Application of Arginase-2 as a target in screening products for diagnosis, prevention and / or treatment of drug-induced renal injury

Abstract

The present invention provides an application of Arginase-2 as a target in screening products for diagnosis, prevention and / or treatment of drug-induced renal injury. The inventors found that in human and animal specimens with drug-induced renal injury, nephrotoxic drugs such as contrast agents or cisplatin can cause a significant increase in the level of Arginase-2 in the blood, and the expression of Arginase-2 protein in the kidneys of animals is significantly upregulated; at the same time, drug inhibition or gene silencing of Arginase-2 expression can significantly increase the urine volume of mice with drug-induced renal injury, reduce blood creatinine levels, alleviate the nitrative stress of renal tubular epithelial cells caused by contrast agents, etc., downregulate HO-1 protein expression, and significantly reverse the apoptosis and ferroptosis of renal tubular epithelial cells caused by contrast agents, etc., thereby using Arginase-2 as an intervention target for screening products for diagnosis, prevention and / or treatment of drug-induced renal injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more specifically, to an application of Arginase-2 as a target in screening, diagnosing, preventing and / or treating products for drug-induced renal injury. Background Art

[0002] Acute kidney injury (AKI) is a major global health problem, associated with high morbidity, increased medical costs, and high mortality. In addition, although AKI is considered a reversible syndrome, it often leads to chronic kidney disease (CKD). Drug-induced renal injury is the main cause of AKI, including the use of contrast agents, cisplatin treatment, etc. Iodine contrast agents (also known as iodine contrast agents, hereinafter referred to as contrast agents) are a class of drugs widely used in clinical radiological procedures to enhance the imaging effect and improve the level of diagnosis and treatment. The global annual usage exceeds 75 million units, mainly used for cardiovascular angiography, urinary tract angiography, other organ cavity contrast agent examinations, and computed tomography (CT) enhancement. However, 11% to 50% of patients may develop contrast-induced nephropathy (CI-AKI) after contrast agent administration, which is defined as a 25% or 44.2 μmol / L increase in serum creatinine compared with baseline within 72 hours after intravascular administration of contrast agent without the influence of surgery, nephrotoxic drugs, etc. Clinical studies have shown that CI-AKI accounts for about 12% of iatrogenic acute kidney injury, resulting in a 4- to 5-fold increase in mortality, a 7% to 31% increase in chronic renal failure and total mortality, and a 5- to 10-fold increase in average hospital stay and socioeconomic burden. CI-AKI has become an important cause of health problems in my country, and the economic burden of the disease is increasing. However, there is still a lack of effective prevention and treatment measures for contrast-induced renal injury. Clinical prevention and treatment measures for CI-AKI, such as hydration therapy and antioxidant therapy, are non-specific and the latest large-scale clinical studies have found that they cannot reduce the incidence of CI-AKI. Therefore, finding specific targets for the development of new drugs for the diagnosis, prevention and / or treatment of drug-induced renal injury such as contrast-induced nephropathy is a technical problem that urgently needs to be solved. Summary of the invention

[0003] Arginase is an enzyme responsible for the urea cycle, which can hydrolyze L-arginine into ornithine and urea. There are two subtypes of arginase, arginase-1 (Arginase-1) is mainly expressed in the liver; arginase-2 (Arginase-2) is mainly expressed in the kidney, especially in the outer medullary stripes of the kidney. Arginase-2 regulates NO production by competing with nitric oxide (NO) synthase (NOS) for the same substrate L-arginine. The inventors found that in human and animal specimens with drug-induced renal injury, nephrotoxic drugs such as contrast agents or cisplatin can cause a significant increase in Arginase-2 levels in the blood, and the expression of Arginase-2 protein in animal kidneys is significantly upregulated. The increase in Arginase-2 levels in renal tissue increases the consumption of L-arginine and leads to a decrease in the L-arginine available to NOS, thereby inducing NOS uncoupling, mediating nitrosative stress, and ultimately damaging renal tissue.

[0004] Based on this, the present invention provides a therapeutic target with potential for treating drug-induced renal injury such as contrast-induced nephropathy, and silences the expression of Arginase-2 through non-specific drug inhibition (nor-NOHA) or gene intervention, alleviates the nitrative stress of renal tubular epithelial cells caused by contrast agents, downregulates HO-1 protein expression, significantly reverses apoptosis and ferroptosis of renal tubular epithelial cells caused by contrast agents, improves the urine volume of mice, and reduces the creatinine level of mice. Specifically, the primary purpose of the present invention is to provide the use of Arginase-2 as a target in screening products for treating drug-induced renal injury.

[0005] The second object of the present invention is to provide the use of Arginase-2 as a target in screening products for preventing drug-induced renal injury.

[0006] The third object of the present invention is to provide the use of Arginase-2 as a target in screening products for alleviating drug-induced renal injury.

[0007] Furthermore, in any of the above schemes, the product includes but is not limited to drugs or biochemical agents. When the product is a drug, the drug may contain one or more pharmaceutically acceptable carriers; and the drug may be further prepared into a corresponding pharmaceutical preparation by conventional methods in the pharmaceutical field; and the drug may also contain one or more other ingredients having the same or similar activity as the present invention, or ingredients having different activity from the present invention, which may enhance the activity described in the above multiple schemes of the present invention, or, in some cases, excipients or other active ingredients may reduce the activity in any of the above schemes of the present invention.

[0008] Furthermore, in any of the above schemes, the drug-induced renal injury is renal injury caused by contrast agents and / or nephrotoxic drugs. Specifically, the contrast agents include but are not limited to iohexol and the like; the nephrotoxic drugs include but are not limited to cisplatin and the like.

[0009] A fourth object of the present invention is to provide the use of Arginase-2 as a target in screening products for diagnosing drug-induced renal injury.

[0010] Specifically, the product includes but is not limited to drugs or biochemical reagents. When the product is a drug, the drug may contain one or more pharmaceutically acceptable carriers; and the drug may be further prepared into a corresponding pharmaceutical preparation by conventional methods in the pharmaceutical field; and the drug may also contain one or more other ingredients having the same or similar activity as the present invention, or ingredients having different activity from the present invention, which may enhance the activity described in the above-mentioned multiple schemes of the present invention, or, in some cases, excipients or other active ingredients may reduce the activity in any of the above-mentioned schemes of the present invention. More specifically, the product includes but is not limited to a kit.

[0011] A fifth object of the present invention is to provide an application of Arginase-2 as a target in screening products for downregulating the expression level of HO-1 or Arginase-2 protein.

[0012] Specifically, the product can target Arginase-2 to perform gene mutation or gene silencing, or the product is a substance that inhibits the activity or expression of Arginase-2. The product can be a drug or a biochemical reagent.

[0013] The sixth object of the present invention is to provide an application of an Arginase-2 inhibitor or a product containing an Arginase-2 inhibitor in preventing drug-induced renal injury.

[0014] The seventh object of the present invention is to provide an Arginase-2 inhibitor or a product containing the Arginase-2 inhibitor for use in treating drug-induced renal injury.

[0015] The eighth object of the present invention is to provide the use of Arginase-2 inhibitors in the preparation of products for treating drug-induced renal injury.

[0016] The ninth object of the present invention is to provide the use of an Arginase-2 inhibitor in the preparation of a product for preventing drug-induced renal injury.

[0017] Specifically, the Arginase-2 inhibitor includes but is not limited to nor-NOHA.

[0018] In some preferred embodiments, in any of the above schemes, the drug-induced renal injury is renal injury caused by contrast agents and / or nephrotoxic drugs; the products include but are not limited to drugs or biochemical agents. When the product is a drug, the drug may contain one or more pharmaceutically acceptable carriers; and the drug may be further prepared into a corresponding pharmaceutical preparation by conventional methods in the pharmaceutical field; and the drug may also contain one or more other ingredients having the same or similar activity as the present invention, or ingredients having different activity from the present invention, which may enhance the activity described in the above multiple schemes of the present invention, or, in some cases, excipients or other active ingredients may reduce the activity in any of the above schemes of the present invention.

[0019] It should be noted that the application in any of the above-mentioned implementation modes does not include the diagnosis and treatment of diseases.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention has discovered a potential target for diagnosing, preventing and / or treating drug-induced renal injury - Arginase-2. In the process of research related to drug-induced renal injury, the present invention has found that the expression of Arginase-2 can be inhibited by drugs or gene silenced, thereby alleviating the nitrification stress of renal tubular epithelial tissue caused by contrast agents and / or nephrotoxic drugs, down-regulating the expression of HO-1 protein, and significantly reversing the apoptosis and ferroptosis of renal tubular epithelial cells caused by contrast agents and / or nephrotoxic drugs. It is proposed to use Arginase-2 as a target for screening products for diagnosing, preventing and / or treating drug-induced renal injury, which provides a new idea for realizing the diagnosis, prevention and / or treatment of drug-induced renal injury, thereby effectively preventing and / or treating drug-induced renal injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Western blot was used to detect the expression level of Arginase-2 protein in HK-2 cells treated with contrast agents;

[0023] Figure 2 The contrast agent under electron microscope causes morphological changes in renal tubular epithelial cells; wherein, Figure A and Figure B are electron microscope images of HK-2 cells in Example 1 without iohexol treatment; Figure C and Figure D are electron microscope images of HK-2 cells in Example 1 treated with iohexol; Figure E and Figure F are images of morphological changes in renal tubular epithelial cells of rats in Example 2 without iohexol intervention (iohexol injection through tail vein); Figure G and Figure H are images of morphological changes in renal tubular epithelial cells of rats in Example 2 treated with iohexol (iohexol injection through tail vein);

[0024] Figure 3This is a HK-2 cell model with Arginase-2 expression knocked down;

[0025] Figure 4 Arginase-2 gene silencing can significantly reverse the reduction of NO production in HK-2 cells caused by iohexol;

[0026] Figure 5 Arginase-2 gene silencing can significantly reverse the increase in 3-NT production in HK-2 cells caused by iohexol;

[0027] Figure 6 Iohexol can cause the decrease of HO-1 mRNA and protein expression in HK-2 cells; Arginase-2 gene silencing can significantly increase the protein expression level of HO-1;

[0028] Figure 7 Arginase-2 gene silencing can significantly reverse the reduction of ATP production and cell viability in HK-2 cells caused by iohexol;

[0029] Figure 8 Arginase-2 inhibitors can significantly increase urine volume and reduce blood creatinine levels in CI-AKI mice.

[0030] Fig. 9 The level of Arginase-2 in the serum of mice with CI-AKI and clinical patients was significantly higher than that in the control group. DETAILED DESCRIPTION

[0031] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the connotation of the present invention, so the present invention is not limited to the specific implementation disclosed below.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0033] In the following examples, unless otherwise specified, the methods used are all conventional methods in the art; the drugs used can be prepared through commercial channels or known public methods.

[0034] The present invention is accomplished through three approaches: cells, animals, and clinical samples.

[0035] Example 1 Cellular pathway

[0036] Normal HK-2 cells and gene silenced HK-2 cells were treated with contrast agent-induced renal tubular epithelial cell injury modeling method. MTT method was used to determine cell viability, and transmission electron microscopy was used to observe the morphological changes of renal tubular epithelial cells; Real-time PCR and Western blot were used to detect changes in the mRNA and protein expression levels of Arginase-2 and HO-1, and nitration stress indicators such as NO, 3-NT, NOS monomer / dimer and apoptosis-related indicators were detected, and ATP levels were determined. The specific implementation method is as follows:

[0037] 1. Establishment of a contrast agent-induced HK-2 cell injury model: Based on the inventors' previous work, the iohexol concentration was determined to be 75 mg (I) / mL and the treatment time was 4 h. The treatment results were as follows: Figure 1 and Figure 2 As shown, Figure 1 It can be seen that iohexol treatment for 4 hours can lead to an increase in the expression level of Arginase-2 protein in HK-2 cells; Figure 2 In the figure, A and B are HK-2 cells incubated without iohexol, and C and D are HK-2 cells incubated with iohexol; Figure 2 In Figures A and B, the cell structure is intact, and the morphology of organelles such as the cell membrane, nucleus and mitochondria is normal; while in Figures C and D, the organelles are obviously swollen: the mitochondria are obviously swollen, the mitochondrial membrane is damaged, the cristae are reduced or disappeared, the endoplasmic reticulum is obviously swollen, and the density of some endoplasmic reticulum breaks increases.

[0038] 2. Establishment of HK-2 cell line with Arginase-2 gene silencing: HK-2 cells were transfected with lentiviral shRNA-Arginase-2 vector to establish HK-2 cell line with Arginase-2 gene silencing; Arginase-2 was detected by Real-time PCR and Western blot to confirm the transfection effect. The processing results were as follows: Figure 3 As shown. Figure 3 , indicating that siRNA3 has the most obvious inhibitory effect on Arginase-2 expression, with an expression rate of about 10%, and the target sequence of Arginase-2SiRNA3 is GAGGGCATATTGTCTATGA. The specific operation is as follows:

[0039] 1) Inoculation of 1×10 6 ~5×10 6 HK-2 cells were cultured in a 6-well plate containing 1863 μL of complete medium;

[0040] 2) Dilute siRNA: dilute 5 μL of 50 nM siRNA stock solution with 120 μL of 1×riboFECTTM CP Buffer and shake gently;

[0041] 3) Preparation of mixed solution: Add 3 μL riboFECTTM CP Reagent, gently pipette to mix, and incubate at room temperature for 0 to 15 minutes to prepare the transfection complex;

[0042] 4) Add the riboFECTTM CP transfection complex to an appropriate amount of complete medium without dual antibodies and mix gently;

[0043] 5) Place the culture plate in a CO2 incubator at 37°C and culture for 24 to 96 hours;

[0044] siRNA was transfected into 6-well plates with riboFECTTM CP Reagent at a transfection concentration of 50 nM.

[0045] 3. Quantitative detection of NO concentration: 1) Detection of intracellular NO concentration using DAF-FMDA fluorescent probe: According to the method of the NO kit (Biyuntian-NO fluorescent probe quantitative detection kit), DAF-FMDA was diluted with the DAF-FMDA diluent provided in the kit at a ratio of 1:1000 to a final concentration of 5 μmol / L; the cell culture medium in the six-well plate was removed and 1 mL of the diluted DAF-FMDA was added; the cells were incubated in a 37°C cell culture incubator for 20 min, and the cells were washed three times with PBS (pH 7.4) to fully remove the DAF-FMDA that had not entered the cells; the Operetta high-content imaging system (PerkinElmer) was used for detection and analysis, with an excitation wavelength of 495 nm and an emission wavelength of 515 nm. Figure 4 The results showed that Arginase-2 gene silencing could significantly reverse the reduction of NO production in HK-2 cells induced by iohexol.

[0046] 4. Real-time PCR and Western blot analysis: Detect changes in HO-1 mRNA gene transcription and 3-NT and HO-1 protein expression levels. The analysis results are as follows: Figure 5 and Figure 6 shown. Figure 5 It showed that Arginase-2 gene silencing could significantly reverse the increase of 3-NT production in HK-2 cells caused by iohexol; Figure 6 It indicates that contrast agents can lead to decreased expression of HO-1 mRNA and protein in HK-2 cells; Arginase-2 gene silencing can significantly upregulate the protein expression level of HO-1.

[0047] 5. Cell proliferation assay (MTT) to detect cell viability, kit to detect cell ATP level: in a 96-well plate, plant 5000 cells / well; add 20μL of MTT solution and 180μL of blank culture medium to each well, and continue incubating in the cell culture incubator for 4h; then add 150μL of Formazan solution to each well, mix well, and measure the absorbance at 490nm with an enzyme reader to detect cell viability; detect ATP level according to the ATP kit operation method; the test results are as follows: Figure 7 shown. Figure 7 It was shown that Arginase-2 gene silencing could significantly reverse the reduction of ATP production and cell viability in HK-2 cells caused by iohexol.

[0048] Example 2 Animal route

[0049] Male C57BL / 6N mice (6-8 weeks) were divided into groups: control group, CI-AKI model group, CI-AKI model group + nor-NOHA (Arginase-2 inhibitor) group. 24h urine was collected in metabolic cages, and urine volume was measured and recorded; orbital blood was collected 24h after modeling to detect renal function evaluation indicators such as blood creatinine and urea nitrogen levels; HE staining and tubular injury scoring were used to evaluate the morphological structure and damage of renal tissue and tubules, and transmission electron microscopy was used to observe the morphological changes of renal tubular epithelial cells; Western blot was used to detect changes in the mRNA and protein expression levels of Arginase-2 and HO-1 in renal tissue and tubules, and nitration stress indicators such as NO, 3-NT, NOS monomer / dimer and apoptosis-related indicators were detected. The specific implementation method is as follows:

[0050] (1) Preparation of control group, CI-AKI mouse model group, CI-AKI model group + nor-NOHA (Arginase-2 inhibitor) group:

[0051] Male C57BL / 6N mice (6-8 weeks) were anesthetized by intraperitoneal injection of 0.6% sodium pentobarbital (80 mg / kg), and then underwent right nephrectomy. Three weeks after the operation, the mice were dehydrated for 48 hours. After 48 hours of dehydration, the mice in the control group were intraperitoneally injected with 100 mg / kg of nor-NOHA (Arginase-2 inhibitor), and 30 minutes later, 10 mL / kg of furosemide was intraperitoneally injected. After an interval of 30 minutes, 15 ml / kg of 0.9% saline was injected into the tail vein; after 48 hours of dehydration, the mice in the CI-AKI model group were intraperitoneally injected with 100 ml / kg of 0.9% saline, and 30 minutes later, 10 mL / kg of furosemide was intraperitoneally injected. After an interval of 30 minutes, 15 mL / kg of iohexol (350 mg (I) / ml) was injected into the tail vein; after 48 hours of dehydration, the CI-AKI model group + nor-NOHA (Arginase-2 inhibitor) group was intraperitoneally injected with 100 mg / kg of nor-NOHA (Arginase-2 inhibitor), and 30 minutes later, 10 mL / kg of furosemide was intraperitoneally injected. After 30 minutes, 15 mL / kg of iohexol (350 mg (I) / ml) was injected into the tail vein. Finally, the mice were placed in metabolic cages and allowed to eat freely. Urine was collected for 24 hours, and the urine volume was measured and recorded. After 24 hours, blood was collected from the eye sockets to test the levels of serum creatinine and urea nitrogen. The kidney tissue was separated and sliced ​​into 3 μm thick sections. The morphology and injury of the kidney tissue and tubules were evaluated by HE staining and tubular injury scoring. The changes in the structure of proximal tubular cells were observed under transmission electron microscopy (magnification, ×5000). Figure 8 shown. Figure 8 The results showed that Arginase-2 inhibitors could significantly increase urine volume and reduce blood creatinine levels in CI-AKI mice; Figure 2 (E, F, G, H) are rats without (E, F) or with (G, H) iohexol intervention (15 mL / kg iohexol was injected into the tail vein for 24 hours). The control group (E, F) had intact cell structure, and the morphology of cell organelles such as cell membrane, cell nucleus and mitochondria was normal (×2000, ×5000); the iohexol group (G, H) had obvious swelling of cell organelles: mitochondria were obviously swollen, mitochondrial membrane was damaged, cristae were reduced or disappeared, endoplasmic reticulum was obviously swollen, and the density of some endoplasmic reticulum ruptures increased. Fig. 9 Figure A in the middle shows that the serum Arginase-2 level in CI-AKI mice was significantly increased compared with the control group.

[0052] Example 3 Clinical specimens

[0053] Venous blood was collected from clinical patients before and 6h, 12h, 24h, 48h, and 72h after contrast imaging, placed in EDTA tubes, centrifuged at 3000r / min for 10min, and the supernatant was stored in a -80℃ refrigerator. The levels of serum creatinine, urea nitrogen, and cystatin C were detected by biochemical automatic analyzer; the level of Arginase-2 was detected by ELISA. The results were as follows: Fig. 9 As shown in Figure B. Fig. 9 Figure B in the middle shows that the serum Arginase-2 level in CI-AKI patients (n=6) was significantly higher than that in non-CI-AKI patients (n=6), and the serum Arginase-2 level in the control group (n=3) who did not use contrast agent but matched clinical characteristics was the lowest.

[0054] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0055] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of Arginase-2 inhibitors in screening products for treating drug-induced renal injury; the drug-induced renal injury is renal injury caused by contrast agents or nephrotoxic drugs.

2. Application of Arginase-2 inhibitors in screening products for preventing drug-induced renal injury; the drug-induced renal injury is renal injury caused by contrast agents or nephrotoxic drugs.

3. Application of Arginase-2 inhibitors in screening products for alleviating drug-induced renal injury; the drug-induced renal injury is renal injury caused by contrast agents or nephrotoxic drugs.

4. Application of Arginase-2 inhibitors in screening products for diagnosing drug-induced renal injury; the drug-induced renal injury is renal injury caused by contrast agents or nephrotoxic drugs.

5. Use of Arginase-2 inhibitors in the preparation of products for treating drug-induced renal injury; the drug-induced renal injury is renal injury caused by contrast agents or nephrotoxic drugs.

6. Use of Arginase-2 inhibitors in the preparation of products for preventing drug-induced renal injury; the drug-induced renal injury is renal injury caused by contrast agents or nephrotoxic drugs.

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