Application of AK4 protein or AK4 gene as target spot in screening medicine for treating chronic kidney disease

Abstract

The invention relates to the technical field of biological medicine, in particular to application of AK4 protein or AK4 gene as a target spot in screening of drugs for treating chronic kidney diseases. By constructing a renal fibrosis model, it is found that the expression level of AK4 protein in the renal fibrosis model is remarkably reduced, and the expression levels of FN, Collagen I, Vimentin and alpha-SMA protein are remarkably increased. The AK4 gene is overexpressed or knocked out in renal tubular epithelial cells, and the fact that the protein expression levels of FN, Vimentin and alpha-SMA can be remarkably increased by the cells with the AK4 gene knocked out is found, and the opposite situation is shown by the cells with the overexpressed AK4 gene. Therefore, the AK4 protein or the AK4 gene can be used as an action target spot for screening the medicine for treating the renal fibrosis; meanwhile, the AK4 protein or an activator of the AK4 gene can be used as a medicine for treating chronic kidney diseases. The invention provides a new way for the treatment of chronic kidney disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to the use of AK4 protein or AK4 gene as a target in screening drugs for the treatment of chronic kidney disease. Background Art

[0002] Adenylate kinase 4 (AK4) protein belongs to the adenylate kinase family, and is mainly located in the mitochondrial matrix, and is a key regulator of cell inflammation, oxidative stress and ATP levels. It has been found that AK4 can participate in the regulation of a variety of cancer diseases, and can accelerate the pathogenesis of serous ovarian cancer and HER2-positive breast cancer by promoting the invasion and proliferation of cancer cells, but its role and mechanism in renal fibrosis are not clear.

[0003] Renal fibrosis (RF) refers to the pathological process in which persistent renal injury or inflammation leads to excessive deposition of extracellular matrix in the kidney, inducing the loss of renal function. In view of the fact that the pathogenesis of renal fibrosis is very complex and has not been fully elucidated, in-depth study of the molecular mechanism of renal fibrosis is of great significance for developing effective intervention strategies and new drug targets for the prevention and treatment of chronic kidney disease (CKD). At present, no technology has been disclosed about the correlation between AK4 and renal injury and renal fibrosis diseases, and there is no report on whether AK4 has a protective effect on renal injury and renal fibrosis. Summary of the Invention

[0004] The purpose of the present invention is to provide the use of AK4 protein or AK4 gene as a target in screening drugs for the treatment of chronic kidney disease, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides the use of AK4 protein or AK4 gene in the preparation of drugs for preventing and / or treating chronic kidney disease.

[0007] Preferably, the chronic kidney disease includes renal fibrosis.

[0008] The present invention provides the use of an agonist of AK4 protein or AK4 gene in the preparation of drugs for preventing and / or treating chronic kidney disease.

[0009] Preferably, the drug achieves the purpose of preventing and / or treating chronic kidney disease by reducing the expression levels of FN protein, Collagen I protein, Vimentin protein and α-SMA protein.

[0010] More preferably, the chronic kidney disease includes renal fibrosis.

[0011] The present invention provides a drug for preventing and / or treating chronic kidney disease, and the drug comprises an agonist of AK4 protein or AK4 gene.

[0012] Preferably, the drug achieves the purpose of preventing and / or treating chronic kidney disease by reducing the expression levels of FN protein, Collagen I protein, Vimentin protein and α-SMA protein.

[0013] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0014] Preferably, the dosage form of the drug is injection, granule, powder or capsule.

[0015] More preferably, the chronic kidney disease includes renal fibrosis.

[0016] The present invention provides the use of AK4 protein or AK4 gene in screening drugs for treating chronic kidney disease.

[0017] The present invention provides a method for screening drugs for treating chronic kidney disease, comprising the step of detecting the expression level of AK4 gene in a subject before and after administration.

[0018] The present invention discloses the following technical effects:

[0019] By constructing a renal fibrosis model, the present invention finds that the expression level of AK4 protein is significantly reduced in the renal fibrosis model, while the expression levels of FN, Collagen I, Vimentin and α-SMA proteins are significantly up-regulated. Based on this, the present invention discovers for the first time that there is a reverse regulation between the expression level of AK4 protein or AK4 gene and renal fibrosis. In order to verify whether AK4 protein or AK4 gene can be used as a target for screening drugs for treating renal fibrosis, in the specific examples of the present invention, the AK4 gene is overexpressed or knocked out in renal tubular epithelial cells, and it is found that knocking out the AK4 gene in the cells will significantly increase the protein expression levels of FN, Vimentin and α-SMA, while overexpressing the AK4 gene in the cells will significantly reduce the protein expression levels of FN, Collagen I, Vimentin and α-SMA induced by TGF-β. Thus, it can be seen that AK4 protein or AK4 gene can be used as a target for screening drugs for treating renal fibrosis; at the same time, an activator of AK4 protein or AK4 gene can be used as a drug for treating chronic kidney disease, especially for treating renal fibrosis. The present invention provides a new way for the treatment of chronic kidney disease. Description of the Drawings

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0021] Figure 1 It shows the expression of AK4 protein in a mouse renal fibrosis model induced by UUO; wherein, A shows the pathological HE staining and Masson staining results of the kidney tissues of the blank control and UUO mice; B shows the tubular injury score; C shows the collagen volume fraction; D shows the protein expression levels of AK4, FN, Collagen I, Vimentin, and α-SMA in the kidney tissues of the control and UUO model mice; E shows the statistical chart of protein expression; * indicates comparison with the control group, *P<0.05, ***P<0.001;

[0022] Figure 2 It shows the expression of AK4 protein in a mouse renal fibrosis model induced by ADE; wherein, A shows the pathological HE staining and Masson staining results of the kidney tissues of the control and ADE model mice; B shows the tubular injury score; C shows the collagen volume fraction; D shows the protein expression levels of AK4, FN, Collagen I, Vimentin, and α-SMA in the kidney tissues of the control and ADE model mice; E shows the statistical chart of protein expression; * indicates comparison with the control group, *P<0.05, ***P<0.001;

[0023] Figure 3 It shows the expression of AK4 protein in a TGF-β-induced renal tubular epithelial cell fibrosis model; wherein, A shows the protein expression levels of AK4, FN, Collagen I, Vimentin, and α-SMA in cells of different groups; B shows the statistical chart of protein expression; * indicates comparison with the control group, *P<0.05, **P<0.01, ***P<0.001;

[0024] Figure 4To investigate the effect of AK4 gene knockout (AK4 KO) on the protein expression of FN, Vimentin, and α-SMA in the TGF-β-induced renal tubular epithelial cell fibrosis model. Among them, A shows the protein expression of FN, Vimentin, α-SMA, and AK4 in the TGF-β-induced renal tubular epithelial cell fibrosis detected by Western blot after AK4 KO; B is the statistical chart of protein expression; *P<0.05, **P<0.01, ***P<0.001;

[0025] Figure 5 To investigate the effect of AK4 gene overexpression (AK4 OE) on the protein expression of FN, Vimentin, and α-SMA in the TGF-β-induced renal tubular epithelial cell fibrosis model. Among them, A shows the protein expression of FN, Vimentin, α-SMA, and AK4 in HK-2 cells induced by TGF-β detected by Western blot before and after AK4 OE intervention; B is the statistical chart of protein expression; *P<0.05, **P<0.01;

[0026] Figure 6 To investigate the effect of AK4 gene overexpression (AK4 OE) on the protein expression of FN, Vimentin, and α-SMA in the UUO-induced mouse renal fibrosis model. Among them, A shows the protein expression of AK4, FN, Vimentin, and α-SMA in the mouse kidney tissue induced by UUO detected by Western blot before and after AK4 OE intervention; B is the statistical chart of protein expression; *P<0.05, **P<0.01, ***P<0.001. Detailed implementation manners

[0027] The various exemplary implementation manners of the present invention will be described in detail below. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0029] Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to those documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0030] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which will be apparent to those skilled in the art. Other embodiments obtained from the description of the present invention will be apparent to those skilled in the art. The description of the present invention and the examples are merely exemplary.

[0031] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] 1. Experimental method:

[0034] (1) Construction and grouping of UUO animal model (renal fibrosis model)

[0035] C57BL / 6 mice were randomly divided into two groups (n = 4): control group and UUO model group.

[0036] UUO model group: Mice were anesthetized by intraperitoneal injection of Zoletil 50 before surgery. After making an incision on the left side of the abdomen, the UUO model was established by ligating the upper and lower one-third of the fallopian tube with 4-0 suture.

[0037] Control group (sham operation group): Mice were anesthetized by intraperitoneal injection of Zoletil 50 before surgery. Only an incision was made on the left side of the abdomen to expose the ureter without ligation.

[0038] Fourteen days after modeling, blood was collected from the abdominal aorta, and mouse kidney tissues were collected.

[0039] (2) Histopathological detection of kidney tissues

[0040] HE staining: Kidney tissues of each group of mice were fixed with 10% formalin, dehydrated, cleared and embedded in paraffin using a paraffin slicer to prepare tissue paraffin sections. After the sections were dewaxed and hydrated conventionally, they were stained with hematoxylin and eosin, and after decolorization and clearing, they were sealed and preserved. The histopathological morphological changes of the kidney tissues were observed using an optical microscope.

[0041] Masson staining: Paraffin sections of kidney tissues from mice in each group were dewaxed, placed in hematoxylin solution for staining, differentiated with acidic ethanol, washed with water and blued back, then placed in Ponceau fuchsin solution and aniline blue solution for staining respectively. After decolorization and transparency, they were sealed with gum. The collagen deposition in kidney tissues was observed under an optical microscope.

[0042] (3) Immunoblotting detection

[0043] Kidney tissues were homogenized and ground in RIPA lysis buffer containing protease and phosphatase inhibitors. After grinding, they were allowed to stand on ice for 20 min, centrifuged at 4 °C and 12,000 rpm for 10 min, and the total protein supernatant was aspirated. Protein quantification was performed by the BCA method. The sample was supplemented with 5× loading buffer, vortexed and mixed well, and the protein was denatured in a 100 °C metal bath. The protein was separated by SDS-PAGE electrophoresis, transferred by wet method and blocked. It was incubated overnight with the corresponding primary antibody on a shaker at 4 °C, and then incubated with the corresponding secondary antibody at room temperature. The protein band images were collected using a full-automatic gel imaging system, and the gray values of the above proteins were semi-quantitatively analyzed using Image J software.

[0044] 2. Experimental results:

[0045] 1. To construct a UUO-induced mouse renal fibrosis model, the pathological results of the mouse kidneys after UUO modeling were detected and analyzed. It was found that compared with the control group of mice, the glomeruli of mice in the UUO model group were atrophied, the lumen was enlarged, the renal tubules were arranged disorderly, and the area of blue collagen fibers increased significantly ( Figure 1 A in). The tubular injury scores (Tubular injury score) and collagen volume fractions (Collagen volume fraction) of the kidneys of mice in each group are shown in Figure 1 B and C in. The results showed that compared with the control group of mice, the tubular injury scores and collagen volume fractions of mice in the UUO model group were significantly increased, indicating the successful construction of the UUO model.

[0046] Compared with the control group, the expression levels of FN, Collagen I, Vimentin, and α-SMA proteins in the kidney tissues of mice in the UUO model group were significantly up-regulated, while the expression level of AK4 protein was significantly decreased ( Figure 1 D-E in).

[0047] Example 2

[0048] 1. Experimental method:

[0049] (1) Construction of an adenine (ADE) animal model (renal fibrosis model) construction and grouping

[0050] C57BL / 6 mice were randomly divided into two groups (n = 4): a control group and an ADE model group.

[0051] ADE model group: After one week of adaptive feeding, the mice in the ADE model group were fed an adenine diet (containing 0.2% adenine + 0.01% fast green) to establish the model.

[0052] Control group: After one week of adaptive feeding, the mice in the control group were fed a diet (containing 0.01% fast green).

[0053] After 4 weeks of modeling, blood was collected from the abdominal aorta, and mouse kidney tissues were collected.

[0054] (2) Histopathological detection of kidney tissues

[0055] Same as Example 1.

[0056] (3) Western blot detection

[0057] Same as Example 1.

[0058] 2. Experimental results:

[0059] An ADE-induced mouse renal fibrosis model was constructed, and the pathological changes of the kidneys of mice in each group were detected. The HE staining results showed that the renal tubular structure of the mice in the control group was normal and arranged orderly; compared with the control group, the renal tissue structure of the mice in the ADE model group was disordered, with vacuole formation, a decrease in the number of glomeruli and atrophy, and dilation and irregular arrangement of renal tubules ( Figure 2 A in); the tubular injury scores and collagen volume fractions of the kidneys of mice in each group are shown in Figure 2 B and C in. The results showed that: compared with the control group mice, the tubular injury scores and collagen volume fractions of the ADE model group mice were significantly increased, indicating the successful construction of the ADE model

[0060] Compared with the control group, the expression levels of FN, Collagen I, Vimentin, and α-SMA proteins in the kidney tissues of ADE model mice were significantly up-regulated, while the expression level of AK4 protein was significantly decreased ( Figure 2 D-E in).

[0061] Example 3

[0062] 1. Experimental method:

[0063] (1) Construction and grouping of a TGF-β-induced renal tubular epithelial cell fibrosis model

[0064] The renal tubular epithelial HK-2 cells were divided into two groups: a control group and a TGF-β model group.

[0065] The method for establishing the TGF-β model is as follows: HK-2 cells in the logarithmic growth phase are taken and seeded in a six-well plate. After the cells adhere to the wall, they are stimulated with recombinant human TGF-β protein (Recombinant Human TGF-β1). The concentration of the recombinant human TGF-β protein (Recombinant Human TGF-β1) is 10 ng / mL, and the dosage is 1 mL. Induction is carried out once; Commercial product: PeproTech recombinant human TGF-β1 (HEK293 derived) / 100-21, PeproTech, SDS-PAGE gel and HPLC analysis purity ≥ 98%; It is carried out with a supporting special solution, and the final concentration is 10 ng / mL). After 48 h, the cell precipitates of each group are collected. At the same time, untreated renal tubular epithelial HK-2 cells are used as the control group to illustrate the success of the model establishment.

[0066] (2) Immunoblotting detection

[0067] Same as Example 1.

[0068] 2. Experimental results:

[0069] Compared with the control group cells, TGF-β (10 ng / mL) stimulation can significantly up-regulate the protein expression of FN, Collagen I, Vimentin and α-SMA in renal tubular epithelial HK-2 cells, and significantly reduce the expression level of AK4 protein ( Figure 3 in A and B).

[0070] Example 4

[0071] 1. Experimental method:

[0072] (1) Construction of AK4 KO stable transfected cells

[0073] CRISPR / Cas9 technology is used to construct AK4 KO stable transfected cells. The sgRNA targeting the AK4 gene is designed using the CRISPR design and construction tool (http: / / chopchop.cbu.uib.no / ) (the nucleotide sequence of the sgRNA is as shown in SEQ ID NO.1, specifically cagtttcaactcaccatca). Through the use of PEI, the Cas9-sgRNA plasmid and the packaging plasmids (psPAX2 and pMD2.G) are used for lentivirus packaging in 293T cells. The packaged lentivirus is transfected into HK-2 cells for 24 h, and AK4 KO stable transfected cells are obtained by screening with puromycin.

[0074] (2) Cell grouping

[0075] The renal tubular epithelial HK-2 cells were divided into four groups: control group, TGF-β model group, AK4 KO group, and AK4 KO + TGF-β group.

[0076] The control group was renal tubular epithelial HK-2 cells without any treatment.

[0077] The TGF-β model group was the TGF-β model cells obtained in Example 3.

[0078] The AK4 KO group was the AK4 KO stable transfected cells obtained in the above steps.

[0079] The AK4 KO + TGF-β group was the AK4 KO stable transfected cells obtained in the above steps induced by TGF-β, and the TGF-β induction method was the same as that in Example 3.

[0080] (3) Immunoblotting detection

[0081] Same as Example 1.

[0082] 2. Experimental results:

[0083] To study whether AK4 has a key regulatory role in renal fibrosis, the present invention constructed HK-2 cells with AK4 gene knockout (AK4KO), and the results are shown in A and B of Figure 4 . The results showed that compared with the control cells, the AK4 KO group significantly increased the protein expression levels of FN, Vimentin, and α-SMA; combined with TGF-β stimulation, AK4 KO further significantly increased the protein expression levels of FN, Vimentin, and α-SMA induced by TGF-β. Thus, it can be seen that AK4 gene knockout (AK4KO) can significantly enhance TGF-β-induced renal tubular epithelial cell fibrosis.

[0084] Example 5

[0085] 1. Experimental method:

[0086] (1) Construction of AK4 OE stable transfected cells

[0087] Order the AK4 OE lentiviral plasmid, and use PEI to package the Cas9-sgRNA plasmid and packaging plasmids (psPAX2 and pMD2.G) into lentivirus in 293T cells. Transfect the packaged lentivirus into HK2 cells for 24 h, and screen with puromycin to obtain AK4 OE stable transfected cells.

[0088] (2) Cell grouping

[0089] The renal tubular epithelial HK-2 cells were divided into four groups: control group, TGF-β model group, AK4 OE group, and AK4 OE + TGF-β group.

[0090] The control group was renal tubular epithelial HK-2 cells without any treatment.

[0091] The TGF-β model group was the TGF-β model cells obtained in Example 3.

[0092] The AK4 OE group was the AK4 OE stable transfected cells obtained in the above steps.

[0093] The AK4 OE + TGF-β group was to induce TGF-β in the AK4 OE stable transfected cells obtained in the above steps, and the TGF-β induction method was the same as that in Example 3.

[0094] (3) Immunoblot detection

[0095] Same as Example 1.

[0096] 2. Experimental results:

[0097] To further verify the key regulatory role of AK4 protein in fibrosis, in this example, HK-2 cells with overexpression of AK4 gene (AK4 OE) were constructed synchronously. The results are shown in Figure 5 A and B as follows. The results showed that compared with the cells in the TGF-β group, AK4 OE would further significantly reduce the protein expression levels of FN, Collagen I, Vimentin, and α-SMA induced by TGF-β. Thus, overexpression of AK4 gene (AK4 OE) could significantly inhibit the fibrosis of renal tubular epithelial cells induced by TGF-β.

[0098] Example 5

[0099] (1) Construction of AK4 gene overexpressing mice

[0100] Order the adeno-associated virus type 9 (AAV9) with overexpression of AK4 gene, and infect the renal tissue of normal mice by renal pelvic injection with AAV9 type adenovirus with overexpression of AK4 gene (AAV9-AK4) and control AAV9 type adenovirus (AAV9-NC) respectively to obtain AK4 gene overexpressing mice.

[0101] (2) Animal grouping

[0102] The mice were divided into four groups: control adenovirus (Control-AAV9-NC) group, control adenovirus + UUO (UUO-AAV9-NC) group, AK4 gene overexpression (Control-AAV9-AK4) group, and AK4 gene overexpression + UUO (UUO-AAV9-AK4) group.

[0103] Control-AAV9-NC group: Normal C57BL / 6 mice were injected with control AAV9 adenovirus (AAV9-NC) into the renal pelvis;

[0104] Control adenovirus + UUO (UUO-AAV9-NC) group: Four weeks after normal C57BL / 6 mice were injected with control AAV9 adenovirus (AAV9-NC) into the renal pelvis, UUO surgical modeling was performed;

[0105] AK4 gene overexpression (Control-AAV9-AK4) group: Normal C57BL / 6 mice were injected with AAV9 adenovirus (AAV9-AK4) overexpressing the AK4 gene into the renal pelvis;

[0106] AK4 gene overexpression + UUO (UUO-AAV9-AK4) group: Four weeks after normal C57BL / 6 mice were injected with AAV9 adenovirus (AAV9-AK4) overexpressing the AK4 gene into the renal pelvis, UUO surgical modeling was performed;

[0107] (3) Western blot detection

[0108] The Western blot detection method was the same as that in Example 1.

[0109] 2. Experimental results:

[0110] To verify the key regulatory role of AK4 in renal fibrosis at the in vivo animal level, in this example, mice overexpressing the AK4 gene (AAV9-AK4) were further constructed, and UUO modeling was performed in parallel. Protein immunoblotting was used to detect the expression level of AK4 protein in normal mice and mice overexpressing the AK4 gene (Control-AAV9-AK4), and to detect the protein expression of FN, Vimentin, and α-SMA in UUO modeling and in mice overexpressing the AK4 gene in UUO mice (UUO-AAV9-AK4). Compared with the Control-AAV9-NC group, overexpression of the AK4 gene (Control-AAV9-AK4) had no significant effect on the protein expression of FN, Vimentin, and α-SMA in the renal tissue of mice (P>0.05); compared with the UUO-AAV9-NC group, overexpression of the AK4 gene (UUO-AAV9-AK4) could significantly reduce the high protein expression of FN, Vimentin, and α-SMA in the renal tissue of UUO mice (P<0.05, P<0.001), as shown in Figure 6 A - B in. The above results indicate that in UUO-induced renal fibrosis in mice, overexpression of the AK4 gene can significantly reduce the protein expression levels of FN, Vimentin, and α-SMA in the renal tissue of UUO mice. Thus, overexpressing the AK4 gene can improve and treat renal fibrosis.

[0111] In summary, the AK4 protein or AK4 gene can be used as a target for screening drugs for preventing and / or treating renal fibrosis, and can also evaluate the effect of treating renal fibrosis by detecting the expression level of the AK4 gene in vivo.

[0112] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of AK4 protein or AK4 gene in the preparation of a medicament for preventing and / or treating chronic kidney disease.

2. The application according to claim 1, wherein The chronic kidney disease includes renal fibrosis.

3. Use of an agonist of AK4 protein or AK4 gene in the preparation of a medicament for preventing and / or treating chronic kidney disease.

4. The application according to claim 4, characterized in that, The medicament achieves the purpose of preventing and / or treating chronic kidney disease by reducing the expression levels of FN protein, Collagen I protein, Vimentin protein and α-SMA protein.

5. A drug for preventing and / or treating chronic kidney disease, characterized in that, The medicament includes an agonist of AK4 protein or AK4 gene.

6. The drug according to claim 5, characterized in that, The medicament achieves the purpose of preventing and / or treating chronic kidney disease by reducing the expression levels of FN protein, Collagen I protein, Vimentin protein and α-SMA protein.

7. The medicament according to claim 5, characterized in that, The medicament further includes pharmaceutically acceptable excipients.

8. The drug according to claim 7, characterized in that, The dosage form of the medicament is injection, granule, powder or capsule.

9. Use of AK4 protein or AK4 gene in screening a medicament for treating chronic kidney disease.

10. A method for screening drugs for treating chronic kidney disease, characterized in that, It includes the step of detecting the expression level of AK4 gene in a subject before and after administration.

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