Use of tiki2 as a target in the preparation of a medicament for preventing and / or treating kidney disease

Abstract

The application discloses application of Tiki2 as a target point in preparation of a medicine for preventing and / or treating kidney diseases, and relates to the technical field of biological medicines.The medicine can promote expression of Tiki2 protein.The inventors find that Tiki2 protein can be used as a treatment target point of acute kidney injury, and increasing expression of Tiki2 protein in renal tubular epithelial cells can treat acute kidney injury.A medicine developed according to the Tiki2 protein can fundamentally slow down and reverse the course of kidney diseases, especially the course of acute kidney injury.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to application of Tiki2 as a target in preparation of a drug for preventing and / or treating kidney diseases. BACKGROUND

[0002] Acute kidney injury (AKI) is a clinical syndrome caused by rapid decline in renal function due to various causes, mainly manifested as elevated serum creatinine and / or reduced urine output. About 5% of hospitalized patients may develop AKI, and the incidence of AKI in intensive care units is as high as 30%. Mild patients only have mild renal damage, and severe patients can have severe renal failure. Since there is still no specific drug for treating AKI, the mortality rate of patients with AKI remains high. AKI is a critical and severe condition in kidney disease. AKI treatment mainly includes early identification and correction of reversible causes, maintenance of internal environment stability, nutritional support, prevention and treatment of complications, and renal replacement therapy. Active treatment of the primary disease, timely detection of risk factors leading to acute renal tubular necrosis and removal are the key to preventing and treating AKI.

[0003] In view of this, the present application is proposed. SUMMARY

[0004] The purpose of the present application is to provide a new application of Tiki2 as a target in the preparation of a drug for preventing and / or treating kidney diseases, thereby providing an effective therapeutic target for specific treatment of AKI.

[0005] The present application is realized in this way:

[0006] In a first aspect, the present application provides an application of Tiki2 as a target in the preparation of a drug for preventing and / or treating kidney diseases, and the drug can promote the expression of Tiki2 protein.

[0007] The WNT signaling pathway plays an important role in the occurrence and development of kidney diseases, and Tiki2 protein is a class of WNT signaling pathway inhibiting molecules. The inventors found that Tiki2 gene is highly expressed in renal proximal tubular epithelial cells, and its expression level in the tubular epithelial cells is negatively correlated with the severity of acute kidney injury. Specific knockout of Tiki2 gene in mouse renal proximal tubular epithelial cells can aggravate the symptoms of acute kidney injury induced by unilateral ischemia-reperfusion injury (UIRI) in mice. The inventors used the method of fluid dynamics to inject Tiki2 expression plasmid into the tail vein, which can relieve the symptoms of acute kidney injury induced by UIRI operation. These research results show that Tiki2 protein can be used as a target for preventing and / or treating kidney diseases, especially as a therapeutic target for acute kidney injury, and increasing the expression of Tiki2 protein in renal tubular epithelial cells may treat acute kidney injury.

[0008] The drug developed for the Tiki2 protein will have the potential to fundamentally slow down and reverse the course of kidney disease, especially the course of acute kidney injury.

[0009] In a preferred embodiment of the application, the kidney disease is acute kidney injury. The acute kidney injury includes acute kidney injury caused by ischemia, infection, drug toxicity, etc.

[0010] In an alternative embodiment, the acute kidney injury is acute kidney injury induced by renal ischemia-reperfusion.

[0011] In an alternative embodiment, the drug can promote the expression of Tiki2 protein in renal tubular epithelial cells.

[0012] In a preferred embodiment of the application, the application includes the effect of reducing renal tubular injury.

[0013] In an alternative embodiment, the expression of a kidney injury molecule is reduced.

[0014] In an alternative embodiment, the expression of the kidney injury molecule Kim1 is reduced.

[0015] In a second aspect, the application also provides a Tiki2 protein up-regulated expression promoter for use in the preparation of a therapeutic drug for kidney disease.

[0016] Any agent or drug that can promote the up-regulated expression of Tiki2 protein can be used as the promoter.

[0017] In an alternative embodiment, the kidney disease is acute kidney injury;

[0018] In an alternative embodiment, the acute kidney injury is acute kidney injury induced by renal ischemia-reperfusion.

[0019] In an alternative embodiment, the promoter can promote the up-regulated expression of Tiki2 protein in renal tubular epithelial cells. The up-regulated expression refers to a slight increase, an increase or a significant increase in the expression level of Tiki2 protein in the renal tubular epithelial cells of a subject with kidney injury disease, compared with the normal subject without kidney injury disease.

[0020] In a third aspect, the application also provides a recombinant vector for treating acute kidney injury, comprising a nucleotide sequence encoding Tiki2 protein, wherein the nucleotide sequence encoding Tiki2 protein is shown in SEQ ID NO. 1.

[0021] The recombinant vector is pCDH-HA-Tiki2.

[0022] In a fourth aspect, the present application further provides a medicament for treating acute kidney injury, which comprises the recombinant vector as described above.

[0023] In a fifth aspect, the present application further provides a use of a recombinant vector in the preparation of a medicament for treating acute kidney injury, which comprises a nucleotide sequence capable of encoding a Tiki2 protein, wherein the nucleotide sequence capable of encoding a Tiki2 protein is as shown in SEQ ID NO. 1. The recombinant vector is pCDH-HA-Tiki2.

[0024] In a preferred embodiment of the present application, the dosage form of the medicament is a tablet, a capsule, a suspension, a solution, an emulsion, a powder, a granule, an injection, a freeze-dried powder injection, a liniment, a coating, a coating film, an ointment, a lotion, a suppository, an aerosol, a spray, a powder spray, an ointment, a plaster, a cataplasm or a patch.

[0025] In an alternative embodiment, the dosage form of the medicament is an injection.

[0026] In a preferred embodiment of the present application, the medicament further comprises a pharmaceutically acceptable additive or excipient.

[0027] In an alternative embodiment, the pharmaceutically acceptable additive or excipient is selected from one or more of the following: a solvent, a buffer, an emulsifier, a suspending agent, a disintegrant, a disintegrating agent, a dispersing agent, a binder, an excipient, a stabilizer, a chelating agent, a diluent, a gelling agent, a preservative, a wetting agent, a lubricant, an absorption delaying agent, a flavoring agent, a sweetening agent, a colorant and a liposome.

[0028] In an alternative embodiment, the medicament further comprises a combination drug, which is for example selected from a fentanyl. The fentanyl includes (-)-THP, (+)-THP and (±)-THP. Studies have shown that fentanyl has a good protective effect on cisplatin-induced kidney injury in mice.

[0029] In a preferred embodiment of the present application, the medicament further comprises a pharmaceutically acceptable salt.

[0030] Suitable pharmaceutically acceptable salts include salts of inorganic acids such as hydrochloric acid, hydrobromic acid and sulfuric acid, and salts of organic acids such as methanesulfonic acid, fumaric acid, maleic acid, acetic acid, oxalic acid, succinic acid, malic acid, tartaric acid, mandelic acid, lactic acid, citric acid, glutamic acid, acetylsalicylic acid, nicotinic acid, aminobenzoic acid, d-acid, hippuric acid, phosphoric acid and aspartic acid. The preferred salt is a hydrochloride or a phosphate.

[0031] Excipients may include, for example: water, ethanol, 2-propanol, glycerol, ethylene glycol, propylene glycol, polyethylene glycol, polypropylene glycol, glucose, fructose, lactose, sucrose, dextran, molasses, starch, modified starch, gelatin, sorbitol, inositol, mannitol, microcrystalline cellulose, methylcellulose, carboxymethyl cellulose, cellulose acetate, shellac, cetyl alcohol, polyvinylpyrrolidone, paraffin wax, wax, natural and synthetic rubber, gum arabic, alginate, dextran, saturated and unsaturated fatty acids, stearic acid, magnesium stearate, zinc stearate, stearic acid Glycerides, sodium lauryl sulfate, edible oils, sesame oil, coconut oil, peanut oil, soybean oil, lecithin, sodium lactate, polyoxyethylene fatty acid esters and polyoxypropylene fatty acid esters, sorbitan fatty acid esters, sorbic acid, benzoic acid, citric acid, ascorbic acid, tannic acid, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, magnesium oxide, zinc oxide, silicon dioxide, titanium dioxide, magnesium sulfate, zinc sulfate, calcium sulfate, potassium carbonate, calcium phosphate, dicalcium phosphate, potassium bromide, potassium iodide, talc, kaolin, pectin, crosspovidone, agar, and bentonite.

[0032] In a preferred embodiment of the present invention, the viral vector is selected from any one of retroviral vectors, adenovirus vectors, adeno-associated virus vectors, and lentiviral vectors.

[0033] In one alternative implementation, the viral vector is selected from adeno-associated virus vectors;

[0034] In one alternative implementation, the adeno-associated virus is selected from any one or more of AAV1 to 13.

[0035] The present invention has the following beneficial effects:

[0036] This invention has discovered a novel therapeutic target for acute kidney injury (AKI)—Tiki2. Experiments have shown that Tiki2 knockout significantly exacerbates AKI symptoms induced by unilateral ischemia-reperfusion injury (UIRI) in mice. Tail vein injection of a Tiki2 expression plasmid using a hydrodynamic approach can alleviate AKI symptoms induced by UIRI surgery. These results indicate that Tiki2 protein can serve as a therapeutic target for AKI, and increasing Tiki2 protein expression in renal tubular epithelial cells may have therapeutic potential for AKI. Drugs developed targeting Tiki2 protein may fundamentally slow down and reverse the progression of kidney diseases, especially AKI. Attached Figure Description

[0037] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be regarded as a limitation on the scope. Other related drawings can also be obtained by those of ordinary skill in the art without creative labor on the basis of these drawings.

[0038] Figure 1 Figure for kidney proximal tubular epithelial cell section results of Tiki2 gene knockout mouse model after sham or UIRI;

[0039] Figure 2 Figure for left kidney PAS staining results and score statistics of 8-12 week-old wild type (Tiki2+ / +) and homozygous (Tiki2- / -) knockout mice after establishing a unilateral kidney ischemia-reperfusion model;

[0040] Figure 3 Figure for immunofluorescence analysis and qPCR quantitative analysis of kidney tubular epithelial cell injury molecule Kim1 of 8-12 week-old wild type (Tiki2+ / +) and homozygous (Tiki2- / -) knockout mice after establishing a unilateral kidney ischemia-reperfusion model;

[0041] Figure 4 Figure for kidney proximal tubular epithelial cell section and injury score statistics of specific Tiki2 knockout mice after sham operation and UIRI operation;

[0042] Figure 5 Figure for immunofluorescence analysis and qPCR quantitative analysis of kidney tubular epithelial cell injury molecule Kim1 of specific Tiki2 knockout mice after sham operation and UIRI operation;

[0043] Figure 6 Figure for left kidney PAS staining results of a unilateral kidney ischemia-reperfusion model after Tiki2 overexpression;

[0044] Figure 7 Figure for immunofluorescence analysis and qPCR quantitative analysis of kidney tubular epithelial cell injury molecule Kim1 of a unilateral kidney ischemia-reperfusion model after Tiki2 overexpression;

[0045] Figure 8 Figure for the results of Western blotting experiment to verify the protein expression level of HA-Tiki2 in mouse kidney. DETAILED DESCRIPTION

[0046] Reference will now be made in detail to embodiments of the application, one or more examples of which are described hereinbelow. Each example is provided as an explanation and not a limitation of the application. Indeed, it will be apparent to one of ordinary skill in the art that numerous modifications and variations of the present application are possible in light of the above teachings without departing from the scope or spirit of the application. For example, features illustrated or described as part of one embodiment can be used with another embodiment to yield a still further embodiment.

[0047] Practicing the present application will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J. E. Coligan et al., eds., 1991), each of which is incorporated herein by reference in its entirety.

[0048] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. If specific conditions are not indicated in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If the manufacturers of the reagents or instruments are not indicated, the conventional products that can be purchased in the market are adopted.

[0049] The features and performances of the present application are further described in detail below in combination with the embodiments.

[0050] Embodiment 1

[0051] In this embodiment, a Tiki2 gene knockout mouse model is constructed by the method of homologous recombination (the mouse model is donated by the laboratory of Professor He Xi of Harvard Medical School), and the DNA sequence coding the bacterial galactosidase gene (LacZ) is used to replace the coding region of the Tiki2 gene of the mouse, so that the Tiki2 gene is knocked out, and the Tiki2 promoter is used to drive the LacZ gene to express galactosidase, and the expression of the Tiki2 gene can be indicated by X-gal staining. The kidneys of 8-12-week-old Tiki2 heterozygotes (+ / -) mice are subjected to sham operation (Sham) or unilateral ischemia-reperfusion operation (UIRI), and the kidneys are taken out 7 days after the operation, and are subjected to OCT embedding, frozen section preparation, X-gal staining and section observation.

[0052] Reference of section results Figure 1 As shown in the figure, the left side (Sham) is the treatment group subjected to the kidney sham operation, and the right side is the unilateral kidney ischemia-reperfusion operation (UIRI, I / R-d7). It is found that the Tiki2 protein is highly expressed in the proximal tubular epithelial cells of the mouse kidney, and the expression of Tiki2 in the renal tubular epithelial cells is significantly reduced after ischemia-reperfusion.

[0053] Embodiment 2

[0054] This embodiment proves that reducing the expression of the Tiki2 gene of the mouse can aggravate the symptoms of acute kidney injury induced by the UIRI operation.

[0055] 8-12-week-old wild-type (Tiki2+ / +) and homozygous (Tiki2- / -) knockout mice are taken to establish a unilateral kidney ischemia-reperfusion model (left kidney ischemia for 30 min, and the body temperature of the mouse is maintained at 37±0.2℃), and the mouse left kidney is taken out for fixation, embedding, sectioning, dewaxing and PAS staining on the seventh day after the operation, and the damage degree of the damaged tubules is scored and counted by histological quantification.

[0056] The results show that the renal tubular damage of the homozygote is significantly increased compared with the wild-type mouse Figure 2 ).

[0057] The expression of the kidney injury molecule Kim1 was detected by immunofluorescence analysis and qPCR quantitative analysis, and the results showed that the expression of Kim1 in homozygous mice was significantly higher than that in wild-type mice Figure 3

[0058] Example 3

[0059] In this example, a Tiki2 conditional knockout mouse model (the mouse model was donated by Professor He Shi's laboratory of Harvard Medical School) was constructed, and was crossed with a kidney tubular epithelial cell-specific Cre transgenic mouse to specifically knockout Tiki2 in the renal proximal tubular epithelial cells. Control mice (Tiki2fl / fl) and Tiki2 knockout mice (Tiki2fl / fl, GGT-Cre) of 8-12 weeks of age were subjected to sham operation and UIRI operation, respectively, according to the method shown in Example 2, and the samples were taken 7 days after the operation and the injured tubules were scored and counted.

[0060] The results showed that specific knockout of Tiki2 in the renal proximal tubular epithelial cells significantly aggravated the symptoms of kidney injury in mice Figure 4 ) and increased the expression of kidney tubular epithelial cell injury molecules caused by UIRI in mice Figure 5

[0061] Example 4

[0062] Wild-type mice of 7-8 weeks of age were divided into two groups, and were injected with pCDH-GFP (a commercially available plasmid) and pCDH-HA-Tiki2 plasmid (DNA encoding HA-TIKI2 fusion protein was inserted into the multiple cloning site of pCDH-GFP using molecular biology methods) (the nucleotide sequence encoding HA-TIKI2 is shown as SEQ ID NO. 1) via tail vein injection, respectively. The unilateral kidney ischemia-reperfusion model was established 24 hours after plasmid injection (left kidney ischemia for 30 minutes, and the body temperature of the mice was maintained at 37±0.2℃), and the samples were taken 7 days after the operation. The kidney injury was observed and quantitatively analyzed. The protein expression of HA-Tiki2 in the mouse kidney was verified by Western blotting.

[0063] It can be seen that overexpression of Tiki2 alleviates kidney tubular epithelial cell injury caused by UIRI in mice. Figure 6

[0064] It can be seen that overexpression of Tiki2 reduces the expression of kidney tubular epithelial cell injury molecules caused by UIRI in mice. Therefore, injection of Tiki2 expression plasmid can significantly alleviate acute kidney injury caused by ischemia-reperfusion. Figure 7

[0065] It can be seen that overexpression of Tiki2 reduces the expression of kidney tubular epithelial cell injury molecules caused by UIRI in mice. Therefore, injection of Tiki2 expression plasmid can significantly alleviate acute kidney injury caused by ischemia-reperfusion. Figure 8 ​​​​It can be seen that HA-Tiki2 was successfully expressed in the kidney of mice. The numbers in the figure represent the mouse number. It can be seen that the expression of HA-TIKI2 in the kidney of 3 mice can be detected by HA antibody.

[0066] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. The present application can be variously changed and modified by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The application of a recombinant vector in the preparation of a drug for treating acute kidney injury, characterized in that, The recombinant vector includes a nucleotide sequence encoding the Tiki2 protein, wherein the nucleotide sequence encoding the Tiki2 protein is shown in SEQ ID NO.1, and the acute kidney injury is acute kidney injury induced by renal ischemia-reperfusion.

2. The application according to claim 1, characterized in that, The recombinant vector is pCDH-HA-Tiki2.

3. The application according to claim 1, characterized in that, The drug is in the form of an injection or a lyophilized powder for injection.

Images