SgRNA for knocking out tmem91 gene and application thereof

Abstract

The application discloses sgRNA for knocking out a TMEM91 gene and application thereof, and relates to the technical field of biological medicines.The sequence of the sgRNA is shown in SEQ ID No.1 or SEQ ID No.2.A gene knockout mouse is constructed according to the TMEM91, and experimental results prove that the sequence can significantly silence the expression of TMEM91.Compared with wild-type mice, the TMEM91 mRNA of the knockout TMEM91 mouse is completely removed, and the TMEM91 gene knockout mouse provides new evidence for the biological function of TMEM91 at the in-vivo level.The application proves for the first time that inhibiting the expression of the TMEM91 gene can enhance kidney injury of the mouse, thereby proving that the TMEM91 is a new target for treating kidney injury, and the application is beneficial to further studying the mechanism of occurrence and development of kidney injury related diseases caused by low expression of the TMEM91.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an sgRNA for knocking out the TMEM91 gene and its application. Background Technology

[0002] TMEM91 (Transmembrane Protein 91) is a transmembrane protein whose function is not fully elucidated. Its encoding gene is located in the 1q21.3 region of human chromosome and is expressed in various tissues, particularly showing high transcriptional levels in the nervous system, retina, and endocrine-related organs. Existing research suggests that TMEM91 may be involved in intracellular vesicle transport, membrane dynamics regulation, or signal transduction processes, and bioinformatics analysis indicates a potential link to autophagy-related pathways. Due to the lack of gene knockout animal models, the physiological function and pathological significance of TMEM91 remain to be explored in depth. Constructing mouse models with TMEM91 deletion or mutation will provide a crucial tool for revealing its biological role and potential disease associations.

[0003] There are currently no reported TMEM91 gene knockout mouse models, their construction methods, or their applications. Summary of the Invention

[0004] To address the technical problems existing in the prior art, this invention provides an sgRNA for knocking out the TMEM91 gene and its application. The technical solution is as follows:

[0005] An sgRNA for knocking out the TMEM91 gene, the sequence of which is shown in SEQ ID No. 1 or SEQ ID No. 2.

[0006] The application of the sgRNA in the method of constructing the TMEM91 gene knockout mouse model.

[0007] A method for constructing a TMEM91 gene knockout mouse model, the method comprising:

[0008] (1) Provide the sgRNA described above;

[0009] (2) Using CRISPR / Cas9 gene editing technology, the sgRNA described in step (1) is injected into mouse zygote cells, so that the TMEM91 gene is knocked out in the cells;

[0010] (3) Cultivate the cells from step (2) to obtain the TMEM91 gene knockout mouse model.

[0011] The application of the sgRNA or the method described herein in the screening of drugs targeting the TMEM91 gene or drugs for treating kidney injury.

[0012] Optionally, the kidney injury includes acute kidney injury.

[0013] Application of reagents that enhance TMEM91 gene expression in the preparation of drugs for treating kidney injury.

[0014] Optionally, the kidney injury includes acute kidney injury.

[0015] A pharmaceutical composition for treating kidney injury, the pharmaceutical composition comprising an agent that enhances the expression of the TMEM91 gene or an agent that enhances the activity of the TMEM91 protein.

[0016] Optionally, the kidney injury includes acute kidney injury.

[0017] The beneficial effects of the technical solutions provided in the embodiments of the present invention include at least the following:

[0018] This invention constructed a gene knockout mouse for TMEM91, and experimental results confirmed that the sequence of this invention can significantly silence the expression of TMEM91. Examination of kidney and brain tissues from the gene knockout mice showed that, compared with wild-type mice, TMEM91 mRNA was completely eliminated in the knockout mice. The TMEM91 gene knockout mice provide new in vivo evidence for the biological function of TMEM91.

[0019] This invention is the first to experimentally demonstrate that inhibiting TMEM91 gene expression can enhance kidney injury in mice, thus proving that TMEM91 is a potential novel target for treating kidney injury, which is beneficial for further research on the pathogenesis and development mechanism of kidney injury-related diseases caused by low TMEM91 expression. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1A This is a schematic diagram of CRISPR / Cas9 gene targeting provided in Embodiment 1 of the present invention;

[0022] Figure 1B This is the target band image obtained by electrophoresis after PCR amplification provided in Example 1 of the present invention;

[0023] Figure 1C This is a graph showing the TMEM91 mRNA detection results in TMEM91-KO mice provided in Example 1 of this invention;

[0024] Figure 2A This is a graph showing the serum creatinine and blood urea nitrogen levels of TMEM91-KO mice provided in Example 2 of this invention;

[0025] Figure 2B This is a PAS staining image of TMEM91-KO mice after kidney injury, provided in Example 2 of this invention. Detailed Implementation

[0026] The technical solution of the present invention will now be described with reference to the accompanying drawings.

[0027] In embodiments of the present invention, words such as "exemplarily," "for example," etc., are used to indicate that something is an example, illustration, or description. Any embodiment or design described as "exemplary" in the present invention should not be construed as being more preferred or advantageous than other embodiments or designs. Specifically, the use of the word "exemplary" is intended to present the concept in a concrete manner. Furthermore, in embodiments of the present invention, the meaning expressed by "and / or" can be both, or either one.

[0028] Given that there is no existing TMEM91 gene knockout mouse model and its application, and no reports on its animal models, this invention aims to address the current situation where there is no TMEM91 animal model and its application.

[0029] In view of the shortcomings of the prior art, the purpose of this invention is to provide a TMEM91 gene knockout mouse model, its construction method and application, in order to solve the problem of the lack of gene knockout animal models and applications.

[0030] The CRISPR / Cas9 gene editing system originates from the adaptive immune mechanisms of bacteria and archaea, used to defend against viral and plasmid invasions. Its core consists of the Cas9 nuclease and programmable single-stranded guide RNA (sgRNA). By designing the sgRNA to complement the target DNA sequence, Cas9 can recognize PAM sequences (usually 5′-NGG-3′) at specific locations and induce DNA double-strand breaks. Then, it can utilize the cell's own non-homologous end joining (NHEJ) or homologous directed repair (HDR) pathways to achieve gene knockout, insertion, or precise editing. With its advantages of simple design, high editing efficiency, low cost, and applicability to various biological systems, CRISPR / Cas9 has become the mainstream technology for constructing gene-modified animal models (such as gene knockout mice), and is especially suitable for direct manipulation in fertilized eggs to quickly obtain precise disease models without foreign sequence residues.

[0031] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0032] Unless otherwise specified, the experimental methods described in the following embodiments are conventional experimental methods well known to those skilled in the art, and are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Where specific conditions are not specified in the experimental methods, they are generally operated under conventional conditions.

[0033] Unless otherwise specified, all materials and reagents described in the following examples are commercially available.

[0034] Example 1: Construction of TMEM91-KO mice

[0035] 1. Material preparation

[0036] Mouse strain: C57BL / 6J, purchased from Jicui Pharmaceutical Co., Ltd.

[0037] Surrogate female mice: of the same strain C57BL / 6J.

[0038] Tools, enzymes, and reagents: Cas9 mRNA in vitro transcription kit, sgRNA purification kit, Eazy-Taq, PMD18-T vector, etc., were all commercially available analytical grade.

[0039] 2. Construction Method

[0040] (2.1) Target design and sgRNA preparation

[0041] 1. Log in to http: / / crispr.mit.edu / and select two high-resolution target sites in the second exon region shared by all transcripts of the mouse TMEM91 gene. The construction strategy is described in [link to strategy]. Figure 1A .from Figure 1A As can be seen, the guide RNA sequences before and after the second exon are designed to achieve gene editing.

[0042] Ultimately, this invention designed and obtained the following two sgRNA1s:

[0043] -sgRNA1: 5'-TGTCCAGAGCCCATAGGCAT-3' (SEQ ID No. 1);

[0044] -sgRNA2: 5'-TGCGCTGGTCTCACTCCAAA-3' (SEQ ID No. 2);

[0045] 2. After synthesizing primers for the above sgRNA sequence, double-stranded DNA templates were obtained by PCR amplification. sgRNA1 and sgRNA2 were transcribed using the T7 in vitro transcription kit, or sgRNA1 and sgRNA2 could be synthesized directly.

[0046] Simultaneously, the Cas9 expression plasmid (available from Beijing Bomei Gene Technology Co., Ltd.) was linearized, and Cas9 mRNA was obtained by in vitro transcription.

[0047] 3. Purify sgRNA1, sgRNA2 and Cas9 mRNA to injection grade (OD260 / 280=1.9-2.1) using an RNA purification kit, and aliquot and store at -80℃.

[0048] (2.2) Microinjection and embryo transfer

[0049] 1. Superovulation in female mice: 6-8 week old female mice were intraperitoneally injected with 10 IU of PMSG, followed by 10 IU of hCG 46-48 hours later.

[0050] 2. In vitro fertilization: Oocytes from the ampulla of the fallopian tube of superovulated female mice were fertilized with sperm from the epididymal tail of male mice on the same day in HTF culture medium for 4-6 hours to obtain single-cell fertilized eggs.

[0051] 3. Microinjection: Mix sgRNA1 and sgRNA2 (25 ng / µL each) with Cas9 mRNA (50 ng / µL) at a volume ratio of 1:1:2; using the Eppendorf FemtoJet micromanipulation system, inject approximately 2 pL of the mixture into the cytoplasm of the fertilized egg.

[0052] 4. Embryo transfer: After injection, surviving embryos are cultured at 37°C and 5% CO2 for 1-2 hours. 20-25 morphologically normal embryos are selected and transferred to the unilateral oviduct of a 0.5 dpc pseudopregnant female mouse. After sealing, the mouse is fed as usual.

[0053] (2.3) Model breeding and genotype identification

[0054] 1. F0 generation: 19-20 days after transplantation, the pups are born, their toes are clipped, and they are numbered as F0 generation.

[0055] 2. Genomic DNA extraction (tail or toe):

[0056] - Lysis buffer: 100mM Tris-HCl pH8.0, 5mM EDTA, 0.5% SDS, 200mM NaCl, proteinase K final concentration 100µg / mL, incubate overnight at 55℃;

[0057] - Phenol / chloroform extraction, ethanol precipitation, and dissolution in 30 µL TE.

[0058] 3. PCR identification:

[0059] Primer:

[0060] F: 5'-CCAGAATTGATCACCTGGGGC-3' (SEQ ID No. 3);

[0061] R: 5'-CACCTCCCTGTTAATACCAACCC-3' (SEQ ID No. 4);

[0062] -System: Template DNA 500ng, primers 0.4 µM each, dNTP 0.25 mM, 10× buffer 5µL, Eazy-Taq 1U, ddH2O to 50µL.

[0063] - Cycle: 94℃ 3min; 94℃ 30s, 60℃ 30s, 72℃ 30s, 35 cycles; 72℃ 8min.

[0064] -Product length: Wild type 1236bp; after knockout, the fragment is shortened by 831bp.

[0065] Experimental results:

[0066] See the experimental results. Figure 1B .exist Figure 1B In the image, each lane represents the PCR identification result of the tail of a mouse labeled with its number. The labels indicate the birth number of each mouse.

[0067] from Figure 1B As can be seen, the wild-type mouse only amplified the full-length 1236bp product, while the TMEM91-KO mouse only amplified the 405bp product. Both sequences of the heterozygous mouse could be amplified. These results indicate that the TMEM91-KO mouse was successfully constructed.

[0068] 4. Sequencing verification: PCR products are directly sent for testing or sequenced after TA cloning. Chromas and DNAMAN are used for comparison to confirm biallelic mutations.

[0069] 5. Passage: Select biallelic mutant F0 and mate with wild-type C57BL / 6J to obtain F1 heterozygotes (+ / -); F1 self-cross, and F2 generation obtained homozygotes (- / -) through Mendelian segregation, i.e. TMEM91-KO mouse model.

[0070] 4. qPCR detection of mRNA expression levels

[0071] (4.1) The tails of KO mice and control mice were lysed, and RNA was extracted using an RNA extraction kit. The RNA content was then determined using a nucleic acid quantification instrument.

[0072] (4.2) Using the extracted RNA, reverse transcription was performed using a reverse transcription kit. The preparation system is shown in Table 1 below:

[0073] Table 1

[0074]

[0075] (4.3) qPCR primer design

[0076] Design qPCR primers for TMEM91:

[0077] Upstream primer sequence: 5'-CACTGAAACTGCGTCTCTGGA-3' (SEQ ID No. 5);

[0078] Downstream primer sequence: 5'-CAAGGCTCAGGTCAGGAAAGT-3' (SEQ ID No. 6);

[0079] (4.4) Prepare the qPCR system

[0080] Table 2

[0081]

[0082] Each sample was added to three sub-wells in a 96-well plate, for a total of 18 wells.

[0083] (4.5) On-machine testing, the program settings are as follows:

[0084] Table 3

[0085]

[0086] The denaturation, annealing / extension were set for 40 cycles. After the denaturation, the temperature was increased to 95°C for 5 seconds and then to 65°C for 5 seconds to terminate the program, thus obtaining the mRNA level of TMEM91 in each sample.

[0087] Experimental results;

[0088] See the experimental results. Figure 1C .from Figure 1C As can be seen, the mRNA level in TMEM91-KO mice was stably knocked out.

[0089] Example 2

[0090] I. Establishment of a mouse model of acute kidney injury (AKI) ischemia-reperfusion (IR):

[0091] 1. Preparation before the experiment

[0092] (1.1) Animals: Male C57BL / 6J mice from the same littermate, 7-9 weeks old, weighing 20-25g, SPF grade (construction method is described above).

[0093] (1.2) Grouping: 4 groups (Sham (control group), Sham+IR (control kidney injury model group), TMEM91-KO, TMEM91-KO+IR), 3 animals in each group, acclimatization feeding 3 days in advance.

[0094] (1.3) Anesthesia system: small animal anesthesia machine, premixed 5% isoflurane + 95% O2 for induction; maintenance flow rate 1.5L / min, maintenance concentration 2%-2.5%.

[0095] (1.4) Instrument sterilization: ophthalmic curved forceps, straight forceps, non-invasive micro-arterial clamps (clamping force 40g), 5-0 silk thread, 6-0 round needle, alcohol cotton balls, 37℃ constant temperature heating pad, timer.

[0096] 2. Surgical Procedure (Two-person operation, completely sterile)

[0097] (2.1) Anesthesia: The mice were placed in an induction box and given 5% isoflurane for 3 minutes. After the righting reflex disappeared, the mice were fixed in a prone position on a 37°C constant temperature plate and the nasal mask was used to maintain 2% isoflurane.

[0098] (2.2) Skin preparation: Shave the hair on both sides of the back and waist area with an electric shaver, and disinfect with iodine solution + 75% alcohol 3 times each.

[0099] (2.3) Incision: With the spine as the midline, make a 1.5cm longitudinal incision downwards from the bilateral costolumbar points (the angle between the lower edge of the 12th rib and the sacrospinal muscle), cut the skin and muscle membrane layer by layer, bluntly separate the muscle layer, and enter the retroperitoneal cavity.

[0100] (2.4) Expose the renal pedicle: Gently separate the fat sac with curved forceps to find the renal arteriovenous bundle (pink pulsation), and use a glass needle to free it for about 3 mm, avoiding damage to the ureter and adrenal branches.

[0101] (2.5) Clamping: The bilateral renal arteries were clamped perpendicular to the renal axis with non-invasive microarterial clamps. The color of the kidneys changed from bright red to pale. The clamping start time was recorded (T=0min).

[0102] (2.6) Temperature control: The mice were kept in a constant temperature plate at 37℃ for 41 minutes. The rectal temperature probe was used to monitor the temperature in real time and maintain it at 36.5-37.5℃.

[0103] (2.7) Reperfusion: At 41 minutes, remove the clamp in the order of "right first, then left". The kidney can be seen to quickly change from pale to bright red, indicating reperfusion. If it does not recover in 10 seconds, apply a wet compress of 37°C saline for 30 seconds.

[0104] (2.8) Suturing: The muscle layer was continuously sutured with a 6-0 round needle, and the skin was sutured intermittently; disinfected with povidone-iodine, and 0.5 mL of 37℃ physiological saline was injected subcutaneously for fluid replacement.

[0105] (2.9) Recovery: Stop anesthesia, place in a 30℃ recovery box, and return to the original cage within 30 minutes when the righting reflex is restored.

[0106] (2.10) End point: Samples were taken 24 hours after re-infusion (see below).

[0107] 3. Postoperative monitoring and endpoint tissue sampling

[0108] (3.1) Observation: Record mental status at 2h, 6h, 12h and 24h after surgery; if death occurs, refrigerate immediately and replace the animal.

[0109] (3.2) Anesthesia: At the end point, 5% isoflurane deep anesthesia was administered, and the righting reflex disappeared.

[0110] (3.3) Blood collection: 0.8-1.0 mL of whole blood was collected by enucleation, and the blood was allowed to stand at room temperature for 15 min. Then it was centrifuged at 3500 r / min at 4℃ for 10 min. 200 µL of the supernatant was collected and stored at -80℃.

[0111] (3.4) Perfusion: Open the thoracic cavity, make an apical incision, puncture the left ventricle with a 25G needle, and rapidly perfuse 20mL of 37℃ physiological saline until the liver is completely pale and there is no blood in both kidneys.

[0112] (3.5) Kidney removal: Quickly remove both kidneys, remove the capsule, and fix them.

[0113] II. Serum urea nitrogen (BUN) and serum creatinine (Scr) detection

[0114] 1. Instruments and reagents

[0115] (1.1) Instrument: Hitachi 7180 fully automated biochemical analyzer, with matching cuvettes and sample trays.

[0116] (1.2) Reagent kit:

[0117] BUN: Urease-UV method reagent (R1 urease 16 kU / L + R2 glutamate dehydrogenase 16 kU / L + NADH 0.3 mmol / L).

[0118] Scr: Picric acid method reagent (R1 0.04% picric acid + 0.3mol / L NaOH; R2 0.04% picric acid + 0.1mol / L phosphate buffer pH 12.0).

[0119] 2. Sample processing

[0120] (2.1) Serum separation: whole blood was left to stand at room temperature for 15 min → centrifuged at 3500 r / min at 4℃ for 10 min → 200 µL of supernatant was aspirated to avoid hemolysis.

[0121] (2.2) Before loading: Centrifuge the serum at 1500 r / min for 2 min to thoroughly remove fiber debris; label the sample cup with a barcode and place at 4℃ for ≤2 h.

[0122] 3. Parameter settings

[0123] (3.1) BUN: Set the machine wavelength to 340nm (main) / 405nm (secondary), 37℃, endpoint method; mix 3µL of sample with 180µL of R1, read A1 after 5min; add 60µL of R2, read A2 after 30s; calculate ΔA.

[0124] (3.2) Scr: Set the machine wavelength to 510nm (main) / 700nm (secondary), 37℃, rate method; 6µL of sample + 180µL of R1, incubate at 37℃ for 5min, record absorbance at 0s and 60s; add 60µL of R2, record ΔA / min in the 60-120s interval.

[0125] 4. Results Output

[0126] 4.1 The instrument automatically displays the concentration, in units of BUN (mmol / L) and Scr (µmol / L).

[0127] 4.2 Export to Excel, use GraphPad Prism to create bar charts, Mean±SEM, t-test or ANOVA.

[0128] Experimental results:

[0129] See the experimental results. Figure 2A .from Figure 2A As can be seen, the serum creatinine and blood urea nitrogen levels in the TMEM91-KO mouse kidney injury model group were significantly increased, and were also significantly higher than those in the wild-type kidney injury model group.

[0130] The results above indicate that TMEM91-KO mice are more sensitive to kidney injury, suggesting that TMEM91 is a potential protective target against kidney injury.

[0131] III. Detailed Procedures for PAS Staining of Kidney Tissue

[0132] 1. Sample preparation

[0133] (1.1) The kidney tissue of mice was fixed in formalin tissue fixative for 48 hours.

[0134] (1.2) Use an automatic dehydrator to dehydrate, clear, and impregnate the tissue with paraffin.

[0135] (1.3) Using an embedding machine, the paraffin-impregnated tissue is retrieved, placed in a mold, and embedded in paraffin.

[0136] (1.4) Using a microtome, cut the embedded wax block into thin slices of 5 μm thickness and place them on a glass slide.

[0137] 2. Reagent preparation

[0138] (2.1) 0.5% periodic acid: 0.5g periodic acid + 100mL double-distilled water, keep at 4℃ away from light, and use within 1 week.

[0139] (2.2) Schiff reagent: Commercial ready-to-use type (Beijing Solarbio), 4℃ protected from light, colorless to light amber, turns red when it becomes ineffective.

[0140] (2.3) Hematoxylin staining solution: Harris hematoxylin, filtered before use.

[0141] (2.4) Hydrochloric acid differentiation solution: 1% HCl and 70% ethanol, freshly prepared.

[0142] 3. Pre-sectioning treatment

[0143] (3.1) Baking: Baking the film in a 60℃ constant temperature oven for 30 minutes to remove excess paraffin.

[0144] (3.2) Dewaxing: 15 min each of xylene I / II / III → 5 min each of gradient ethanol (100%→95%→80%→70%→50%) → 3 min of running water rinsing.

[0145] 4. Staining procedure (room temperature, humidified chamber, protected from light)

[0146] (4.1) Oxidation: 0.5% periodic acid for 8 min → rinse with running water for 5 min → rinse with PBS for 3 × 3 min.

[0147] (4.2) Staining: Add Schiff reagent to completely cover the tissue and place it in a humidified chamber in the dark for 20 minutes (after which the tissue will be visible to the naked eye as purplish-red).

[0148] (4.3) Rinsing: Rinse slowly with running water for 10 minutes to remove unbound dye.

[0149] (4.4) Counterstaining: hematoxylin for 2 min → running water for 1 min.

[0150] (4.5) Differentiation: 1% hydrochloric acid alcohol for 30s → running water for 1min.

[0151] (4.6) Return to blue: tap water overflow for 15 minutes.

[0152] (4.7) Dehydration: 50%→70%→80%→95%→100% each for 2 min → xylene I / II each for 5 min.

[0153] (4.8) Covering: Add neutral resin to the coverslip, avoiding air bubbles, and let it air dry at room temperature for 24 hours.

[0154] 4. Result Interpretation and Photographing

[0155] (4.1) Glycogen and mucopolysaccharides are bright purple-red; the nucleus is blue.

[0156] (4.2) Take photos with an upright microscope at 200× / 400×.

[0157] Experimental results:

[0158] See the experimental results. Figure 2B .from Figure 2B The results show that TMEM91-KO mice exhibited more severe kidney damage, with reduced peritubular villi and the appearance of tubular casts in some renal tubules compared to wild-type mice. These results indicate that TMEM91 has a protective effect against kidney injury in animal models.

[0159] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An sgRNA for knocking out the TMEM91 gene, characterized in that, The sequence of the sgRNA is shown in SEQ ID No. 1 or SEQ ID No.

2.

2. The application of sgRNA in the method for constructing a TMEM91 gene knockout mouse model according to claim 1.

3. A method for constructing a TMEM91 gene knockout mouse model, characterized in that, The method includes: (1) Provide the sgRNA according to claim 1; (2) Using CRISPR / Cas9 gene editing technology, the sgRNA described in step (1) is injected into mouse zygote cells, so that the TMEM91 gene is knocked out in the cells; (3) Cultivate the cells from step (2) to obtain the TMEM91 gene knockout mouse model.

4. The use of the sgRNA according to claim 1 or the method according to claim 3 in the method of screening drugs targeting the TMEM91 gene or drugs for treating kidney injury.

5. The application according to claim 4, characterized in that, The kidney injury mentioned includes acute kidney injury.

6. Application of reagents that enhance TMEM91 gene expression in the preparation of drugs for treating kidney injury.

7. The application according to claim 6, characterized in that, The kidney injury mentioned includes acute kidney injury.

8. A pharmaceutical composition for treating kidney injury, characterized in that, The pharmaceutical composition includes reagents that enhance TMEM91 gene expression or reagents that enhance TMEM91 protein activity.

9. The pharmaceutical composition according to claim 8, characterized in that, The kidney injury mentioned includes acute kidney injury.

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