] (3) Slices are stained with Schiff's solution for 10-30 minutes and then washed with water. The time of this process must be strictly controlled. If the time is too long, the basement membrane of the tissue specimen will be stained too darkly. If the time is too short, the basement membrane of the tissue specimen will be stained too lightly.

[0064] (4) Stain the cell nucleus with hematoxylin for 1-2 min and wash with water.

[0065] (5) Slice and place in 1% hydrochloric acid ethanol for a few seconds, then wash with water. This process should be short, as too long a time will easily cause the tissue specimen to fade.

[0066] (6) The slices will turn blue in ammonia water for a few seconds.

[0067] (7) Rinse the sections with running water and observe under a microscope. The cell nuclei are stained blue and the basement membrane is stained pink.

[0068] (8) Dehydration with gradient ethanol, clearing with xylene, and sealing with resin.

[0069] PAS-positive substances (polysaccharides and glycogen) appear red, and nuclei appear blue.

[0070] 10. Masson staining of mouse kidney tissue sections

[0071] The staining kit (catalog no: HT15–1KT, Sigma-Aldrich) was used for staining. The specific method was according to the instructions of the kit. The collagen aggregated in the interstitium was stained with aniline blue. Ten non-overlapping fields of view in the renal cortex area were randomly selected for each renal tissue specimen under a microscope at 400×. The percentage of interstitial fibrosis area in the selected renal tissue area was analyzed using Image Proplus 6.0, and the average percentage of renal fibrosis area of ​​each specimen was calculated.

[0072] Example 1 Epirubicin reduces renal tubular lipid deposition and renal tubular damage caused by unilateral ureteral ligation (UUO)

[0073] CD-1 male mice (Zhejiang Weitong Lihua) aged 2 months and weighing 20-22g were selected and divided into a control group and an Epirubicin (MCE, HY-13624) treatment group. The Epirubicin treatment group received a single intraperitoneal injection of Epirubicin (1.5 mg / kg) dissolved in DMSO and saline daily, while the control group mice were injected with an equal volume of 0.9% sodium chloride solution daily. After 3 consecutive days of injection, unilateral ureteral ligation (UUO) was performed as an obstructive nephropathy injury model. After the operation, a single intraperitoneal injection of 0.9% sodium chloride solution or Epirubicin (1.5 mg / kg) was continued every other day. On the 10th day after surgery, both groups of mice were killed, and the contralateral and UUO renal tissues were collected. The contralateral kidneys and UUO renal tissues of the control group and Epirubicin group were stained with oil red, and the results are as follows: Figure 1 As shown in A; the triglyceride (TG) content in the contralateral kidney and UUO kidney tissues of the two groups of mice was detected respectively. The results are shown in Figure 1 As shown in B; Glycogen (PAS) staining and Masson staining were performed on the contralateral kidney and UUO kidney tissues of the control group and Epirubicin group. Figure 1 As shown in C.

[0074] Result analysis: Figure 1 A and 1B show that the lipid content in the renal tissue of the UUO side of the mice in the Epirubicin treatment group was significantly reduced compared with that in the control group. Figure 1 C shows that the tubular structure of the UUO side of the kidney tissue in the Epirubicin-treated mice was more complete than that in the control group. Figure 1 C and Figure 1 D shows that the level of interstitial fibrosis in the renal tissue of the UUO side of the mice treated with Epirubicin was lower. That is, Epirubicin can reduce lipid accumulation in renal tubular epithelial cells in the kidney tissue and alleviate tubular damage and the progression of interstitial fibrosis.

[0075] Example 2 Epirubicin can inhibit TGF-β1-induced NRK-52E tubular epithelial cell damage and lipid accumulation

[0076] Rat kidney epithelial cell line (NRK-52E, purchased from ATCC, USA) was used and divided into a control group (TGF-β) and an Epirubicin treatment group (TGF-β+Epirubicin). Epirubicin (final concentration of 5 μM) and an equal volume of DMSO were added for 12 hours, followed by BODIPY staining, and the number of lipid droplets in each cell of the two groups was counted. The results are shown in Figure 2. Figure 2As shown in A and 2B. In NRK-52E cells, the cells were divided into a control group (not treated with TGF-β) and a TGF-β treated group. Transforming growth factor-β (TGF-β, RD Tech., catalog number 240-B-010) with a final concentration of 2 ng / mL and an equal volume of DMSO were added to the cells. Each group of cells was further divided into two groups, and Epirubicin (final concentration of 5 μM) and an equal volume of DMSO were added for 12 hours. The triglyceride (TG) content was detected, and the results are shown in Figure 2 C. In NRK-52E cells, they were divided into a control group (not treated with TGF-β) and a TGF-β1-treated group. TGF-β with a final concentration of 2 ng / mL and an equal volume of DMSO were added to the two groups of cells for 48 hours. In the last 12 hours, Epirubicin was added to the two groups of cells at different working final concentrations (0, 0.5, 1, 5, 10 μM). The expression of p-ACC1 and ACC1 in each group of cells was detected by Western blot. The results are shown in Figure 2 As shown in D. In NRK-52E cells, TGF-β with a final concentration of 2 ng / mL was pre-treated for 48 hours as the experimental group, and the untreated NRK-52E cells were used as the control group (veh). Epirubicin with a final concentration of 1 μM and 5 μM and an equal volume of DMSO (veh) were added in the last 12 hours, and then MTT was used to detect cell activity. The results are shown in Figure 2 E. NRK-52E cells were treated with TGF-β at a final concentration of 2 ng / mL for 48 hours. Epirubicin was added to the cells at different final concentrations (0, 0.5, 1, 5, 10, 50 μM) for the last 12 hours. The expression of B56δ in the cells was then detected by Western blot. The results are shown in Figure 2 As shown in F.

[0077] Result analysis: Figure 2 The results of A, 2B, and 2C showed that 5 μM Epirubicin significantly reduced the lipid production of tubular cells induced by TGF-β, and the triglyceride content was significantly decreased. Figure 2 D The results showed that under TGF-β treatment conditions, 5 μM Epirubicin could increase the expression level of p-ACC1 in tubular cells, and the increasing effect became more obvious with the increase of Epirubicin dose, showing a dose-dependent manner. Figure 2 The results in E showed that the cell activity was increased after treatment with 1μM and 5μM Epirubicin, and no toxic side effects were produced on tubular cells. Figure 2F The results showed that Epirubicin could significantly inhibit the high expression of B56δ caused by TGF-β, and the inhibitory effect became more significant with the increase of Epirubicin dose. That is, in the NRK-52E cell line, Epirubicin could reduce lipid deposition by targeting B56δ and thus slow down the progression of renal tubular injury.

[0078] Example 3: Increased expression of B56δ in various chronic kidney diseases

[0079] In this example, clinical samples were tested, and human kidney tissue puncture specimens with renal tubular damage such as IgA nephropathy, diabetic nephropathy, membranous nephropathy and chronic interstitial nephritis were collected for B56δ immunohistochemical staining to observe expression and distribution. The results are as follows: Figure 3 A and 3B. 2-month-old CD-1 male mice weighing 20-22 g were selected for UUO surgery. The mice were killed on days 0, 3, 7, and 10 after surgery, and the contralateral kidney and UUO kidney tissues were collected. Western blot was used to detect the expression of B56δ in the UUO kidney tissue on days 0, 3, 7, and 10 after UUO surgery. The results are shown in Figure 3 As shown in C.

[0080] Result analysis: Figure 3 A The results showed that B56δ increased in a variety of clinical diseases accompanied by renal tubular damage. Figure 3 B. Figure 3 C The results show that B56δ increased significantly in the kidneys of mice after UUO surgery. That is, B56δ expression increased in mouse obstructive nephropathy and various chronic kidney diseases in humans. Therefore, the conclusion drawn in Example 1 is that the administration of EPI to inhibit B56δ can reduce lipid accumulation in the kidneys of mice with UUO. Similarly, it is speculated that the administration of Epirubicin may inhibit the expression of B56δ in the kidneys of chronic kidney diseases such as human IgA nephropathy, diabetic nephropathy, membranous nephropathy and chronic interstitial nephritis, thereby reducing lipid accumulation and extracellular matrix deposition in the kidneys, thereby slowing down the progression of the disease.

Claims

1. The use of epirubicin in the preparation of drugs for treating obstructive nephropathy or other chronic kidney diseases.

2. The use according to claim 1, characterized in that: The obstructive nephropathy includes obstructive nephropathy renal tubular damage.

3. The use according to claim 2, characterized in that: The obstructive nephropathy renal tubular damage includes obstructive nephropathy caused by unilateral ureteral ligation.

4. The use according to claim 1, characterized in that: The use includes reducing intratubular lipid accumulation caused by mild unilateral ureteral ligation.

5. The use according to claim 1, characterized in that: The application includes reducing lipid accumulation caused by activation of the de novo fatty acid synthesis pathway in tubular cells after unilateral ureteral ligation.

6. The use according to claim 1, characterized in that: The application includes inhibiting B56δ and promoting the increase of ACC1 phosphorylation level.

7. The use according to claim 1, characterized in that: The medicine includes a single or compound preparation.

8. The use according to claim 1, characterized in that: The dosage form of the drug includes injection, soft capsule, suppository or aerosol.

9. The use according to claim 1, characterized in that: The other chronic kidney diseases also include IgA nephropathy, diabetic nephropathy, membranous nephropathy or chronic interstitial nephritis.

10. Application of epirubicin in the preparation of small molecule inhibitors of B56δ protein.

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