Application of Nit2 as a renal injury marker and therapeutic target

Abstract

The present invention discloses Nit2 as a diagnostic marker and new therapeutic target for ischemia-reperfusion-induced acute kidney injury (IR-AKI), and the use of a Nit2 activator in the preparation of a drug for alleviating IR-AKI. The present invention first discovered that Nit2 expression is reduced in IR-AKI, and knocking out Nit2 aggravates renal injury. Accordingly, overexpression of Nit2 reduces tubular epithelial cell damage under hypoxia-reoxygenation conditions. Furthermore, a small molecule activator ciclopirox olamine (CPX) that promotes Nit2 expression was screened and obtained, and its effect of alleviating IR-AKI was evaluated in mice, indicating that Nit2 is expected to be a target for IR-AKI, and its activator has the potential to be a drug for treating IR-AKI.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical biotechnology and relates to the application of Nit2 as a kidney injury marker and a therapeutic target, and specifically relates to the application of Nit2 as an acute kidney injury marker for preparing products for diagnosing acute kidney injury and as a therapeutic target for preparing drugs for alleviating / treating acute kidney injury. Background Art

[0002] Nit2 belongs to the 10th branch of the Nitrilase superfamily and is evolutionarily conservative, but its function has long been unknown. In 2004, it was even listed by researchers as the fourth of the "Top Ten Unknown Proteins with Unknown Functions to be Researched", but research progress in this field has been very limited in the past two decades. Nit2 is expressed in various organs of the human body, but the role of Nit2 in kidney disease, especially AKI, has not been reported. Summary of the invention

[0003] In order to overcome the defects of the prior art, the first object of the present invention is to provide the use of Nit2 as an acute kidney injury marker, wherein the Nit2 can be used as an acute kidney injury marker for diagnosing acute kidney injury and can also be used to evaluate the prognosis of acute kidney injury.

[0004] The second object of the present invention is to provide the use of Nit2 as an acute kidney injury marker in the preparation of an acute kidney injury diagnostic product.

[0005] The third object of the present invention is to provide the use of Nit2 as a drug target in the development, design, and screening of drugs for alleviating or treating acute kidney injury.

[0006] The fourth object of the present invention is to provide use of a Nit2 activator in the preparation of a drug for alleviating or treating acute kidney injury.

[0007] A fifth object of the present invention is to provide use of ciclopirox olamine CPX as a Nit2 activator in a drug for alleviating or treating acute kidney injury.

[0008] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0009] The present invention first provides the use of Nit2 as an acute kidney injury marker, wherein the amino acid sequence of Nit2 is shown in SEQ ID No. 1. The gene library sequence number is: BC020620.1.

[0010] MTSFRLALIQLQISSIKSDNVTRACSFIREAATQGAKIVSLPECFNSPYGAKYFPEYAEKIPGESTQKLSEVAKECSIYLIGGSIPEEDAGKLYNTCAVFGPDGTLLAKYRKIHLFDIDVPGKITFQESKTLSPGDSF STFDTPYCRVGLGICYDMRFAELAQIYAQRGCQLLVYPGAFNLTTGPAHWELLQRSRAVDNQVYVATASPARDDKASYVAWGHSTVVNPWGEVLAKAGTEEAIVYSDIDLKKLAEIRQQIPVFRQKRSDLYAVEMKKP

[0011] The Nit2 can be a Nit2 protein or an mRNA sequence encoding a Nit2 protein.

[0012] The amino acid sequence includes the amino acid sequence shown in SEQ ID No.1, or an amino acid sequence having the same function as SEQ ID No.1 formed by substitution, deletion and / or addition of the amino acid sequence shown in SEQ ID No.1.

[0013] The acute renal injury is caused by ischemia-reperfusion, cisplatin, contrast media or nephrotoxic antibiotics.

[0014] The marker is a serum diagnostic marker, a plasma diagnostic marker or a tissue cell diagnostic marker.

[0015] The present invention uses the single-cell transcriptomics public database (http: / / humphreyslab.com / SingleCell / ) for bioinformatics analysis. Omics data suggest that Nit2 is mainly expressed in tubular cells in the kidney, and its expression decreases in the state of acute kidney injury (IR-AKI) caused by ischemia-reperfusion. Next, the detection of Nit2 expression in clinical AKI renal biopsy specimens found that Nit2 expression did decrease. Further in vitro and in vivo experiments also showed that with the extension of renal ischemia time in mice (15, 20 or 30 minutes), the expression of Nit2 in the renal cortex gradually decreased. Similarly, the expression of Nit2 also decreased in HK-2 cells with hypoxia and reoxygenation. It shows that Nit2 is downregulated in renal tubular epithelial cells in the IR-AKI state and is positively correlated with injury. Therefore, Nit2 can be used as an acute kidney injury marker for the diagnosis of acute kidney injury, and can also be used to evaluate the prognosis of acute kidney injury.

[0016] The present invention provides use of a reagent for detecting Nit2 in preparing a product for diagnosing acute kidney injury.

[0017] The reagents may be primers for specifically amplifying Nit2 and / or antibody probes for specifically recognizing Nit2.

[0018] The product can be a kit, a chip or a nucleic acid membrane strip.

[0019] The kit comprises an antibody against Nit2 protein, and the antibody is a monoclonal antibody or a polyclonal antibody against Nit2 protein.

[0020] The detection method of the kit includes colloidal gold immunochromatography, immunoblotting, immunohistochemistry, immunofluorescence, flow cytometry, enzyme-linked immunosorbent assay, nucleic acid probe method or qPCR method.

[0021] Furthermore, Nit2 can be used as a drug target in the development, design, and screening of drugs for alleviating or treating acute kidney injury.

[0022] Since Nit2 expression is downregulated in renal tubular epithelial cells in the IR-AKI state and is positively correlated with injury, it can be seen that acute kidney injury can be alleviated by increasing the expression level of Nit2 in renal tubular epithelial cells in the IR-AKI state, and Nit2 agonists can increase the expression level of Nit2 in renal tubular epithelial cells in the IR-AKI state, and therefore, can be used to prepare drugs for alleviating or treating acute kidney injury.

[0023] The drug for alleviating or treating acute kidney injury includes a gene therapy drug for overexpression of Nit2 or a Nit2 agonist.

[0024] Nit2 agonists include chemical drugs, peptide drugs or protein drugs that target and promote the expression level or activity of Nit2.

[0025] The chemical drug may be ciclopirox olamine (CPX).

[0026]

[0027] Beneficial technical effects of the present invention:

[0028] The present invention uses bioinformatics to analyze the single-cell transcriptome database, as well as detect renal biopsy specimens of AKI patients, mice and cell AKI models, and the expression of Nit2 is reduced. It shows that Nit2 can be used as a diagnostic marker for AKI. It is of great significance to the screening of AKI. In addition, by screening Nit2 agonists, the Nit2 expression level of patients with acute kidney injury can be increased, thereby alleviating or treating acute kidney injury. Therefore, Nit2 agonists can be used to alleviate or treat acute kidney injury. The present invention further provides a Nit2 agonist ciclopirox olamine that promotes Nit2 expression level or activity, which can significantly alleviate or treat acute kidney injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the expression level of Nit2 in IR-AKI state.

[0030] Figure 2 This is a graph showing that knockout of Nit2 aggravates renal injury indicators in AKI caused by ischemia-reperfusion in mice.

[0031] Figure 3 The effect of overexpression of Nit2 on HK-2 tubular epithelial cell damage induced by hypoxia and reoxygenation.

[0032] Figure 4 This is the effect of ciclopirox olamine CPX on the expression level of Nit2.

[0033] Figure 5 The effect of ciclopirox olamine CPX on IR-AKI in mice. DETAILED DESCRIPTION

[0034] Example 1 Nit2 expression is downregulated in renal tubular epithelial cells in IR-AKI state.

[0035] In order to clarify the expression of Nit2 in ischemia-reperfusion-induced acute kidney injury (IR-AKI), we first used the single-cell transcriptomics public database (http: / / humphreyslab.com / SingleCell / ) to perform bioinformatics analysis on the "11. Mouse IRI Kidney (Sham, 4hrs, 12hrs, 2days, 14days, and 6weeks)" omics dataset. The results are as follows Figure 1 As shown:

[0036] Among them, A: Bioinformatics analysis showed that Nit2 was significantly decreased in the IR-AKI state, and the data came from http: / / humphreyslab.com / SingleCell / kidney single cell data.

[0037] B: Expression of Nit2 in renal biopsy specimens of AKI patients and statistical results of the percentage of Nit2-positive areas in renal tubules; Note: The scale of the left large icon is 200 μm, and the scale of the right small icon is 50 μm. Using t-test, compared with the control group, P<0.0001(n=5).

[0038] C: The renal cortex of the mice was obtained after renal ischemia for different time periods (15, 20 and 30 minutes) and reperfusion for 3 days to detect the protein expression level of Nit2.

[0039] D: HK-2 cells were deprived of oxygen for 24 hours and then reoxygenated for 1, 2, and 3 hours, and the protein expression level of Nit2 was detected.

[0040] like Figure 1 In A, omics data suggest that Nit2 is mainly expressed in tubular cells in the kidney, and its expression decreases in IR-AKI. Next, following clinical research ethics, clinical AKI renal biopsy specimens were obtained and tested to find that Nit2 expression was indeed decreased ( Figure 1 B in the figure).

[0041] Subsequently, 20 10-week-old C57BL / 6 mice were randomly divided into 4 groups, with 5 mice in each group. The 4 groups were: sham operation control group, model group (ischemia 15 minutes reperfusion group, ischemia 20 minutes reperfusion group, and ischemia 30 minutes reperfusion group).

[0042] The surgical model was established as follows: after the mice were anesthetized, they were placed in a thermostatic incubator. After the temperature reached 37°C, an opening was made at the angle between the left side of the mouse spine and the lower edge of the costal arch, about 2-3 cm and parallel to the spine. The opening was made to expose the left kidney to the outside of the body, and the kidney was kept moist. The fascia and fat beside the renal artery and vein were separated with fine forceps, and the artery and vein were clamped with hemostats for 15, 20 or 30 minutes. During the clamping process, the mice were placed in a 37°C incubator, and the left kidney and hemostats were put back into the body to avoid kidney damage. After the clamping was completed, the hemostats were released and the wound was sutured. The mice were placed in a 37°C incubator for 1 hour to wake them up, and then returned to the cage. Five mice were taken as a group at 15, 20 and 30 minutes, respectively, and recorded as the 15-minute ischemia reperfusion group, the 20-minute ischemia reperfusion group, and the 30-minute ischemia reperfusion group, respectively. At the same time, the treatment process of the sham operation group was the same as that of the model group, except that the left renal artery and vein were not clamped after the left kidney was exposed. The kidneys were taken out to detect the expression of related proteins on the 3rd day after the operation. The results showed that the expression of Nit2 in the renal cortex gradually decreased ( Figure 1 C in.

[0043] Similarly, human renal tubular epithelial cells HK-2 were treated with hypoxia and reoxygenation. HK-2 cells were cultured with DMEM / F12 medium without FBS and placed in a 1% O2, 5% CO2, 37°C three-gas incubator for 24 hours of hypoxia, and then supplemented with 2% FBS and placed back in a 37°C, 5% CO2 cell incubator for reoxygenation for 1 hour, 2 hours, and 3 hours. Western blotting results showed that Nit2 expression also decreased in HK-2 cells ( Figure 1 D in.

[0044] The above results indicate that Nit2 expression is downregulated in renal tubular epithelial cells in IR-AKI state and is positively correlated with injury.

[0045] Example 2 Knockout of Nit2 aggravates renal injury under ischemia-reperfusion state.

[0046] Nit2 knockout mice (C57BL / 6JCya-Nit2 em1 / Cya) was purchased from Saiye Biotechnology Co., Ltd., product number S-KO-10339.

[0047] A unilateral renal ischemia-reperfusion (IR-AKI) model was established using knockout mice (hereinafter referred to as KO): the mice were anesthetized and placed in a constant temperature incubator. After the temperature reached 37 ℃, an opening was made at the angle between the left side of the mouse spine and the lower edge of the costal arch, about 2-3 cm and parallel to the spine. The opening was made to expose the left kidney to the body, and the kidney was kept moist. The fascia and fat beside the renal artery and vein were separated with fine forceps, and the artery and vein were clamped with hemostats for 30 minutes. During the clamping process, the mice were placed in a 37 ℃ incubator, and the left kidney and hemostats were put back into the body to avoid kidney damage. After 30 minutes, the hemostats were released and the wound was sutured. The mice were placed in a 37 ℃ incubator for 1 hour to wake them up, and then put back into the cage (modeling group). At the same time, the sham operation group (WT group) was treated in the same way as the modeling group, except that the left renal artery and vein were not clamped after the left kidney was exposed. In order to collect blood for serum creatinine at the time of sacrifice, the contralateral kidney was removed 48 hours after surgery to eliminate the renal compensation effect. The animals were sacrificed 72 hours after surgery, blood was collected from the heart for serum creatinine measurement, and kidney tissue was taken for testing. Figure 2 As shown:

[0048] Wherein, A: mouse creatinine value.

[0049] B: Western blotting to detect the changes in renal cortical related protein expression in Nit2 knockout (KO) mice and wild-type (WT) mice after IR-AKI (n=3). Note: t-test was used to compare WT with KO group. P<0.05.

[0050] Figure 2 A in the figure shows that the serum creatinine value of mice in the KO group after IR-AKI was higher than that in the WT group, indicating that their renal function was more severely impaired. At the same time, protein immunoblotting was used to detect the expression level of KIM-1, a kidney injury marker, in the renal cortex. The results showed that the expression of KIM-1 in mice in the KO group after IR-AKI was higher than that in the WT group ( Figure 2 B in the figure).

[0051] The above results indicate that knocking out Nit2 aggravates kidney damage in the IR-AKI state.

[0052] Example 3 Overexpression of Nit2 in HK-2 renal tubular epithelial cells can alleviate the damage caused by hypoxia and reoxygenation.

[0053] The Nit2 expression vector was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number HG23517-UT. Overexpression was achieved in human renal tubular epithelial cells HK-2 by transient transfection. 24 hours after transfection, a culture process of 24 hours of hypoxia and 6 hours of reoxygenation was performed to simulate renal ischemia-reperfusion AKI. Specifically as follows: 24 hours after transfection, the complete culture medium containing 10% fetal bovine serum (FBS) was discarded and replaced with DMEM / F12 culture medium without FBS and placed in a 1% O2, 5% CO2, 37°C three-gas incubator for 24 hours of hypoxia, then supplemented with 2% FBS and placed back in a 37°C, 5% CO2 cell culture incubator for 6 hours of reoxygenation. The results of protein immunoblotting are shown in Figure 3 As shown, the results showed that overexpression of Nit2 could alleviate cell damage caused by hypoxia and reoxygenation.

[0054] Example 4 The promoting effect of CPX on the expression level of Nit2.

[0055] The dual luciferase reporter gene (purchased from GeneCare GV715 lentiviral vector) containing the Nit2 promoter was used to construct a stable HK-2 cell line. The cells were cultured in 96-well plates and lysed after 48 hours of drug treatment to detect the activities of firefly luciferase and Renilla luciferase. DMSO was used as a solvent control. The results are shown in Figure 4 As shown:

[0056] Among them, A: Dual luciferase reporter gene assay investigated the concentration dependence of CPX on Nit2 transcriptional activity.

[0057] B: Western blotting was used to examine the effect of CPX treatment on Nit2 protein expression levels in HK-2 cells.

[0058] Figure 4 A in the figure shows that ciclopirox olamine (CPX) can significantly increase promoter activity at 5-10 μM, and the higher the concentration, the stronger the activity. In addition, ordinary HK-2 cells were seeded in 6-well plates, adhered overnight, and then treated with DMSO, 5 μM and 10 μM CPX for 48 hours, respectively. After the cells were lysed, the expression of related proteins was detected by western blotting.

[0059] Figure 4 Panel B shows that CPX treatment of HK-2 cells for 48 hours increased Nit2 expression.

[0060] Example 5 The improving and therapeutic effects of CPX on ischemia-reperfusion acute kidney injury.

[0061] The modeling method of ischemia-reperfusion acute renal injury is the same as that in Example 2.

[0062] After anesthesia, the mice were placed in a constant temperature incubator. When the temperature reached 37°C, an opening was made at the angle between the left side of the mouse spine and the lower edge of the costal arch, about 2-3 cm and parallel to the spine. The opening exposed the left kidney to the outside of the body, kept the kidney moist, and the fascia and fat beside the renal artery and vein were separated with fine forceps, and the artery and vein were clamped with hemostatic forceps for 30 minutes. During the clamping process, the mice were placed in a 37°C incubator, and the left kidney and hemostatic forceps were put back into the body to avoid kidney damage. After 30 minutes, the hemostatic forceps were released and the wound was sutured. The mice were placed in a 37°C incubator for 1 hour to wake them up, and then returned to the cage (modeling group).

[0063] Experimental groups: divided into 4 groups, namely sham operation + solvent control group, sham operation + CPX group, IR-AKI + solvent control group, IR-AKI + CPX group, 5 mice in each group, a total of 20 mice.

[0064] Implementation method: 20 8-week-old male C57BL6J mice were taken and treated differently according to the groups. CPX was administered prophylactically, that is, the first administration was given 24 hours before the renal ischemia-reperfusion surgery. Specifically, the mice in the sham operation + solvent control group were first intraperitoneally injected with saline solvent control, and 24 hours later, the skin was surgically incised to expose the kidneys; the mice in the sham operation + CPX group were first intraperitoneally injected with 10 mg / kg of CPX, and 24 hours later, the skin was surgically incised to expose the kidneys; the mice in the IR-AKI + solvent control group were first intraperitoneally injected with saline solvent control, and 24 hours later, the IR-AKI surgery caused renal ischemia for 30 minutes; the mice in the IR-AKI + CPX group were first intraperitoneally injected with 10 mg / kg of CPX, and 24 hours later, the IR-AKI surgery caused renal ischemia for 30 minutes.

[0065] The corresponding solvent or CPX treatment was given 2 hours before surgery, 24 hours after surgery, and 48 hours after surgery. The animals were killed 72 hours after surgery, and blood was collected and fresh cortical kidney tissue was collected for subsequent lysis and collection of tissue protein. Figure 5 As shown:

[0066] A: Effect of ciclopirox olamine CPX on serum creatinine in the sham operation group and the acute kidney injury group.

[0067] B: Western blotting was used to detect the expression of KIM-1, a renal injury marker.

[0068] The results showed that in the IR-AKI mouse model, preventive administration of CPX 24 hours in advance could reduce serum creatinine (p = 0.05). Figure 5 As shown in A. In addition, protein level detection also showed that CPX could reduce the expression of KIM-1, a renal injury marker after IR-AKI ( Figure 5 The above results preliminarily suggest that CPX can alleviate renal injury caused by IR-AKI.

Claims

1. Application of ciclopirox olamine in the preparation of Nit2 agonists.

2. The use of ciclopirox olamine in the preparation of drugs for alleviating or treating acute kidney injury caused by ischemia-reperfusion.

Images