Use of DNA-dependent protein kinase specific inhibitor in preparation of drugs to prevent and treat renal fibrosis

A protein kinase, renal fibrosis technology, applied in the field of medicine

Pending Publication Date: 2019-09-06
NANJING CHILDRENS HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the technical problems existing in the prior art, the present invention provides the use of a DNA-dependent protein kinase activity inhibitor in the preparation and prevention of renal fibrosis-related diseases. The DNA-dependent protein kinase activity in

Method used

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  • Use of DNA-dependent protein kinase specific inhibitor in preparation of drugs to prevent and treat renal fibrosis
  • Use of DNA-dependent protein kinase specific inhibitor in preparation of drugs to prevent and treat renal fibrosis
  • Use of DNA-dependent protein kinase specific inhibitor in preparation of drugs to prevent and treat renal fibrosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Materials and methods

[0022] 1) Materials and reagents

[0023] Inhibitor NU7441 was purchased from Selleck Company, and mTOR, p-mTOR, a-SMA and PCNA antibodies were purchased from CellSignaling Technology Company. FN, DNA-PK and p-DNA-PK antibodies were purchased from ABCAM Company. β-actin was purchased from Nanjing Biotech Company. Fluorescent secondary antibodies were purchased from Invitrogen. TGFβ1 was purchased from R&D Company. Secondary antibodies and other reagents required for Western Blot were purchased from Sigma.

[0024] 2) Cell culture and treatment

[0025] Human renal tubular epithelial cells (HK2) and rat fibroblasts (NRK49F) were cultured in DMEM / F12 medium containing 10% fetal bovine serum, 0.5% penicillin and streptomycin at 37°C, 5% carbon dioxide and 95% air. In order to study the protective effect and mechanism of NU7441 on renal fibrosis caused by TGFβ1, we changed the medium to serum-free medium and added NU7441 at an appropriate conc...

Embodiment 2

[0037] Example 2 Cytotoxicity experiment of NU7441

[0038] The human renal tubular epithelial cell line (HK2) was applied, spread in a 96-well plate, and cultured in 100 μL medium. Cells were cultured at 37°C for 24 hours in a CO2 incubator. NU7441 (prepared in complete medium) at final concentrations (100nM, 500nM, 1000nM, 5000nM, 10000nM and 20000nM) was added to the plate, and the culture plate was incubated in the incubator for 24 hours. Add 10 µL of CCK-8 solution to each well of the 96-well plate using a pipette. Taking care not to introduce air bubbles in the wells, incubate the plate in the incubator for 2 h. Absorbance at 450 nm was measured using a microplate reader. The result is as figure 1 shown. The results showed that NU7441 below 1000nM (1μM) did not cause direct toxic side effects on renal tubular epithelial cells.

Embodiment 3

[0039] Example 3 NU7441 affects the activation of rat long fiber cells induced by transforming growth factor (TGFβ1)

[0040] Rat fibroblasts (NRK49F) were cultured in vitro. After the cells were pretreated with NU7441 for 1 hour, they were treated with TGFβ1 (5ng / ml) for 24 hours to prepare samples. Western blot was used to detect the expression of fibrosis-related proteins in NRK49F. It was found that NU7441 Treatment can significantly inhibit the synthesis of fibrotic proteins such as α-SMA and Fibronectin induced by TGFβ1 ( figure 2 ); further, in NRK49F cells, NU7441 treatment can significantly inhibit the synthesis of cell proliferation-related protein PCNA ( figure 2 ); Western blot results showed that NU7441 could inhibit DNA-PKcs kinase activity by inhibiting the phosphorylation of DNA-PKcs ( figure 2 ). In summary, DNA-PKcs-specific inhibitor NU7441 can inhibit the phosphorylation of DNA-PKcs, thereby inhibiting the activation of NRK49F cells stimulated by TGFβ1...

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Abstract

The invention belongs to the field of pharmacy and particularly relates to use of a DNA-dependent protein kinase specific inhibitor in the preparation of drugs to prevent and treat renal fibrosis. Furthermore, the DNA-dependent protein kinase activity inhibitor is NU7441. The DNA-PK activity inhibitor herein is a DNA-PK catalytic subunit selective inhibitor, which helps relieve renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction, and improve the renal functionality. The NU7441 inhibitor can inhibit DNA-PK activation specifically so as to inhibit abnormal activation ofmTOR, inhibit the epithelial-mesenchymal transition of renal tubular epithelial cells, inhibit the collagen generation of renal tubular epithelial cells and mesenchymal fibroblasts under TGF-beta 1 stimulation, improve renal pathological injury and renal function, and improve renal tubulointerstitial fibrosis. The effective clinical drugs to prevent and treat renal fibrosis-related diseases are provided herein.

Description

technical field [0001] The invention belongs to the field of medicine, and specifically relates to the use of a DNA-dependent protein kinase (DNA-dependent protein kinase, DNA-PK) specific inhibitor NU7441 in the preparation of drugs for preventing and treating renal fibrosis-related diseases. Background technique [0002] With the development of society, chronic kidney disease (CKD) has become a public health issue of global concern. A survey in 2012 showed that in my country, the prevalence of CKD was about 10.8%, and the total number of people was nearly 120 million. Since the course of CKD is mostly continuous progression, and eventually develops to end-stage renal disease (ESRD), the only alternative treatment is dialysis or kidney transplantation. The treatment cost of ESRD is expensive. Statistics show that in the ten years from 2000 to 2010, the global medical expenses for dialysis were as high as 1.1 trillion US dollars, while the proportion of dialysis is increasi...

Claims

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Application Information

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IPC IPC(8): A61K31/5377A61P13/12
CPCA61K31/5377A61P13/12
Inventor 杨运文游然于婧张爱华贾占军刘素雯高慧萍王佩培
Owner NANJING CHILDRENS HOSPITAL
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