Application of HS148 in preparation of medicine for treating acute kidney injury

Abstract

The invention discloses application of HS148 in preparation of a medicine for treating acute kidney injury. The application discovers that HS148 improves renal tubular epithelial cell apoptosis by inhibiting the protein level of cell death protein kinase 3 (Dapk3), further relieves acute kidney injury induced by cis-platinum, and may become an effective clinical medicine for preventing and treating AKI.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and more specifically to the use of HS148 in the preparation of medicaments for the treatment of acute kidney injury. Background Technology

[0002] Acute kidney injury (AKI) is a clinical syndrome characterized by a rapid decline in renal function, with high incidence and mortality rates that are increasing annually. A cross-sectional study in 2013 showed that the detection rate of AKI in hospitalized patients in my country was as high as 2%, the in-hospital mortality rate was as high as 12.4%, and 19% to 31% of surviving patients eventually progressed to chronic kidney disease or end-stage renal disease. Due to the complex etiology and unclear pathogenesis of AKI, there are currently no satisfactory treatments, and severely ill patients often require renal replacement therapy, placing a significant economic burden on families and society. Renal tubular epithelial cell (RTEC) damage is the main pathological basis of AKI. In the disease state, RTEC, based on its metabolic characteristics and unique anatomical structure, is more susceptible to damage from ischemia / reperfusion (I / R), nephrotoxins, etc., than other types of kidney cells, leading to acute tubular necrosis and renal dysfunction. Therefore, exploring the molecular mechanisms of renal tubular epithelial cell injury and finding effective intervention targets are current research hotspots.

[0003] HS148 is a selective cell death protein kinase 3 (Dapk3) inhibitor, also known as 2-((1-(3-fluorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-D]pyrimidin-6-yl)thio)butyramide, with CAS number 1892595-16-2. Its molecular formula is as follows: .

[0004] Currently, the application of HS148 is focused on the cardiovascular field, especially exploring the role of DAPK3 in hypertension and vascular function regulation. No association has been found between HS148 and acute kidney injury. Summary of the Invention

[0005] Purpose of the invention: This invention provides a new pharmaceutical use for HS148, specifically disclosing the use of HS148 in the preparation of a medicament for treating acute kidney injury.

[0006] To address the aforementioned technical problems, this invention discloses the use of HS148 in the preparation of a medicament for treating acute kidney injury.

[0007] Specifically, the acute kidney injury is cisplatin-induced acute kidney injury.

[0008] HS148 inhibits the levels of serum creatinine (SCr), blood urea nitrogen (BUN), and kIM-1.

[0009] At the same time, HS148 alleviates the degree of renal tubular damage and inhibits renal tubular epithelial cell apoptosis.

[0010] HS148 is the main active ingredient of the drug, which also includes a pharmaceutically acceptable carrier.

[0011] Furthermore, the drug dosage form is any one of tablets, capsules, or liquid preparations.

[0012] In some embodiments, the drug is administered orally or intravenously.

[0013] Beneficial Effects: This invention discloses a novel use of HS148 in the preparation of drugs for treating acute kidney injury. HS148 not only improves renal function but also reduces renal pathological damage. Furthermore, HS148 can improve cisplatin-induced acute kidney injury and significantly inhibit the upregulation of the inflammatory marker IL-1β in cisplatin-induced acute kidney injury. This application preliminarily verifies that HS148 improves acute kidney injury by inhibiting Dapk3 protein levels and apoptosis. Attached Figure Description

[0014] Figure 1 HS148 showed that it significantly improved renal dysfunction and renal tubular injury in cisplatin-induced acute kidney injury; Figure 2 HS148 showed that it significantly inhibited apoptosis in cisplatin-induced acute kidney injury; Figure 3 The expression changes of HS148 target Dapk3 in cisplatin-induced acute kidney injury were shown. Figure 4 The results showed that in an in vitro model of renal tubular epithelial cells, Dapk3 deficiency significantly reduced cisplatin-induced renal tubular epithelial cell apoptosis. Detailed Implementation

[0015] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0016] The C57BL / 6 mice used in the following examples were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and were housed in an SPF-grade barrier environment at the Experimental Animal Center of Nanjing Medical University. They had free access to water, were fed standard rodent food, and were kept at a constant temperature and humidity with a 12 / 12h circadian rhythm.

[0017] The experimental methods used in the following examples are as follows: (a) Materials and reagents: HS148 was purchased from MCE (USA); Dapk3 antibody was purchased from Abcam; KIM-1 antibody was purchased from R&D Systems; Cleaved caspase3 was purchased from CST; GAPDH antibody was purchased from Proteintech; fluorescent secondary antibody was purchased from Beyotime; and apoptosis detection kit was purchased from B&D.

[0018] (II) Cell Culture and Treatment: Mouse renal tubular epithelial cells (mPTCs) were cultured in DMEM / F12 medium containing 10% fetal bovine serum, 0.5% penicillin, and streptomycin at 37°C, with 5% carbon dioxide and 95% air. To investigate the role of Dapk3 in cisplatin-induced renal tubular epithelial cell damage, mPTCs were transfected with a small interfering agent or plasmid of Dapk3 when they reached 50% confluence, followed by cisplatin treatment for 24 h. Cells were then collected for Western blot analysis and flow cytometry analysis to detect apoptosis.

[0019] (III) Cisplatin-induced acute kidney injury mouse model and experimental grouping: Mice were randomly divided into three groups: a blank control group (vehicle), a cisplatin injection model group (cisplatin), and a cisplatin + HS148 group (cisplatin + HS148). HS148 was dissolved in Cremophor EL + Saline and administered intraperitoneally at a dose of 10 mg / kg. Mice in the control group or HS148 treatment group received intraperitoneal injections of either the solvent or HS148 once daily, starting 12 hours before cisplatin injection. Cisplatin model mice received a single intraperitoneal injection of 20 mg / kg cisplatin, while mice in the control group received the same dose of saline. After 72 hours, all mice were euthanized, and kidney and blood samples were collected. All animal experiments were conducted in accordance with the Chinese Regulations on the Management and Use of Laboratory Animals.

[0020] (iv) Kidney function tests: After centrifugation, serum components were collected from mouse blood samples, and serum creatinine and blood urea nitrogen levels were measured using a fully automated biochemical analyzer at Nanjing Children's Hospital.

[0021] (v) Western blot: Cell and tissue samples were added with an appropriate amount of protein lysis buffer, centrifuged at 12,000 rpm for 15 min in a pre-cooled centrifuge at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA kit. 5xSDS loading buffer was added proportionally, mixed well, and then boiled in a metal bath at 100°C for 5 min. A 10% polyacrylamide gel was prepared using the YAG PAGE gel rapid preparation kit. After loading, the gel was incubated at a constant voltage of 80V for 30 min and then at 120V for 1 h until the bromophenol blue indicator reached the bottom of the separating gel. The gel was then transferred to a PVDF membrane using a constant current of 30 mA wet transfer for 1.5 h. The PVDF membrane was blocked in 5% skim milk for 1 h and then incubated with primary antibody (4°C, shaker overnight). Secondary antibody was added (Fibronectin (FN, Abcam, 1:1000) and GAPDH (Proteintech, 1:1000) for 1 h. The gel was then developed using ECL chemiluminescence buffer on a gel imaging system, and semi-quantitative protein band analysis was performed using ImageJ.

[0022] (vi) Real-time quantitative PCR (RT-qPCR): TRIzol was added to tissue samples to extract total RNA. After determining the RNA concentration, it was reverse transcribed into cDNA. RT-qPCR was performed using the Roche SYBR Green method, with a reaction volume of 10 μL. (vii) PAS glycogen staining: Tissues were fixed in 4% paraformaldehyde and routinely dehydrated and embedded. Sections were dewaxed to 4 μm in distilled water. The sections were rinsed with tap water for 2-3 min, then twice with distilled water, placed in an oxidizing agent, and incubated at room temperature for 8 min. The sections were then rinsed once with tap water, followed by two more washes with distilled water. Schiff staining solution was then added to the samples, and the samples were incubated in a dark place at room temperature for 25 min. The sections were rinsed with tap water for 10 min, stained with hematoxylin for 1 min, differentiated in acidic differentiation solution for 5 s, and rinsed with tap water for 15 min to allow the samples to regain their blue color. The sections underwent routine dehydration treatment stepwise, and finally mounted with neutral resin.

[0023] (viii) TUNEL immunofluorescence staining: Tissues were fixed in 4% paraformaldehyde and routinely dehydrated and embedded; sections were dewaxed to 4μm and purified to distilled water; sample pretreatment: 2 mg / mL Proteinase K solution was diluted to 20 µg / mL with 1×PBS, and 100 µL of the diluted Proteinase K solution was added to each sample, incubated at room temperature for 20 min for permeabilization; labeling and detection: 5×Equilibration Buffer was diluted to 1×Equilibration Buffer with double-distilled water. 100 µL of 1×Equilibration Buffer was added to each sample, and equilibration was carried out at room temperature for 20-30 min. During equilibration, the labeling solution was prepared in the dark according to the table below. After equilibration, the liquid on the sample was aspirated, and 50 µL of TdT incubation buffer was added. An appropriate amount of water was added to the dark box, the slide was placed in it, the box was closed, and it was incubated at 37℃ for 60 min. The liquid on the sample was aspirated, and the sample was rinsed with 1×PBS 3 times for 5 min each time. Add 50 µL of DAPI to each sample for nucleus staining, incubate at room temperature in the dark for 10 min, rinse with 1×PBS three times for 5 min each time. Aspirate the liquid from the sample, mount with anti-fluorescence quencher, store overnight at 4°C in the dark, and observe and photograph using a laser confocal microscope.

[0024] (ix) Statistical Analysis: Data are expressed as mean ± SD. One-way ANOVA was used for comparisons among multiple groups, and t-tests were used for comparisons between two groups. A p-value < 0.05 was considered statistically significant.

[0025] Example 1: Effects of HS148 drug intervention on the kidneys of mice with cisplatin-induced acute kidney injury.

[0026] To evaluate the role of HS148 in protecting against cisplatin-induced acute kidney injury, we constructed a cisplatin-induced acute kidney injury model in mice, dividing them into three groups: WT+vehicle group, WT+cis group, and WT+cis+HS148 group. We measured kidney injury-related biochemical indicators in mouse serum. 72 hours after intraperitoneal injection of cisplatin to induce the model, serum creatinine and blood urea nitrogen levels were significantly elevated, the early-stage specific biomarker KIM-1 was significantly upregulated, and kidney pathological damage, such as tubular vacuolar degeneration and cast formation, was also more severe. However, after HS148 treatment, the corresponding kidney damage and renal function indicators in mice were significantly reduced. Figure 1 A-1D). Furthermore, the renal tubular injury score also suggests that HS148 treatment can improve cisplatin-induced renal pathological damage (A-1D). Figure 1 (E-1F). Therefore, HS148 can not only improve renal function but also reduce renal pathological damage. These results indicate that HS148 can improve cisplatin-induced acute kidney injury.

[0027] To further demonstrate the role of HS148 in protecting against cisplatin-induced acute kidney injury, we examined apoptosis and inflammation-related markers in acute kidney injury. TUNEL immunofluorescence staining results showed a significantly increased proportion of apoptotic cells in mice with cisplatin-induced acute kidney injury, while the proportion of apoptotic cells significantly decreased after HS148 treatment, indicating that HS148 can inhibit cisplatin-induced apoptosis of renal tubular epithelial cells. Figure 2 A-2B). RT-qPCR results showed that HS148 treatment significantly inhibited the upregulation of IL-1β, an inflammatory marker in cisplatin-induced acute kidney injury. Figure 2 C).

[0028] Example 2: Changes in the expression of cell death protein kinase 3 (Dapk3) in acute kidney injury.

[0029] To evaluate the role of HS148 target Dapk3 in acute kidney injury, we searched databases and found that The Human Protein Atlas database showed high expression of Dapk3 in the kidney. Figure 3 A); Immunofluorescence staining showed that Dapk3 was localized in renal tubular epithelial cells ( Figure 3 B); Western blot results showed that in the cisplatin-induced mouse model of acute kidney injury, the expression level of Dapk3 protein was significantly decreased compared with the control group (B). Figure 3 C).

[0030] Example 3: Effects of cell death protein kinase 3 (Dapk3) intervention on cisplatin-induced renal tubular epithelial cells.

[0031] To further verify the role of HS148 in renal tubular epithelial cells, we cultured renal tubular epithelial cells in vitro. First, we knocked down Dapk3 expression based on siDapk3 small interference, and then stimulated the cells with cisplatin (5 μg / ml) for 24 h before collecting the cells. Figure 4 A-4B Western blot results validated the efficiency of knocking down Dapk3. Figure 4 C-4D Western blot results showed that after cisplatin induction, cleaved caspase 3, an apoptosis-related marker of renal tubular epithelial cells, increased significantly, while cleaved caspase 3 decreased significantly after Dapk3 deletion. Figure 4 E-4F flow cytometry results showed that cisplatin-induced apoptosis in renal tubular epithelial cells was significantly increased, while knockdown of Dapk3 expression significantly reduced apoptosis. Subsequently, we overexpressed Dapk3 plasmid in renal tubular epithelial cells in vitro, stimulated them with cisplatin (5 μg / ml) for 24 h, and then harvested the cells. Figure 4G-4H Western blot results validated the Dapk3 overexpression efficiency. Figure 4 I-4J Western blot results showed that overexpression of Dapk3 significantly enhanced the expression of cleaved caspase 3 protein in cisplatin-induced renal tubular epithelial cells. Figure 4 K-4L flow cytometry results showed that overexpression of Dapk3 could increase the level of cisplatin-induced apoptosis.

[0032] In summary, this invention provides the use of the Dapk3 inhibitor HS148 in the preparation of a drug for alleviating cisplatin-induced acute kidney injury-related symptoms. The drug is administered via intraperitoneal injection at a mouse dose of 10 mg / kg, and improves acute kidney injury by inhibiting Dapk3 protein levels and apoptosis.

[0033] This invention provides an idea and method for the application of HS148 in the preparation of drugs for treating acute kidney injury. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. The application of HS148 in the preparation of drugs for the treatment of acute kidney injury, wherein, The structural formula of HS148 is: 。 2. The application according to claim 1, characterized in that, The acute kidney injury mentioned is cisplatin-induced acute kidney injury.

3. The application according to claim 1, characterized in that, HS148 inhibits the expression level of serum creatinine (SCr).

4. The application according to claim 1, characterized in that, HS148 inhibits the expression level of blood urea nitrogen (BUN).

5. The application according to claim 1, characterized in that, HS148 inhibits the expression level of KIM-1.

6. The application according to claim 1, characterized in that, HS148 alleviates the degree of renal tubular damage.

7. The application according to claim 1, characterized in that, HS148 inhibits apoptosis of renal tubular epithelial cells.

8. The application according to claim 1, characterized in that, HS148 is the main active ingredient of the drug, which also includes a pharmaceutically acceptable carrier.

9. The application according to claim 1, characterized in that, The drug dosage form is any one of tablets, capsules, or liquid preparations.

10. The application according to claim 1, characterized in that, The drug can be administered orally or intravenously.

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