Use of RINL antagonist in prevention and treatment of hyperuricemia

Abstract

The invention belongs to the field of biological medicine, and relates to application of an RINL antagonist in prevention and treatment of hyperuricemia. Specifically, the invention provides an application of an RINL gene or an encoding protein thereof, and the RINL gene or the encoding protein thereof: (1) is used as a marker for diagnosis, detection or prognosis of hyperuricemia; (2) preparing a reagent or a kit for diagnosing or detecting hyperuricemia; or (3) the compound is used for preparing a medicine for preventing and / or treating hyperuricemia. The RINL provided by the invention is an important related gene in the occurrence and development process of hyperuricemia, and is also a potential hyperuricemia treatment site. RINL expression is inhibited, and the uric acid excretion capacity of cells and mice is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of a RINL antagonist in the prevention and treatment of hyperuricemia. Background Art

[0002] Hyperuricemia (HUA) is a metabolic disease caused by purine metabolism disorder, characterized by elevated uric acid levels in the blood. Under normal circumstances, uric acid is a product of purine metabolism in the human body and is excreted from the body through the kidneys. When uric acid is produced excessively or excreted insufficiently, the uric acid concentration in the blood will increase, exceeding its solubility in the blood, thereby forming urate crystals, which may lead to health problems such as gout. When gout attacks, patients will experience severe pain, and it is called the "king of pain". Clinically, it manifests as acute onset of joint swelling and pain, often accompanied by elevated blood uric acid levels. The latest epidemiological data show that the incidence of gout is increasing year by year worldwide, affected by multiple factors such as race, genes, age, gender, diet, occupation, environment and education level. Gout attacks seriously affect the patient's work and quality of life, and bring great mental stress and economic burden to patients and society. Therefore, it is particularly important to study the pathogenesis of gout and develop effective prevention and treatment drugs.

[0003] At present, clinical drugs for hyperuricemia are mainly divided into drugs that promote uric acid metabolism (benzbromarone, probenecid), drugs that inhibit uric acid production (allopurinol, febuxostat), drugs that alkalinize urine (sodium bicarbonate) and drugs that promote uric acid dissolution (citrate preparations). At present, as the prevalence of HUA increases year by year, more and more different uric acid-lowering drugs are needed to meet the treatment needs of different populations.

[0004] Considering the diversity of causes of hyperuricemia and the lack of a cure, there is an urgent need to develop new and more effective treatments. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the purpose of the present disclosure is to provide a use of a RINL antagonist in the prevention and treatment of hyperuricemia.

[0006] In order to achieve the above objectives, the present disclosure adopts the following specific scheme:

[0007] In one aspect, the present disclosure provides a use of a RINL gene or a protein encoded therein, wherein the RINL gene or the protein encoded therein is: (1) used as a marker for diagnosing, detecting or prognosing hyperuricemia; (2) used to prepare a reagent or kit for diagnosing or detecting hyperuricemia; or (3) used to prepare a drug for preventing and / or treating hyperuricemia. In another aspect, the present disclosure provides a RINL antagonist, wherein the RINL antagonist specifically targets the nucleotide sequence shown in SEQ ID NO.7 or specifically binds to the amino acid sequence shown in SEQ ID NO.8, and has any of the following functions:

[0008] (1) Inhibit RINL gene expression;

[0009] (2) Deletion or silencing of the RINL gene;

[0010] (3) Antagonize RINL protein.

[0011] In another aspect, the present disclosure provides a composition comprising the aforementioned RINL antagonist, and optionally a pharmaceutically acceptable carrier or excipient.

[0012] In another aspect, the present disclosure provides a method for inhibiting RINL expression in a cell, the method comprising the following steps:

[0013] The aforementioned RINL antagonist or the aforementioned composition is delivered to cells to inhibit the expression of the RINL gene in the cells.

[0014] In another aspect, the present disclosure provides a use of the aforementioned use, the aforementioned RINL antagonist and / or the aforementioned composition in the preparation of a medicament for preventing and / or treating hyperuricemia in an individual.

[0015] In another aspect, the present disclosure provides a method for preventing and / or treating hyperuricemia, comprising administering an effective amount of the aforementioned RINL antagonist and / or the aforementioned composition to a subject in need thereof.

[0016] In another aspect, the present disclosure provides the aforementioned RINL antagonist and / or the aforementioned composition for use in preventing and / or treating hyperuricemia.

[0017] The present disclosure has at least the following beneficial effects:

[0018] 1. The applicant unexpectedly discovered that RINL is an important gene involved in the development of HUA and a potential treatment site for HUA.

[0019] 2. The applicant found that knocking down / knockout of RINL significantly enhanced the uric acid excretion capacity of cells and mice, and designed a series of effective RINL antagonists.

[0020] 3. The applicant discovered that RINL protein has the potential to inhibit uric acid excretion. By constructing a RINL (P139S) point mutation stably overexpressing HK2 cell lines, it was demonstrated that RINL (P139S) point mutation inhibits the degradation of RINL protein in cells, increases the level of the protein in cells, and further inhibits uric acid excretion in HK2 cells.

[0021] 4. By constructing RINL knockout and tubule-specific knockout mice, the applicant verified that RINL knockout can promote uric acid excretion in the renal tubules of mice and reduce uric acid levels in mice. Similarly, RINL knockout also promotes renal uric acid excretion in HUA mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It shows that RINL is a related pathogenic gene of HUA. Figure 1 A is the pedigree of the HUA family in southern Fujian. Figure 1 B is the Sanger gene sequencing of family members. Figure 1 C is the comparison of RINL protein sequence / mutation site between humans and other species, Figure 1 D shows the location of RINL expression in healthy kidneys detected by immunohistochemistry.

[0023] Figure 2 It was shown that RINL knockdown promoted uric acid excretion in HK2 cells. Figure 2 A is a comparison of the effects of three RINL siRNAs knocking down RINL in HK2 cells. Figure 2 B is the detection of extracellular uric acid levels in HK2 cells after treatment with si-RINL2. Figure 2 C and Figure 2 D is the detection of intracellular uric acid excretion protein ABCG2 level after HK2 cells were treated with si-RINL2; GAPDH was used as a reference protein. The data presented here represent at least three independent experiments. All data are expressed as SEM ± mean, and P values ​​were determined by unpaired t-test. *P < 0.05.

[0024] Figure 3 It was shown that RINL knockout promoted uric acid secretion in HK2 cells, Figure 3 A is the 7-day cell viability test of HK2 cells with control (LentiV2) and RINL knockout (LentiV2-RINL gRNA), Figure 3 B is the detection of extracellular uric acid levels in control (LentiV2) and RINL knockout (LentiV2-RINL gRNA) HK2 cells under normal (and induced HUA cell model) conditions. Figure 3C is the detection of intracellular uric acid levels in control (LentiV2) and RINL knockout (LentiV2-RINL gRNA) HK2 cells under normal (and induced HUA cell model) conditions. Figure 3 D is the immunoblotting detection of the levels of uric acid excretion protein OAT3 / ABCG2 in control (LentiV2) and RINL knockout (LentiV2-RINL gRNA) HK2 cells under normal conditions. Figure 3 E is the immunoblotting detection of the levels of uric acid excretion protein OAT3 / ABCG2 in the control (LentiV2) and RINL knockout (LentiV2-RINL gRNA) HK2 cells under the conditions of induced HUA cell model. Figure 3 F and Figure 3 G is immunofluorescence detection of intracellular RINL (F) and uric acid excretion protein ABCG2 (G) levels in HK2 cells with control (LentiV2) and RINL knockout (LentiV2-RINL gRNA); the data presented here represent at least three independent experiments. All data are expressed as SEM ± mean, and P values ​​were determined by unpaired t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0025] Figure 4 It was shown that RINL overexpression inhibited uric acid secretion in HK2 cells. Figure 4 A is the 7-day cell viability test of HK2 cells with control (pLV) and RINL stably overexpressed (pLV-RINL). Figure 4 B is the detection of extracellular uric acid levels in control (pLV) and RINL stably overexpressed (pLV-RINL) HK2 cells under normal (and induced HUA cell model) conditions. Figure 4 C is the detection of intracellular uric acid levels in control (pLV) and RINL stably overexpressed (pLV-RINL) HK2 cells under normal (and induced HUA cell model) conditions. Figure 4 D is the immunoblotting detection of the levels of uric acid excretion protein OAT3 / ABCG2 in HK2 cells with control (pLV) and RINL stably overexpressed (pLV-RINL) under normal conditions. Figure 4 E is the immunoblotting detection of the levels of uric acid excretion protein OAT3 / ABCG2 in HK2 cells with control (pLV) and RINL stably overexpressed (pLV-RINL) under the conditions of induced HUA cell model. Figure 4F and 4G immunofluorescence detection of intracellular RINL (F) and uricosuric acid excretion protein ABCG2 (G) levels in HK2 cells with control (pLV) and RINL stably overexpressed (pLV-RINL); the data presented here represent at least three independent experiments. All data are expressed as SEM ± mean, and P values ​​were determined by unpaired t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0026] Figure 5 The RINL (P139S) point mutation further inhibits uric acid secretion in HK2 cells. Figure 5 A is the detection of Rinl mRNA levels in HK2 cells with control (pLV), RINL stably overexpressed cells, and RINL (P139S) point mutation stably overexpressed cells. Figure 5 B and 5C are the detection of extracellular (B) and intracellular (C) uric acid levels in HK2 cells with control (pLV), RINL stably overexpressed, and RINL (P139S) point mutation stably overexpressed under normal conditions. Figure 5 D and 5E are the detection of extracellular (D) and intracellular (E) uric acid levels in HK2 cells with control (pLV), RINL stably overexpressed, and RINL (P139S) point mutation stably overexpressed under the conditions of induced HUA cell model. Figure 5 F is to detect the intracellular RINL and uricosuric acid excretion protein ABCG2 levels of HK2 cells with control (pLV), RINL stable overexpression and RINL (P139S) point mutation stable overexpression. The data presented here represent at least three independent experiments. All data are expressed as SEM ± mean, and P values ​​are determined by unpaired t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, #P<0.05, ##P<0.01.

[0027] Figure 6 For wild type (RINL + / + ) and RINL knockout (RINL - / - ) Detection of postnatal developmental indicators in mice. Figure 6 A- Figure 6 H is the detection of wild type (RINL + / + ) and RINL knockout (RINL - / - ) The time of developmental indicators such as auricle separation, body hair, lower tooth eruption, upper tooth eruption, eye opening, ear opening, crawling, and flat turning in newborn mice. n = 35-50. All data are expressed as SEM ± mean, and P values ​​were determined by unpaired t test. *P < 0.05, **P < 0.01.

[0028] Figure 7The complete knockout of RINL promoted the excretion of uric acid in the kidneys of mice and reduced the level of uric acid in the mice. Figure 7 A is the construction strategy of RINL knockout mice. Figure 7 B is wild type (RINL + / + ) and RINL knockout (RINL - / - ) Mouse genotype identification, DNA gel electrophoresis, Figure 7 C is the immunoblot detection of 8-month-old wild-type (RINL + / + ) and RINL knockout (RINL - / - ) Mouse kidney RINL protein, Figure 7 D is immunofluorescence detection of 8-month-old wild-type (RINL + / + ) and RINL knockout (RINL - / - ) Mouse kidney RINL protein, Figure 7 E is the qPCR detection of 8-month-old wild-type (RINL + / + ) and RINL knockout (RINL - / - ) Rin1 mRNA level in mouse kidney, Figure 7 F is immunohistochemical detection of 8-month-old wild-type (RINL + / + ) and RINL knockout (RINL - / - ) Mouse kidney RINL, Figure 7 G and 7H are for detecting wild type (RINL + / + ) and RINL knockout (RINL - / - ) Serum uric acid levels in male (G) / female (H) mice at 2, 4, 8, 12, and 16 months of age. The data presented here represent at least three independent experiments. All data are expressed as SEM ± mean, and P values ​​were determined by unpaired t-test. *P<0.05, **P<0.01.

[0029] Figure 8 This is the result of the decrease in uric acid levels in mice after RINL complete knockout. Figure 8 A~8F are 8-month-old wild-type (RINL + / + ) and RINL knockout (RINL - / - ) Detection and statistics of body weight (A), kidney-to-body weight ratio (B), serum creatinine (C), serum urea nitrogen (D), urea nitrogen / creatinine (E), and serum uric acid (F) levels in male mice. Figure 8 G is 8-month-old wild type (RINL + / + ) and RINL knockout (RINL - / - ) Male mouse kidney sections were stained with HE, and the tubular spaces were counted. Figure 8 H is an 8-month-old wild type (RINL + / + ) and RINL knockout (RINL - / - ) Detection of interleukin-1β in male mouse kidneys, Figure 8 I is 8-month-old wild type (RINL + / + ) and RINL knockout (RINL - / - ) Detection of uric acid excretion protein OAT3 / ABCG2 levels in male mouse kidneys. The data presented here represent at least three independent experiments. All data are expressed as SEM ± mean, and P values ​​were determined by unpaired t-test. *P<0.05, **P<0.01.

[0030] Fig. 9 The results of the test show that RINL promotes renal uric acid excretion in HUA mice. Fig. 9 A-9B is a test for 2-month-old wild (RINL + / + ) and RINL knockout (RINL - / - ) Serum uric acid levels on day 14 of HUA model establishment in male and female mice. Fig. 9 C-9D was used to detect 2-month-old wild (RINL + / + ) and RINL knockout (RINL - / - ) Serum uric acid levels on day 28 of HUA model establishment in male and female mice, Fig. 9 E is the detection of 2-month-old wild (RINL + / + ) and RINL knockout (RINL - / - ) Levels of uric acid excretion proteins OAT1 / OAT3 in kidneys of normal / HUA male mice; the data presented here represent at least three independent experiments. All data are expressed as SEM ± mean, and P values ​​were determined by unpaired t-test. *P<0.05, **P<0.01.

[0031] Fig.10 Tubule-specific knockout of RINL promotes renal uric acid excretion in male mice. Fig.10 A is the construction strategy of RINL conditional knockout mice. Fig.10 B is the detection of Rinl mRNA levels in kidneys of 4-month-old control (Control) and RINL tubule-specific knockout (Cko) male mice. Fig.10 D-10F is the body weight (D), serum uric acid (E) and serum creatinine (F) levels of 6-month-old control (Control) and RINL tubule-specific knockout (Cko) male mice; (GJ) Body weight (G), kidney-to-body weight ratio (H), serum uric acid (I) and serum creatinine (J) levels of 8-month-old control (Control) and RINL tubule-specific knockout (Cko) male mice. Fig.10 K and 10L are the urine uric acid (K) and creatinine (L) levels of 8-month-old control (Control) and RINL renal tubule-specific knockout (Cko) male mice. Fig.10M and 10N are the levels of uric acid excretion fraction (FEUA) (M) and urinary uric acid excretion rate (N) in 8-month-old control (Control) and RINL tubule-specific knockout (Cko) male mice. The data presented here represent at least three independent experiments. All data are expressed as SEM ± mean, and P values ​​were determined by unpaired t-test. *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0032] I. Terminology

[0033] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present disclosure belongs.

[0034] As used herein, the articles "a" and "an" refer to one or more than one (ie, at least one) of the grammatical object to which the article refers. For example, "an element" means one element or more than one element.

[0035] As used herein, the term "RINL" stands for Ras And Rab Interactor Like, which is a Ras and Rab interactor-like protein that participates in a variety of biological processes in cells, especially related to vesicle-mediated transport and the regulation of Rab proteins. The protein expressed by RINL can activate the Rab5 subfamily small GTPase, thereby playing a role in endocytosis. RINL is expressed in a variety of tissues, especially in the thymus and spleen, where the expression level is high.

[0036] As used herein, the term "OAT3" is organic anion transporter 3, which is a membrane transporter belonging to the SLC22A family and is responsible for the transmembrane transport of a variety of endogenous and exogenous compounds. OAT3 is mainly expressed in the basolateral membrane of the proximal tubules of the kidney. OAT3 mediates the transport of organic anions and participates in the secretion, reabsorption and distribution of a variety of drugs in the kidney. In addition, OAT3 is also involved in the excretion of uric acid, which plays an important role in maintaining blood uric acid levels and preventing hyperuricemia.

[0037] As used herein, the term "ABCG2" refers to ATP-binding cassette transporter G2, also known as Breast Cancer Resistance Protein (BCRP), which is a member of the ATP-binding cassette (ABC) transporter family. ABCG2 is expressed in a variety of tissues, including the intestine, liver, kidney, placenta, and blood-brain barrier. It is a multidrug resistance protein that can pump a variety of drugs from inside the cell to outside the cell, thereby reducing the concentration of drugs inside the cell, causing tumor cells to develop resistance to certain chemotherapy drugs. In addition, it is also involved in the transport of endogenous metabolites, including the excretion of uric acid. ABCG2 is expressed at the brush membrane end of the proximal tubules of the kidney and is involved in the secretion of urate, while also regulating uric acid levels outside the kidney. Mutations in the ABCG2 gene or changes in expression levels may affect the balance of uric acid metabolism, leading to increased or decreased blood uric acid levels, which are associated with the risk of hyperuricemia and gout.

[0038] As used herein, the term "uric acid" is a colorless, odorless crystal with a chemical formula of C5H4N4O3, which is the final product when the human body and other mammals metabolize purines. Purines are components of nucleic acids. When cells divide and die, purines are released and eventually metabolized into uric acid. Uric acid is mainly produced in the human body through purine metabolism in the liver and excreted from the body by the kidneys. Under normal circumstances, the production and excretion of uric acid are balanced to maintain blood uric acid levels within a relatively stable range. When uric acid is overproduced or underexcreted in the body, blood uric acid levels rise, leading to hyperuricemia.

[0039] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such names include progeny thereof. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It should also be understood that all progeny may not be exactly identical in terms of DNA content, due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, this is clear from the context.

[0040] As used herein, RNA interference (RNAi) is a sequence-specific RNA targeting process that provides a method for directly knocking down and effectively silencing any gene containing a homologous sequence. The gene regulation mechanism of RNAi involves the expression of non-coding silencing RNA molecules that are complementary to the mRNA molecules transcribed from the target gene. The complementary RNA molecules are expressed as long double-stranded RNA (dsRNA), which are then cut into small interfering RNA (siRNA) molecules of 19-27 nucleotides (nt) by RNase III / helicase protein Dicer, with a 2nt overhang at the 3' end. Afterwards, siRNA is incorporated into a ribonuclease protein complex known as RNA-induced silencing complex (RISC). One chain of siRNA still associates with RISC to guide the complex toward the complementary target RNA. The endonuclease digestion target mRNA guided by this siRNA leads to the truncation and inactivation of the targeted RNA. The degradation of the targeted mRNA leads to a reduction in the translation of the produced protein. In this way, siRNA-guided RNAi can effectively silence or knockout targeted genes without mutating or altering the genomic DNA sequence.

[0041] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and where it does not occur.

[0042] "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other expression elements may be supplied by a host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus) incorporating a recombinant polynucleotide.

[0043] "Administering," "giving," and "treating" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administering," "giving," and "treating" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering," "giving," and "treating" also mean in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnosis, a combination composition, or by another cell. "Treatment," when applied to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0044] "Treatment" means administering an internal or external therapeutic agent to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Generally, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in the patient or population being treated, either by inducing regression of such symptoms or inhibiting the progression of such symptoms to any clinically unmeasurable degree. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether a disease symptom has been alleviated may be assessed by any clinical test method commonly used by a physician or other health care professional to assess the severity or progression of the symptom. Although the embodiments of the present disclosure (e.g., treatment methods or articles) may not be effective in alleviating the target disease symptoms that every patient has, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test. The term "treating" an individual suffering from a disease or disease condition means that the symptoms of the individual are partially or completely alleviated, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing underlying diseases and / or preventing symptom deterioration or disease development. Treatment also includes any pharmaceutical use of any chimeric antigen receptor provided and the compositions provided herein.

[0045] The term "consisting essentially of" or variations thereof as used throughout the specification and claims means including all recited elements or groups of elements, and optionally including other elements of similar or different properties to the recited elements, which other elements do not significantly alter the basic or novel properties of a specified dosing regimen, method, or composition.

[0046] As used herein, the term "antagonist" refers to a substance that can act at the nucleic acid or protein level to reduce or inhibit the expression or activity of RINL. It includes but is not limited to shRNA, siRNA, miRNA, dsRNA, small molecule compounds, stRNA, aptamers, or antibodies or their antigen-binding fragments.

[0047] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphipathic compounds, plasmids and viruses. Therefore, the term "vector" includes an autonomously replicating plasmid or virus. The term should also be interpreted to include non-plasmid and non-viral compounds that promote the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0048] As used herein, the terms "patient," "subject," "individual," and the like are used interchangeably herein and are intended to include living organisms (e.g., mammals) in which an immune response can be elicited. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.

[0049] As used herein, the term "kit" is any product (e.g., a package or container) containing at least one reagent (e.g., a therapeutic agent, a probe, a small molecule, etc.) for specific detection and / or therapeutically affecting the expression of the markers described in the present invention. The kit can be promoted, distributed or sold as a whole for performing the method described in the present invention. The kit may contain one or more reagents, which are necessary for the expression of the composition used in the method described in the present invention. In certain embodiments, the kit may further contain a reference standard, for example, a nucleic acid encoding a protein that does not affect or regulate a signaling pathway that controls an immune response, cell growth, division, migration, survival or apoptosis. A person skilled in the art can envision many such control proteins, including but not limited to: common molecular tags (e.g., green fluorescent protein and β-galactosidase); proteins that are not included in the classification of any pathway including cell growth, division, migration, survival or apoptosis by GeneOntology reference; or ubiquitous housekeeping proteins. The reagents in the kit may be provided by a single container, or by a mixture of two or more reagents in a single container. In addition, explanatory materials describing the use of the composition in the kit may be included.

[0050] As used herein, the term "prognosis" refers to the act of predicting the course and outcome of a disease in advance. More specifically, the course of a disease after treatment may vary depending on the patient's physiological or environmental conditions, and it can be interpreted as all actions that predict the course of a disease after treatment, taking into account the patient's overall condition. For the purposes of this disclosure, prognosis prediction can be interpreted as predicting the disease-free survival rate of gout patients by predicting the course of the disease and the condition after treatment of gout patients.

[0051] As used herein, the terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, substance or composition as described herein that is effective to achieve a particular biological result or provide a therapeutic or prophylactic benefit.

[0052] Abbreviations

[0053] Abbreviations English full name Chinese terminology RINL Ras and Rab interactor like Ras and Rab interactor-like protein ABCG2 ATP binding cassette subfamily G member 2 ATP-binding cassette subfamily G member 2 UA Uric acid Uric acid OAT3 Organic anion transporter 3 Organic anion transporter 3

[0054] II. Detailed description of specific implementation scheme

[0055] In one aspect, the present disclosure provides a use of a RINL gene or a protein encoded therein, wherein the RINL gene or the protein encoded therein is: (1) used as a marker for diagnosing, detecting or prognosing hyperuricemia; (2) used for preparing a reagent or kit for diagnosing or detecting hyperuricemia; or (3) used for preparing a drug for preventing and treating hyperuricemia.

[0056] On the other hand, the present disclosure provides a RINL antagonist, wherein the RINL antagonist specifically targets the nucleotide sequence shown in SEQ ID NO.7 or specifically binds to the amino acid sequence shown in SEQ ID NO.8, and has any of the following functions:

[0057] (1) Inhibit RINL gene expression;

[0058] (2) Deletion or silencing of the RINL gene;

[0059] (3) Antagonize RINL protein.

[0060] In some embodiments, the RINL antagonist is selected from shRNA, siRNA, miRNA, dsRNA, stRNA, a small molecule compound, an aptamer, or an antibody or an antigen-binding fragment thereof.

[0061] In some embodiments, the antibody or antigen-binding fragment thereof is selected from a whole antibody, a F(ab)-fragment, a F(ab)2-fragment, a single-chain antibody, a chimeric antibody, a bivalent antibody-construct, a synthetic antibody, a bispecific single-chain antibody or a cross-cloned antibody.

[0062] In some embodiments, the RINL antagonist is an anti-RINL antibody.

[0063] In some embodiments, the RINL antagonist is a siRNA comprising a sense strand and an antisense strand, which are 15-30 base pairs in length, and the antisense strand comprises a complementary region complementary to an mRNA encoding RINL.

[0064] In some embodiments, the nucleotide sequence of the siRNA is selected from any one of the following combinations of sense strands and antisense strands:

[0065] (1) a sense strand having the sequence shown in SEQ ID NO.1, and an antisense strand having the sequence shown in SEQ ID NO.2; or

[0066] (2) a sense strand having a sequence as shown in SEQ ID NO.3, and an antisense strand having a sequence as shown in SEQ ID NO.4; or

[0067] (3) A sense strand having a sequence as shown in SEQ ID NO.5, and an antisense strand having a sequence as shown in SEQ ID NO.6.

[0068] In some embodiments, the siRNA comprises a sense strand as shown in SEQ ID NO.3, and an antisense strand as shown in SEQ ID NO.4.

[0069] In some embodiments, the sense strand and the antisense strand of the siRNA are further modified, and the modification is selected from: one or more of: 2'-methoxyethyl modification, 2'-methoxy modification, 2'-deoxy-2'-fluoro modification, thiophosphate modification, adenosine-2' phosphate modification, uridine-2' phosphate modification, guanosine-2' phosphate modification, guanosine-diol nucleic acid modification, adenosine-diol nucleic acid modification, N-(2,3-dihydroxypropyl)-3,5-bis(trifluoromethyl)benzamide modification or 5'-phosphate mimetic modification.

[0070] In some embodiments, at least one nucleotide of the siRNA is conjugated to one or more targeting ligands.

[0071] In some embodiments, the targeting ligand is conjugated to the sense strand of the siRNA.

[0072] In some embodiments, the targeting ligand is conjugated to any nucleotide of the sense strand of the siRNA.

[0073] In some embodiments, the targeting ligand is conjugated to the 5' end of the sense strand of the siRNA.

[0074] In some embodiments, the targeting ligand is conjugated to the 3 '-end of the sense strand of the siRNA.

[0075] In some embodiments, the targeting ligand comprises a protein, a carbohydrate, or a lipid.

[0076] In some embodiments, the protein is selected from the group consisting of a natural protein, a synthetic polyamino acid, and an antibody.

[0077] In some embodiments, the natural protein is selected from human serum albumin, low-density lipoprotein, or globulin.

[0078] In some embodiments, the synthetic polyamino acid is selected from polylysine, poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactic acid-co-ethylene glycol) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer, polyethylene glycol, polyvinyl alcohol, polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer or polyphosphazene.

[0079] In some embodiments, the carbohydrate is selected from dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, amino sugars, or hyaluronic acid.

[0080] In some embodiments, the lipid is selected from a fatty acid, a sterol, or a phospholipid.

[0081] In some embodiments, the fatty acid is selected from capric acid, caprylic acid, lauric acid, palmitic acid, myristic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, or eicosenoic acid.

[0082] In some embodiments, the sterol is selected from cholesterol, cholesteryl, cholestanol, stigmasterol, cholestanoic acid, or ergosterol.

[0083] In some embodiments, the phospholipid is selected from di-hexadecyl-rac-glycerol or triethyl-amine 1,2-di-O-hexadecyl-rac-glycerol-3-hydrogen phosphonate.

[0084] In some embodiments, the targeting ligand comprises an aminosugar.

[0085] In some embodiments, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

[0086] In some embodiments, the GalNac moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

[0087] In some embodiments, the targeting ligand has the following structure:

[0088]

[0089] In some embodiments, the targeting ligand is conjugated to the sense strand of the siRNA at the 3'-end via a linker to form the following conjugate:

[0090]

[0091] X may be selected from S or O.

[0092] In some embodiments, the RINL antagonist can promote the expression of OAT3 and ABCG2 proteins and increase uric acid excretion.

[0093] In another aspect, the present disclosure provides nucleic acids encoding the aforementioned RINL antagonists.

[0094] In another aspect, the present disclosure provides an expression vector comprising the aforementioned nucleic acid.

[0095] In another aspect, the present disclosure provides a cell comprising the aforementioned nucleic acid and an expression vector.

[0096] In another aspect, the present disclosure provides a composition comprising the aforementioned RINL antagonist, and optionally a pharmaceutically acceptable carrier or excipient.

[0097] In some embodiments, the content of the RINL antagonist in the composition is 1% to 99%.

[0098] In some embodiments, the composition further comprises a second portion of a drug, wherein the drug is selected from one or more of allopurinol, febuxostat, benzbromarone, probenecid, sodium bicarbonate, citric acid preparations, and combinations thereof.

[0099] In some embodiments, the composition is formulated for ocular, vaginal, rectal, intranasal, transdermal, subcutaneous administration, intravenous infusion, intraarterial, translymphatic, intrabronchial, intrapleural, intraperitoneal, cerebrospinal or intramuscular injection, pulmonary, intrathecal or intraventricular administration; preferably subcutaneous administration.

[0100] In another aspect, the present disclosure provides a method for inhibiting RINL expression in a cell, the method comprising the following steps:

[0101] The aforementioned RINL antagonist or the aforementioned composition is delivered to cells to inhibit the expression of the RINL gene in the cells.

[0102] In another aspect, the present disclosure provides a use of the aforementioned use, the aforementioned RINL antagonist and / or the aforementioned composition in the preparation of a medicament for preventing and / or treating hyperuricemia in an individual.

[0103] In another aspect, the present disclosure provides a method for treating hyperuricemia by administering a therapeutically effective amount of the aforementioned RINL antagonist and / or the aforementioned composition to a subject.

[0104] On the other hand, the present disclosure provides a method for detecting the prognosis of hyperuricemia by detecting the expression and mutation of RINL in a subject.

[0105] Experimental Materials and Methods

[0106] Cell culture and transfection

[0107] HK2 (human renal cortical proximal tubule epithelial cells) were purchased from Procell Life Science Co., Ltd. (Wuhan, China) and cultured in DMEM / F12 medium (Pricella, Catalog No. PM150312) supplemented with 10% fetal bovine serum (VivaCell, Catalog No. C04001) and 1% penicillin / streptomycin (Solarbio, Catalog No. P8420 / S8290) in an incubator containing 5% CO2.

[0108] Transient transfection was performed using Lipofectamine 2000 (Thermo, Cat. No. 11668-019).

[0109] HK2 cells were transiently transfected with si-NC and si-RINL by co-incubation with Lipofectamine 2000. The sequence of si-RINL is as follows:

[0110] Construction of conditional knockout mice

[0111] Mice were housed in a standard environment characterized by a 12-h light / dark cycle, a temperature of 22–25°C, 40–60% humidity, and free access to food and water. All animals were housed in a specific pathogen-free facility. Male DB / M and DB / DB mice aged 12 weeks were from Cavens Biological (China). C57BL / 6J (B6) mice were from Vital River Laboratories (China).

[0112] By using the high-performance Crisper strategy to knock out a large fragment of the mouse RINL gene (such as Figure 7a). Subsequently, a mating was performed with B6 animals once to obtain RINL heterozygous mice, which were then mated to generate RIN homozygous mice. For RINL KO genotype detection, PCR was performed using the following primers: F: 5'-ctacccatgtccggatctcc-3' (SEQ ID NO: 9), R: 5'-acggtcacagagattagggc-3' (SEQ ID NO: 10), RINL KO mice showed a 759 bp band, while wild-type mice had no band. PCR was performed using the following primers: F: 5'-ATGAGGGAATGGATGAACGG-3' (SEQ ID NO: 11), R: 5'-TCTGGAGGTCAGTGAGGAGC-3' (SEQ ID NO: 12), RINL KO mice had no band, while wild-type mice showed a 434 bp band.

[0113] To generate RINL loxP / loxP mice, a targeting vector was first constructed to insert two loxP sites at both ends of the fourth exon of RINL. Subsequently, a mating was performed with B6 animals once to obtain RINL loxP / + mice, which were then mated to generate RINL loxP / loxP mice. For RINL loxP genotype detection, PCR was performed using the following primers: F1: 5'-ATTTCCTGTAGCTCAGCACACTAT-3' (SEQ ID NO: 13), R1: 5'-CAGATGTTTCTTTCTGCCTGCTTG-3' (SEQ ID NO: 14); F2: 5'-ATTTCCTGTAGCTCAGCACACTAT-3' (SEQ ID NO: 15), R2:: 5'-CAGATGTTTCTTTCTGCCTGCTTG-3' (SEQ ID NO: 16), RINL loxP mice showed a 370 bp band, while the wild type showed a 300 bp band.

[0114] RINL loxP / loxP mice and Pax8-Cre-ER T2 Mating of mice to generate tubule-specific RINL knockout mice Pax8-Cre-ER T2 + / RINL loxP / loxP (RINL cKO). Heterozyme with Pax8-Cre-ER T2 Positive siblings served as controls. PCR amplification for Pax8-Cre-ER T2For transgene, the primer sequences were 5'-ATGCCCACCAAAGTCATCAGTGTAG--3' (SEQ ID NO: 17) and 5'-CCAGTCCCAGGTACTGACAGAG-3' (SEQ ID NO: 18), respectively.

[0115] Construction of knockout and overexpression stable cell lines

[0116] LentiV2-RINLgRNA and pLV-RINL plasmids were constructed and co-transfected with PMDL, VSVG and REV plasmids into 293T cells for 24 hours using lip2000 to package the virus and obtain the virus suspension. The virus suspension was used to infect the target HK2 cells for 8 hours, and finally, after puro screening, RINL knockout and overexpression HK2 cell lines were obtained.

[0117] Immunohistochemical staining

[0118] Paraffin-embedded kidney sections were dewaxed and dehydrated through a series of xylene and alcohol gradient concentrations. Antigen retrieval was performed by autoclaving at 100°C for 5 min in sodium citrate buffer (pH 6.0). After this, the sections were treated with 3% hydrogen peroxide for 10 min to inhibit endogenous catalase activity. After that, the sections were pre-incubated with 5% goat serum for 1 hour and then incubated with anti-RINL antibody (1:200, Abcam, cat. no. ab243527) at 4°C overnight. The sections were then incubated with horseradish peroxidase-conjugated secondary antibody AffiniPure Goat Anti-Rabbit IgG (H+L) (Bioss) at 37°C for 1 hour and then subsequently treated with streptavidin HRP AffiniPure goat anti-rabbit IgG (H+L) (Boster, cat. no. BA1039). The expression of RINL in the kidney was analyzed using a Zeiss microscope (Oberkochen, Germany).

[0119] Immunofluorescence staining

[0120] After 24 hours of cell treatment, specific HK2 cells cultured in 24-well plates were fixed with 4% paraformaldehyde. Afterwards, they were blocked with 3% FBS; or paraffin-embedded kidney sections were dewaxed and dehydrated through a series of xylene and alcohol gradient concentrations. Antigen retrieval was performed by autoclaving at 100°C for 5 min in sodium citrate buffer (pH 6.0).

[0121] Cells or tissues were incubated with anti-hRINL, hABCG2, and mRINL antibodies at 4°C overnight (1:100, ABclonal, Wuhan, China, catalog #A17110). 555-conjugated goat anti-rabbit IgG (H+L) (1:400, ABclonal, Wuhan, China, catalog #AS057) was incubated at 37°C for 1 hour. Subsequently, cell nuclei were stained with DAPI (Sigma, Milwaukee, USA, catalog #D5942) for 5 min in the dark. Immunofluorescence images were collected and processed by Zen Software (Oberkochen, Germany).

[0122] Serum biochemical analysis

[0123] The concentrations of UA, CRE (creatinine), BUN (blood urea nitrogen), and XOD in serum were assessed using commercially available test kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

[0124] Western blot analysis

[0125] Kidney tissue or cells were homogenized in RIPA buffer containing 0.1% phenylmethylsulfonyl fluoride (Solarbio, Catalog No. R0010) and then centrifuged at 12000r / min for 20min at 4°C. The supernatant was collected and the protein concentration was determined using a BCA protein assay kit (Yamei, Catalog No. ZJ101). The protein sample was mixed with 5× loading buffer at a ratio of 4:1 and then boiled for 10min. Afterwards, the mixture was separated using 8%–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride membrane (Merck, Catalog No. IPVH00010) for 2 hours.

[0126] After transfer, the membrane was incubated with 5% skim milk dissolved in TBST for 1 hour. After that, the membrane was incubated at 4°C for 12 hours with antibodies targeting RINL (1:200, Abcam, Catalog No. ab243527), ABCG2 (CST, Catalog No. 42078), and OAT3 (Proteintech, Catalog No. A3119) at a dilution of 1:1000 each.

[0127] After three TBST washes, the membrane was incubated with secondary antibodies AffiniPure Goat Anti-Mouse IgG (H+L) (Boster, Catalog No. BA1038) and AffiniPure Goat Anti-Rabbit IgG (H+L) (Boster, Catalog No. BA1039) at room temperature at a dilution of 1:10000 for 12 hours. Subsequently, the membrane was imaged using an enhanced chemiluminescence kit (Advansta, Catalog No. K-12045-D50) and grayscale scanning analysis was performed using Image J software.

[0128] Example

[0129] A further understanding of the present disclosure may be obtained by reference to some specific embodiments given herein, which are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure in any way. Obviously, various modifications and variations may be made to the present disclosure without departing from the essence of the present disclosure, and therefore, these modifications and variations are also within the scope of the present application. The clinical trial data used in the present disclosure have been obtained with the knowledge and consent of the subjects.

[0130] Example 1: RINL is a gene associated with HUA

[0131] The applicant screened a HUA family in southern Fujian, collected whole blood from HUA patients and healthy people in the family, and performed whole exome sequencing on the whole blood of family members. The applicant unexpectedly found that the diseased members all had heterozygous mutations in the RINL gene, while the healthy members did not have this mutation, see Figure 1 A and Figure 1 B. At the same time, by comparing the RINL amino acid sequences of different species, it was found that the amino acid site corresponding to the mutation site is conserved in multiple species. Figure 1 C.

[0132] At the same time, since the kidney is the main organ for uric acid excretion and the renal tubule is the main site for uric acid excretion and reabsorption, the applicant obtained paraffin-embedded samples of healthy human kidneys from the Fifth Hospital of Xiamen. After sectioning and immunohistochemical staining, it was found that RINL was expressed to a certain extent in the renal tubules. The results are shown in Figure 1 D, therefore, this also suggests that RINL may affect renal tubular uric acid excretion.

[0133] The results of whole exome sequencing, protein sequence alignment and immunohistochemistry for the expression location of RINL indicate that RINL may be a potential HUA-related gene and may affect renal tubular uric acid excretion. That is, RINL may be a new target related to uric acid excretion.

[0134] RINL (Ras And Rab Interactor Like) is a protein-coding gene that participates in a variety of biological processes in cells as a guanine nucleotide exchange factor (GEF), especially related to vesicle-mediated transport and the regulation of Rab proteins.

[0135] Example 2: RINL knockdown promotes uric acid excretion in HK2 cells

[0136] In order to further explore the specific effect of the RINL gene on uric acid excretion, the following cell experiments were performed in this example.

[0137] Considering that renal proximal tubular epithelial cells are the main site of uric acid excretion, the human renal proximal tubular epithelial cell line (HK2) was selected as a model cell in this embodiment, and three groups of siRNA targeting RINL were designed. The sequence information is shown in Table 1 below. RINL was knocked down using siRNA by liposome transfection. HK2 cells were lysed to collect proteins, and the expression level of RINL in the cells was analyzed by immunohistochemistry. The results, as shown in 2A, showed that siRNA significantly reduced the expression of RINL in the model cells. After another siRNA, siRINL-2, was selected to knock down RINL in HK2 cells by liposome transfection, the cell culture fluid was collected and the uric acid level therein was detected by a uric acid detection kit (Nanjing Jiancheng). The applicant found that as the expression of RINL in the cells decreased, the uric acid level in the cell culture fluid increased significantly, which also suggested that the level of uric acid secreted by HK2 cells increased significantly, see. Figure 2 B. Correspondingly, after RINL knockdown, the expression level of uric acid excretion protein ABCG2 in HK2 cells increased significantly, see Figure 2 C, D.

[0138] Table 1 Sequence information of siRINL

[0139]

[0140] The above results indicate that RINL is an effective target for promoting uric acid excretion in renal tubular epithelial cells. Knocking down RINL significantly increases the expression of uric acid excretion protein ABCG2, and reduces the intracellular uric acid content by promoting uric acid excretion outside the cells, indicating that RINL plays an important physiological role in the physiological process of uric acid excretion.

[0141] Example 3: RINL knockout promotes uric acid secretion in HK2 cells

[0142] In this example, a RINL-knockout HK2 cell line was constructed and combined with a HUA cell model to further explore the effect of RINL on uric acid excretion.

[0143] Using human renal proximal tubule epithelial cell line (HK2) as model cells, a RINL knockout cell model was constructed, and its cell activity was tested by MTT cytotoxicity assay. The experimental results are shown in Figure 3A, it can be seen that the knockout of RINL will not have a significant effect on the cell activity state. The normal and RINL knockout HK2 cell lines were used to construct the HUA cell model (the cells were treated with 2.5mM adenosine for 24h, and then 0.005U / LXOD was added to treat the cells for 8h), the cell culture medium was collected, and the uric acid level in the culture medium was detected by a uric acid detection kit (Nanjing Jiancheng). At the same time, the cells were collected, resuspended with PBS, and the cells were shaken and broken. After centrifugation at 12000 rpm for 15min, the supernatant was collected, and the uric acid level in the supernatant was detected by a uric acid detection kit (Nanjing Jiancheng). At the same time, the sample protein level was detected and corrected using the protein level. Results are shown in Figure 3 B and 3C, compared with the control group, the extracellular uric acid level was significantly increased and the intracellular uric acid level was significantly decreased after RINL knockout.

[0144] Normal and RINL knockout HK2 cell lines were used to construct HUA cell models, and the cells were broken to collect intracellular proteins. The levels of intracellular uric acid excretion proteins OAT3 and ABCG2 were detected by immunoblotting. Figure 3 As shown in D and 3E, the level of uric acid excretion protein in HK2 cells with RINL knockout was significantly increased, which also indicates that RINL knockout promotes the excretion of cellular uric acid.

[0145] In addition, immunofluorescence was performed on normal and RINL knockout HK2 cell slides to detect the intracellular RINL and ABCG2 levels. Figure 3 As shown in F and 3G, the results showed that the level of intracellular uric acid excretion protein ABCG2 was significantly increased after RINL knockout.

[0146] The above results indicate that RINL knockout can promote uric acid excretion in human renal proximal tubular epithelial cells.

[0147] Example 4: RINL overexpression inhibits uric acid secretion in HK2 cells

[0148] In this example, the effect of RINL on uric acid excretion was further explored by constructing a RINL-overexpressing HK2 cell line and combining it with a HUA cell model.

[0149] A cell model with stable overexpression of RINL was constructed and its cell activity was tested by MTT cytotoxicity assay. The experimental results are shown in Figure 4A, it can be seen that the overexpression of RINL will not have a significant effect on the cell activity state. The HUA cell model was constructed using normal and RINL overexpressing HK2 cell lines (the cells were treated with 2.5mM adenosine for 24h, and then 0.005U / L XOD was added to treat the cells for 8h), the cell culture medium was collected, and the uric acid level in the culture medium was detected using a uric acid detection kit (Nanjing Jiancheng). At the same time, the cells were collected, resuspended with PBS, and the cells were shaken and broken. After centrifugation at 12000 for 15min, the supernatant was collected, and the uric acid level in the supernatant was detected using a uric acid detection kit (Nanjing Jiancheng). At the same time, the sample protein level was detected and corrected using the protein level. Results are shown in Figure 4 B and 4C, compared with the control group, the extracellular uric acid level of RINL-overexpressing cells was significantly increased, and the intracellular uric acid level was significantly decreased.

[0150] Normal and RINL overexpressing HK2 cell lines were used to construct HUA cell models, and the intracellular proteins were collected by crushing the cells. The levels of intracellular uric acid excretion proteins OAT3 and ABCG2 were detected by immunoblotting. Figure 4 As shown in D and 4E, the level of uric acid excretion protein in HK2 cells overexpressing RINL was significantly increased, which also indicates that RINL overexpression inhibits the excretion of cellular uric acid.

[0151] Immunofluorescence was performed on normal and RINL-overexpressing HK2 cell slides to detect the levels of intracellular RINL and ABCG2. Figure 4 As shown in F and 4G, the results showed that the level of intracellular uric acid excretion protein ABCG2 was significantly increased after RINL overexpression.

[0152] In summary, the experimental results indicate that RINL overexpression inhibits uric acid excretion in human renal proximal tubular epithelial cells.

[0153] Example 5: RINL (P139S) point mutation inhibits uric acid secretion in HUA cell model

[0154] In this example, a RINL (P139S) point mutation stably overexpressing HK2 cell line was constructed to explore the effect of RINL point mutation on uric acid excretion in renal proximal tubular epithelial cells.

[0155] Flag-RINL and Flag-RINL(P139S) point mutation stably overexpressing HK2 cell lines were constructed by lentiviral packaging and infection. Figure 5 As shown in A, mRNA level detection found that the Flag-RINL (P139S) mRNA level was significantly increased after overexpression, but there was no significant difference compared with Rinl mRNA in Flag-RINL overexpressing cells, suggesting that the RINL (P139S) point mutation has no effect on the transcription of this gene.

[0156] Normal, RINL overexpressing, and RINL (P139S) point mutation overexpressing HK2 cell lines were used to construct HUA cell models (cells were treated with 2.5 mM adenosine for 24 h, and then 0.005 U / L XOD was added to treat the cells for 8 h). The cell culture medium was collected, and the uric acid level in the culture medium was detected using a uric acid detection kit (Nanjing Jiancheng). At the same time, the cells were collected, resuspended with PBS, and the cells were shaken and broken. After centrifugation at 12,000 rpm for 15 min, the supernatant was collected, and the uric acid level in the supernatant was detected using a uric acid detection kit (Nanjing Jiancheng). At the same time, the sample protein level was detected and corrected using the protein level. Figure 5 As shown in BE, compared with HK2 cells overexpressing RINL, the intracellular uric acid excretion protein level was significantly reduced after RINL (P139S) point mutation, the extracellular uric acid level showed a more significant decrease, and the intracellular uric acid level was more significantly increased.

[0157] Normal, RINL overexpressing, and RINL (P139S) point mutation overexpressing HK2 cell lines were used, and the intracellular proteins were collected by cell disruption. The levels of intracellular RINL and uric acid excretion protein ABCG2 were detected by immunoblotting. Figure 5 F shows that compared with HK2 cells with RINL overexpression, the level of RINL in cells after RINL(P139S) point mutation increased significantly, and the level of uric acid excretion protein ABCG2 decreased significantly. This also shows that after RINL(P139S) point mutation, the protein is not easily degraded, allowing it to exist more stably in cells and play a role in inhibiting uric acid excretion.

[0158] The above results show that the RINL (P139S) point mutation inhibits the degradation of RINL protein in cells, increases the level of the protein in cells, and further inhibits the excretion of uric acid in HK2 cells.

[0159] Example 6: RINL knockout promotes uric acid excretion in mouse kidneys and reduces uric acid levels in mice

[0160] In this example, RINL knockout mice were constructed to explore the effect of RINL on uric acid excretion at the in vivo level.

[0161] The CRISPR-Cas9 method was used to construct mice with systemic knockout of the RINL large fragment, and the flowchart is shown in the figure. Figure 7 As shown in A. After obtaining enough pups, the postnatal developmental indicators of the mice were tested, such as Figure 6 A- Figure 6As shown in Figure H, there is no significant difference between wild-type and RINL knockout mice in terms of auricle separation, body hair, lower tooth eruption, upper tooth eruption, eye opening, ear opening, crawling, and flat turning, which indicates that RINL knockout has no significant effect on the development of mice. Since the gender characteristics of newborn mice cannot be seen as female or male, the detection of developmental indicators is conducted on all offspring of wild-type and knockout mice. Figure 7 As shown in B, the genotypes of wild-type and RINL homozygous knockout male mice were obtained by clipping the toes of mice. The kidney tissues of 8-month-old wild-type and RINL knockout male mice were detected by qPCR, immunoblotting, immunofluorescence, and immunohistochemistry. Figure 7 As shown in C to F, RINL expression was not found in the kidneys of RINL knockout mice. The applicant used a uric acid detection kit (Nanjing Jiancheng) to detect the serum uric acid levels of male and female mice at 2, 4, 8, 12, and 16 months of age, and found that compared with wild-type mice, the serum uric acid levels of RINL knockout mice were significantly reduced with age. Figure 7 G and 7H, which also shows that RINL knockout can reduce the uric acid level in mice. The applicant collected blood and kidney samples from 8-month-old mice and tested the mouse body weight, kidney-to-body weight ratio, serum uric acid, serum creatinine, and serum urea nitrogen levels. Figure 8 As shown in A to 8F, there was no significant difference in body weight, kidney-to-body ratio, and serum creatinine (detected by Nanjing Jiancheng Company's creatinine detection kit) levels between wild-type and knockout mice. Compared with wild-type mice, serum urea nitrogen (detected by Nanjing Jiancheng Company's creatinine detection kit) and uric acid (detected by Nanjing Jiancheng Company's creatinine detection kit) levels in RINL knockout mice were significantly reduced. Consistent with previous results, this indicates that RINL knockout can significantly reduce uric acid levels in mice without affecting the development of mice. Kidney damage in 8-month-old wild-type and RINL knockout male mice was detected by sectioning and HE staining, as shown in Figure 8 As shown in G, there was no obvious damage in the kidneys of knockout mice. The levels of interleukin-1β in the kidneys of 8-month-old wild-type and RINL knockout male mice were detected by ELISA kit. Figure 8 As shown in H, there is no obvious inflammation in the kidneys of RINL knockout mice. The levels of uric acid excretion protein in the kidneys of 8-month-old wild-type and RINL knockout male mice were detected by immunoblotting. Figure 8 As shown in I, compared with wild-type mice, the level of renal uric acid excretion protein OAT3 / ABCG2 in RINL knockout mice was significantly increased, which also indicates that RINL knockout can promote renal uric acid excretion in mice.

[0162] The above results show that complete knockout of RINL can promote uric acid excretion in the mice's kidneys without affecting their normal development.

[0163] Example 7: RINL knockout promotes renal uric acid excretion in HUA mice

[0164] In this example, a 2-month-old wild (RINL + / + ) and RINL knockout (RINL - / - ) HUA model of male and female mice was established to detect the effect of RINL on uric acid excretion in HUA mice.

[0165] HUA was induced in mice by intraperitoneal injection of potassium oxyphosphate (PO) and free drinking of 10% fructose water. PO was dissolved in normal saline and intraperitoneally injected into mice at a dose of 350 mg / kg per day for 28 days. The control group of mice was treated with normal saline (solvent). The serum uric acid levels of mice were measured on days 14 and 28. Fig. 9 As shown in A-9D, RINL knockout significantly reduced serum uric acid levels in HUA mice, regardless of female or male mice. The kidneys of mice were collected on day 29, and the levels of uric acid excretion proteins OAT3 / OAT1 in the kidneys of male mice were detected by immunoblotting. Fig. 9 As shown in E, consistent with the serum results, the levels of uric acid excretion proteins OAT3 / OAT1 in the mice kidneys were significantly increased after RINL knockout.

[0166] The above results indicate that RINL knockout can rescue the drug-induced increase in serum uric acid levels and make mice less susceptible to HUA.

[0167] Example 8: Tubule-specific knockout of RINL promotes uric acid excretion in mouse kidneys

[0168] In this example, RINL renal tubular specific knockout mice were constructed to further explore the effect of RINL on renal tubular uric acid excretion.

[0169] Construction of C57 / BL6 mice with specific knockout of renal tubule RINL (RINL Cko) Fig.10 As shown in A, the applicant inserted loxP sites at both ends of exon 4 of the RINL gene and expressed the loxP sites by interacting with Pax8 CreER T2 The mice were crossed and combined with tamoxifen to obtain RINL renal tubule-specific knockout mice. Fig.10 As shown in B, the applicant induced renal tubule-specific knockout of the RINL gene in male mice by injecting 75 mg / kg of tamoxifen daily (for 7 consecutive days) when the mice were 4 months old. Fig.10 As shown in C, after induction, kidney samples of some mice were collected and the level of Rinl mRNA was detected. The results showed that the level of Rinl mRNA in the kidneys of RINL renal tubule-specific knockout mice was significantly reduced, indicating that the renal tubular RINL gene was knocked out. Fig.10As shown in D-10F, the mice were weighed at 6 months of age and their serum uric acid and creatinine levels were tested by taking blood samples from the jaw. It was found that RINL renal tubule-specific knockout mice also significantly reduced uric acid and creatinine levels in mice.

[0170] The mice were killed at 8 months old, and their blood and kidney tissues were collected for testing, and the results were similar to those at 6 months old, such as Fig.10 As shown in G-10J, compared with control mice (RINL not knocked out in the renal tubules), RINL cKO mice had no significant differences in body weight and kidney-to-body ratio, but RINL cKO mice had significantly reduced serum uric acid and creatinine levels. Fig.10 As shown in K and 10L, urine tests of control and RINLcKO mice showed that urine uric acid and creatinine levels were significantly increased after RINL renal tubule-specific knockout. The uric acid excretion fraction (FEUA = (urine uric acid * serum creatinine) / (serum uric acid * urine creatinine) * 100%) and urine uric acid excretion rate (UUE = urine uric acid / urine creatinine) were calculated based on the test results. Fig.10 M and 10N, the levels of FEUA and UUE in RINL cKO mice were significantly increased. The results of serum and urine tests suggest that RINL tubule-specific knockout promotes the excretion of uric acid from the body through urine. The levels of renal uric acid excretion proteins in 8-month-old control and RINL cKO mice were detected by immunoblotting. Fig.10 As shown in Figure O, compared with control mice, the level of renal uric acid excretion protein OAT3 / ABCG2 in RINL cKO mice was significantly increased, which also indicates that tubular-specific knockout of RINL can promote renal uric acid excretion in mice.

[0171] The above results show that RINL can regulate renal tubular uric acid excretion by regulating the level of renal tubular uric acid excretion protein, and tubular-specific knockout of RINL promotes renal uric acid excretion.

[0172] In summary, the experimental results show that RINL is a HUA-related gene that significantly affects the excretion of uric acid in the renal tubules. Knockdown or knockout of RINL promotes uric acid secretion in HK2 cells. In mice, full knockout of RINL promotes uric acid excretion in the kidneys of mice and reduces uric acid levels in mice. For HUA mice, RINL knockout promotes uric acid excretion in the kidneys of HUA mice.

[0173] The above is only an embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and equivalent changes made by using the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

[0174] Table 2 Sequence information

[0175]

[0176]

Claims

1. Use of the RINL gene or its encoded protein, wherein the RINL gene or its encoded protein: (1) is used as a marker for diagnosis, detection or prognosis of hyperuricemia; (2) is used to prepare a reagent or kit for diagnosis or detection of hyperuricemia; or (3) is used to prepare a drug for prevention and / or treatment of hyperuricemia.

2. A RINL antagonist, wherein The RINL antagonist specifically targets the nucleotide sequence shown in SEQ ID NO.7 or specifically binds to the amino acid sequence shown in SEQ ID NO.8, and has any of the following functions: (1) Inhibit RINL gene expression; (2) Deletion or silencing of the RINL gene; (3) Antagonize RINL protein.

3. The RINL antagonist according to claim 2, wherein The RINL antagonist is selected from shRNA, siRNA, miRNA, dsRNA, stRNA, small molecule compounds, aptamers, antibodies or antigen-binding fragments thereof; Preferably, the antibody or antigen-binding fragment thereof is selected from the group consisting of a whole antibody, a F(ab)-fragment, a F(ab)2-fragment, a single-chain antibody, a chimeric antibody, a bivalent antibody-construct, a synthetic antibody, a bispecific single-chain antibody or a cross-cloned antibody; Preferably, the RINL antagonist is an anti-RINL antibody.

4. The RINL antagonist according to claim 2 or 3, wherein The RINL antagonist is a siRNA, the siRNA comprises a sense strand and an antisense strand, and the length of the siRNA is 15-30 base pairs, the antisense strand comprises a complementary region complementary to the mRNA encoding RINL; Preferably, the nucleotide sequence of the siRNA is selected from any one of the following combinations of sense strands and antisense strands: (1) a sense strand having the sequence shown in SEQ ID NO.1, and an antisense strand having the sequence shown in SEQ ID NO.2; or (2) a sense strand having a sequence as shown in SEQ ID NO.3, and an antisense strand having a sequence as shown in SEQ ID NO.4; or (3) A sense strand having a sequence as shown in SEQ ID NO.5, and an antisense strand having a sequence as shown in SEQ ID NO.

6.

5. The RINL antagonist according to claim 4, wherein The sense strand and antisense strand of the siRNA are further modified, and the modification is selected from: one or more of 2'-methoxyethyl modification, 2'-methoxy modification, 2'-deoxy-2'-fluoro modification, thiophosphate modification, adenosine-2' phosphate modification, uridine-2' phosphate modification, guanosine-2' phosphate modification, guanosine-diol nucleic acid modification, adenosine-diol nucleic acid modification, N-(2,3-dihydroxypropyl)-3,5-bis(trifluoromethyl)benzamide modification or 5'-phosphate mimetic modification.

6. The RINL antagonist according to claim 4 or 5, wherein At least one nucleotide of the siRNA is conjugated to one or more targeting ligands; Preferably, the targeting ligand is conjugated to the sense strand of the siRNA; Preferably, the targeting ligand is conjugated to any nucleotide of the sense strand of the siRNA; Preferably, the targeting ligand is conjugated to the 5' end of the sense strand of the siRNA; Preferably, the targeting ligand is conjugated to the 3'-end of the sense strand of the siRNA; Preferably, the targeting ligand comprises a protein, a carbohydrate or a lipid.

7. A composition comprising the RINL antagonist according to any one of claims 2 to 6, and optionally a pharmaceutically acceptable carrier or excipient.

8. The composition according to claim 7, wherein The composition further comprises a second part of drugs, which are selected from allopurinol, febuxostat, benzbromarone, probenecid, sodium bicarbonate, citric acid preparations or any combination thereof.

9. A method for inhibiting RINL expression in a cell, the method comprising the following steps: The RINL antagonist according to any one of claims 2 to 6 or the composition according to any one of claims 7 to 8 is delivered to a cell to inhibit the expression of the RINL gene in the cell.

10. Use of the RINL antagonist according to any one of claims 2 to 6 and / or the composition according to claim 7 or 8 in the preparation of a medicament for preventing and / or treating hyperuricemia in an individual.

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