Use of RINL gene and antagonist thereof in diagnosis and treatment of hyperuricemia

Abstract

Use of the RINL gene and an antagonist thereof in the diagnosis and treatment of hyperuricemia. Use of the RINL gene or the encoded protein thereof. The RINL gene or the encoded protein thereof is: (1) used as a marker for the diagnosis, detection, or prognosis of hyperuricemia; (2) used for preparing a reagent or kit for diagnosing or detecting hyperuricemia; or (3) used for preparing a drug for preventing and / or treating hyperuricemia. RINL is an important relevant gene in the development and progression of hyperuricemia, and is also a potential treatment site for hyperuricemia. Inhibiting the expression of RINL significantly improves the ability of cells and mice to excrete uric acid.

Description

Use of RINL gene and its antagonists in the diagnosis and treatment of hyperuricemia

[0001] Cross-references to related applications

[0002] This disclosure claims priority to Chinese Patent Application No. 2024110402743, filed on July 31, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure pertains to the field of biomedicine and specifically relates to the use of the RINL gene and its antagonists in the diagnosis and treatment of hyperuricemia. Background Technology

[0004] Hyperuricemia (HUA) is a metabolic disease caused by purine metabolism disorder, characterized by elevated uric acid levels in the blood. Normally, uric acid is a product of purine metabolism and is excreted through the kidneys. When uric acid production is excessive or excretion is insufficient, the concentration of uric acid in the blood rises, exceeding its solubility in the blood, thus forming urate crystals, which may lead to health problems such as gout. Gout attacks cause severe pain, often referred to as the "king of pains," clinically manifesting as acute, episodic joint swelling and pain, frequently accompanied by elevated blood uric acid levels. Recent epidemiological data shows that the incidence of gout is increasing worldwide, influenced by factors such as race, genes, age, sex, diet, occupation, environment, and education level. Gout attacks severely impact patients' work and quality of life, placing immense psychological and economic burdens on patients and society. Therefore, research into the pathogenesis of gout and the development of effective preventative and therapeutic drugs are particularly important.

[0005] Currently, clinical medications for hyperuricemia are mainly divided into drugs that promote uric acid metabolism (benzbromarone, probenecid), drugs that inhibit uric acid production (allopurinol, febuxostat), drugs that alkalize urine (sodium bicarbonate), and drugs that promote uric acid dissolution (citric acid preparations). With the increasing prevalence of hyperuricemia (HUA) each year, there is a growing need for more diverse uric acid-lowering drugs to meet the treatment needs of different populations.

[0006] Given the diverse causes of hyperuricemia and the lack of a cure, there is an urgent need to develop new and more effective treatment methods. Summary of the Invention

[0007] In order to address the problems existing in the prior art, the purpose of this disclosure is to provide the use of the RINL gene and its antagonists in the diagnosis and treatment of hyperuricemia.

[0008] To achieve the above objectives, this disclosure adopts the following specific solutions:

[0009] In one aspect, this disclosure provides a use of the RINL gene or its encoded protein, wherein the RINL gene or its encoded protein is used as a biomarker for the diagnosis, detection or prognosis of hyperuricemia; (2) for the preparation of reagents or kits for the diagnosis or detection of hyperuricemia; or (3) for the preparation of drugs for the prevention and / or treatment of hyperuricemia.

[0010] On the other hand, this disclosure provides an RINL antagonist for the prevention and / or treatment of hyperuricemia, 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:

[0011] (1) Inhibit RINL gene expression;

[0012] (2) Cause the RINL gene to be deleted or silenced;

[0013] (3) Antagonize RINL protein.

[0014] On the other hand, this disclosure provides a composition comprising the aforementioned RINL antagonist, and optionally a pharmaceutically acceptable carrier or excipient.

[0015] On the other hand, this disclosure provides a method for inhibiting RINL expression in cells, the method comprising the following steps:

[0016] The aforementioned RINL antagonist or the aforementioned composition is delivered to cells to inhibit the expression of the RINL gene in the cells.

[0017] On the other hand, this disclosure provides the use of the aforementioned method, the aforementioned RINL antagonist, and / or the aforementioned composition in the preparation of a medicament for the prevention and / or treatment of hyperuricemia.

[0018] On the other hand, this disclosure provides a method for preventing and / or treating hyperuricemia, comprising administering a therapeutically effective amount of the aforementioned RINL antagonist and / or the aforementioned composition to a subject in need.

[0019] On the other hand, this disclosure provides the aforementioned RINL antagonists and / or the aforementioned compositions for the prevention and / or treatment of hyperuricemia. This disclosure has at least the following beneficial effects:

[0020] 1. The applicant unexpectedly discovered that RINL is an important related gene in the development and progression of HUA, and also a potential therapeutic site for HUA.

[0021] 2. The applicant found that knocking down / knockout RINL significantly improved the uric acid excretion capacity of cells and mice, and designed a series of RINL antagonists with good effects.

[0022] 3. The applicant discovered that the RINL protein has the potential to inhibit uric acid excretion. By constructing a stable overexpression HK2 cell line with the RINL (P139S) point mutation, it was demonstrated that the RINL (P139S) point mutation inhibits the degradation of the RINL protein in cells, thereby increasing the intracellular level of the protein and further inhibiting uric acid excretion in HK2 cells.

[0023] 4. By constructing RINL knockout mice and renal tubule-specific knockout mice, the applicant verified that RINL knockout can promote renal tubular uric acid excretion and reduce uric acid levels in mice. Similarly, RINL knockout also promoted renal uric acid excretion in HUA mice. Attached Figure Description

[0024] Figure 1 shows that RINL is a pathogenic gene associated with HUA. Figure 1A is a pedigree chart of HUA families in southern Fujian. Figure 1B shows the sanger gene sequencing of family members. Figure 1C shows the comparison of RINL protein sequences / mutation sites between humans and other species. Figure 1D shows the location of RINL expression in healthy kidneys detected by immunohistochemistry.

[0025] Figure 2 illustrates how RINL knockdown promotes uric acid excretion from HK2 cells. Figure 2A compares the effects of three RINL siRNAs on RINL knockdown in HK2 cells. Figure 2B shows the extracellular uric acid level after si-RINL2 treatment of HK2 cells. Figures 2C and 2D show the intracellular uric acid excretion protein ABCG2 level after si-RINL2 treatment of HK2 cells; 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-tests. *P < 0.05.

[0026] Figure 3 shows that RINL knockout promotes uric acid secretion in HK2 cells. Figure 3A shows the cell viability of HK2 cells (control, LentiV2, and RINL knockout (LentiV2-RINL gRNA)) after 7 days. Figure 3B shows the extracellular uric acid levels of HK2 cells (control, LentiV2, and RINL knockout (LentiV2-RINL gRNA)) under normal (and induced HUA cell model) conditions. Figure 3C shows the intracellular uric acid levels of HK2 cells (control, LentiV2, and RINL knockout (LentiV2-RINL gRNA)) under normal (and induced HUA cell model) conditions. Figure 3D shows the levels of the uric acid excretion protein OAT3 / ABCG2 in HK2 cells (control, LentiV2, and RINL knockout (LentiV2-RINL gRNA)) under normal conditions, as detected by Western blotting. Figure 3E shows the levels of the uric acid excretion protein OAT3 / ABCG2 in HK2 cells (control, LentiV2, and RINL knockout (LentiV2-RINL gRNA)) under normal conditions, as detected by Western blotting. The levels of uric acid excretion protein OAT3 / ABCG2 in HK2 cells (using gRNA) under induced HUA cell model conditions are shown in Figures 3F and 3G. These figures illustrate the intracellular RINL (F) and RINL knockout (LentiV2-RINL gRNA) levels in HK2 cells under immunofluorescence assays. Data presented here represent at least three independent experiments. All data are expressed as SEM ± mean, and p-values ​​were determined by unpaired t-tests. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0027] Figure 4 shows the inhibition of uric acid secretion in HK2 cells by RINL overexpression. Figure 4A shows the cell viability of HK2 cells after 7 days in control (pLV) and RINL-stably overexpressed (pLV-RINL) cells. Figure 4B shows the extracellular uric acid levels in control (pLV) and RINL-stably overexpressed (pLV-RINL) HK2 cells under normal (and induced HUA cell model) conditions. Figure 4C shows the intracellular uric acid levels in control (pLV) and RINL-stably overexpressed (pLV-RINL) HK2 cells under normal (and induced HUA cell model) conditions. Figure 4D shows the immunoblotting results for the control (pLV) cell. The levels of uric acid excretory protein OAT3 / ABCG2 in HK2 cells with stable RINL overexpression (pLV-RINL) under normal conditions are shown in Figure 4E. Figure 4F and 4G show the intracellular RINL (F) and uric acid excretory protein ABCG2 (G) levels in HK2 cells with stable RINL overexpression (pLV-RINL) and control (pLV) cells under induced HUA cell model conditions, as detected by Western blotting. 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-tests. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0028] Figure 5 shows the further inhibition of uric acid secretion by HK2 cells by the RINL (P139S) point mutation. Figure 5A shows the detection of Rinl mRNA levels in HK2 cells with control (pLV), stable RINL overexpression, and stable RINL (P139S) point mutation overexpression. Figures 5B and 5C show the extracellular (B) and intracellular (C) uric acid levels in HK2 cells with control (pLV), stable RINL overexpression, and stable RINL (P139S) point mutation overexpression under normal conditions. Figures 5D and 5E show the extracellular (D) and intracellular (E) uric acid levels in HK2 cells with control (pLV), stable RINL overexpression, and stable RINL (P139S) point mutation overexpression under induced HUA cell model conditions. Figures 5F-5H show the intracellular RINL and uric acid excretion protein ABCG2 levels in HK2 cells with control (pLV), stable RINL overexpression, and stable RINL (P139S) point mutation overexpression. The data provided here represent at least three independent experiments. All data are expressed as SEM ± mean, and p-values ​​were determined by unpaired t-tests. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, #P<0.05, ##P<0.01.

[0029] Figure 6 shows the wild type (RINL)+ / + ) and RINL knockout (RINL - / - Postnatal developmental markers in mice were assessed. Figures 6A-6H show the results for wild-type (RINL) mice. + / + ) and RINL knockout (RINL - / - The timeframes at which newborn mice exhibited developmental milestones such as ear separation, body fur, lower tooth eruption, upper tooth eruption, eye opening, ear opening, crawling, and righting. n = 35-50. All data are expressed as SEM ± mean, and p-values ​​were determined by unpaired t-tests. *P < 0.05, **P < 0.01.

[0030] Figure 7 shows that RINL knockout promotes renal uric acid excretion and reduces uric acid levels in mice. Figure 7A shows the strategy for constructing RINL knockout mice, and Figure 7B shows the wild-type (RINL) knockout mice. + / + ) and RINL knockout (RINL - / - DNA gel electrophoresis images for mouse genotyping, Figure 7C shows the immunoblotting detection of 8-month-old wild-type mice (RINL). + / + ) and RINL knockout (RINL - / - RINL protein in mouse kidneys, Figure 7D shows the immunofluorescence detection of wild-type RINL protein in 8-month-old mice. + / + ) and RINL knockout (RINL - / - RINL protein in mouse kidneys, Figure 7E shows the qPCR detection of wild-type RINL protein in 8-month-old mice. + / + ) and RINL knockout (RINL - / - Rinl mRNA levels in the kidneys of mice. Figure 7F shows the immunohistochemical detection of Rinl mRNA levels in 8-month-old wild-type mice. + / + ) and RINL knockout (RINL - / - ) mouse kidney RINL, Figures 7G and 7H show the detection of 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. Data presented here represent at least three independent experiments. All data are expressed as SEM ± mean, and p-values ​​were determined by unpaired t-tests. *P < 0.05, **P < 0.01.

[0031] Figure 8 shows the reduction in uric acid levels in mice after complete RINL knockout. Figures 8A-8F show the results of 8-month-old wild-type (RINL) mice. + / + ) and RINL knockout (RINL - / - The detection and statistical analysis of body weight (A), kidney-to-body weight ratio (B), serum creatinine (C), serum urea nitrogen (D), urea nitrogen / creatinine ratio (E), and serum uric acid (F) levels in male mice were performed. Figure 8G shows the results for 8-month-old wild-type (RINL) mice. + / +) and RINL knockout (RINL - / - Kidney sections from male mice were stained with hematoxylin and eosin (HE), and the intertubular spaces were counted. Figure 8H shows the kidney sections from 8-month-old wild-type mice (RINL). + / + ) and RINL knockout (RINL - / - Interleukin-1β (IL-1β) assays in the kidneys of male mice. Figures 8I and 8J show the results for 8-month-old wild-type mice (RINL). + / + ) and RINL knockout (RINL - / - Detection of uric acid excretion protein OAT3 / ABCG2 levels in the kidneys of male mice. Data presented here represent at least three independent experiments. All data are expressed as SEM ± mean, and p-values ​​were determined by unpaired t-tests. *P<0.05, **P<0.01.

[0032] Figure 9 shows the results of RINL promoting uric acid excretion in the kidneys of HUA mice. Figures 9A-9B show the results of RINL-induced uric acid excretion in 2-month-old wild-type mice. + / + ) and RINL knockout (RINL - / - Serum uric acid levels in male and female mice on day 14 of HUA model establishment. Figures 9C-9D show the detection of uric acid levels in 2-month-old wild-type mice (RINL). + / + ) and RINL knockout (RINL - / - Serum uric acid levels in male and female mice on day 28 of HUA model establishment. Figure 9E shows the detection of uric acid levels in 2-month-old wild-type mice (RINL). + / + ) and RINL knockout (RINL - / - Normal / HUA male rat renal uric acid excretion protein OAT1 / OAT3 levels; 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-tests. *P<0.05, **P<0.01.

[0033] Figure 10 shows how RINL tubule-specific knockout promotes uric acid excretion in the kidneys of male mice. Figures 10A and 10B illustrate the construction strategy for RINL conditional knockout mice. Figure 10C shows the kidney rinl cells of 4-month-old control and RINL tubule-specific knockout (Cko) male mice. mRNA level detection: Figures 10D-10F show 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; Figures 10G-10J show the 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; Figures 10K and 10L show the urinary uric acid (K) and creatinine (L) levels of 8-month-old control (Control) and RINL tubule-specific knockout (Cko) male mice; Figures 10M and 10N show the urinary fractional excretion (FEUA) (M) and urinary uric acid excretion rate (N) levels of 8-month-old control (Control) and RINL tubule-specific knockout (Cko) male mice. Figures 10O and 10P show the expression levels and quantitative data of proteins (RINL, OAT3, ABCG2). 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-tests. *P<0.05, **P<0.01. Detailed Implementation

[0034] I. Terminology

[0035] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0036] The articles “a” and “a kind” used in this article refer to one or more (i.e., at least one) grammatical objects. For example, “a kind of element” means one element or more elements.

[0037] As used herein, the term "RINL" stands for Ras and Rab Interactor Like, referring to Ras and Rab interaction factor-like proteins that participate in various biological processes within cells, particularly in vesicle-mediated transport and the regulation of Rab proteins. RINL-expressed proteins can activate Rab5 subfamily small GTPases, thereby playing a role in endocytosis. RINL is expressed in various tissues, with particularly high levels in the thymus and spleen.

[0038] As used in this article, the term "OAT3" stands for Organic Anion Transporter 3, a membrane transporter belonging to the SLC22A family, responsible for the transmembrane transport of various endogenous and exogenous compounds. OAT3 is primarily expressed on the basolateral membrane of the proximal tubules of the kidney. OAT3 participates in the secretion, reabsorption, and distribution of various drugs in the kidneys by mediating the transport of organic anions. Furthermore, OAT3 is involved in uric acid excretion, playing a crucial role in maintaining serum uric acid levels and preventing hyperuricemia.

[0039] As used in this article, the term "ABCG2" refers to ATP-binding cassette transporter G2, also known as breast cancer resistance protein (BCRP), a member of the ATP-binding cassette (ABC) transporter family. ABCG2 is expressed in various tissues, including the intestine, liver, kidneys, placenta, and blood-brain barrier. It is a multidrug resistance protein that pumps various drugs from inside cells to outside cells, thereby reducing intracellular drug concentrations and leading to resistance in tumor cells to certain chemotherapeutic drugs. Furthermore, it is involved in the transport of endogenous metabolites, including uric acid excretion. ABCG2 is expressed at the brush membrane end of the proximal tubules of the kidney, participating in urate secretion and also regulating uric acid levels extrarenally. Mutations in the ABCG2 gene or changes in its expression levels can affect uric acid metabolism homeostasis, leading to elevated or decreased serum uric acid levels, and are associated with the risk of hyperuricemia and gout.

[0040] As used in this article, the term "uric acid" is a colorless, odorless crystalline solid with the chemical formula C5H4N4O3, and is the final product of purine metabolism in humans and other mammals. Purines are components of nucleic acids; they are released during cell division and death and are eventually metabolized into uric acid. In the human body, uric acid is mainly produced through purine metabolism in the liver and excreted 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 there is excessive production or insufficient excretion of uric acid in the body, blood uric acid levels rise, leading to hyperuricemia.

[0041] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include their progeny. Therefore, the words “transformation” and “transformed cell” include primary test cells and cultures derived from them, regardless of the number of transfections. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in terms of DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells. Where different names are used, the context will be clear.

[0042] As used in this article, RNA interference (RNAi) is a sequence-specific RNA targeting process that provides a way to directly knock down and effectively silence 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 growing double-stranded RNA (dsRNA), which is then cleaved by the RNase III / helicase protein Dicer into small interfering RNA (siRNA) molecules of 19–27 nucleotides (nt), with a 2 nt overhang at the ' end. The siRNA is then incorporated into a ribonuclease-protein complex called the RNA-induced silencing complex (RISC). One strand of the siRNA remains associated with the RISC to guide the complex toward the complementary target RNA. This siRNA-directed endonuclease digests the target mRNA, resulting in truncation and inactivation of the targeted RNA. The degradation of the targeted mRNA leads to reduced translation of the resulting protein. In this way, siRNA-directed RNAi can effectively silence or knock out the targeted gene without mutating or altering the genomic DNA sequence.

[0043] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0044] "Expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector contains 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 clomids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus) incorporating recombinant polynucleotides.

[0045] When applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, "application," "giving," and "treatment" refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Application," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Application," "giving," and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. When "treatment" is applied to humans, veterinary, or research subjects, it refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0046] "Treatment" means administering an oral or topical therapeutic agent to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more symptoms of the disease, whether by inducing the regression of these symptoms or inhibiting their progression to any clinically unmeasurable degree. The amount of therapeutic agent that effectively relieves any specific disease symptom (also called the "therapeutic effective dose") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, as well as the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating the symptoms of the target disease present in every patient, they should reduce the symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student 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 "treatment" for an individual suffering from a disease or disease condition indicates that the individual's symptoms are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing underlying disease and / or preventing the worsening of symptoms or the development of disease. Treatment also includes any chimeric antigen receptors provided and any pharmaceutical use of the compositions provided herein.

[0047] The term “consistently of” or variations thereof, used throughout the specification and claims, means that all said elements or groups of elements are included, and optionally include other elements that are similar to or different in nature from said elements, which do not significantly alter or introduce new properties to the specified dosing regimen, method or composition.

[0048] 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. This includes, but is not limited to, shRNA, siRNA, miRNA, dsRNA, small molecule compounds, stRNA, aptamers, or antibodies or their antigen-binding fragments.

[0049] A "vector" is a composition of substances containing isolated nucleic acids and capable of delivering those isolated nucleic acids into cells. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, etc.

[0050] As used herein, the terms “patient,” “subject,” “individual,” etc., are used interchangeably and are intended to include any living organism (e.g., a mammal) that can elicit an immune response. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species.

[0051] As used herein, the term "kit" is any article (e.g., a package or container) containing at least one reagent (e.g., a therapeutic agent, probe, small molecule, etc.) for the specific detection and / or therapeutic effect on the expression of the biomarkers described in this invention. The kit may be promoted, distributed, or sold as a whole for performing the methods described in this invention. The kit may contain one or more reagents essential for the expression of the composition used in the methods described in this invention. In some embodiments, the kit may further contain reference standards, such as nucleic acids encoding proteins that do not affect or regulate signaling pathways controlling immune responses, cell growth, division, migration, survival, or apoptosis. Many such control proteins are conceivable to those skilled in the art, including but not limited to: common molecular tags (e.g., green fluorescent protein and β-galactosidase); proteins not included in any classification including cell growth, division, migration, survival, or apoptosis by GeneOntology reference; or ubiquitous housekeeping proteins. The reagents in the kit may be provided in a single container or in a mixture of two or more reagents in a single container. Additionally, explanatory material describing the use of the compositions in the kit may be included.

[0052] 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 disease after treatment may vary depending on the patient's physiological or environmental condition, and it can be interpreted as all actions that predict the course of disease after treatment, taking into account the patient's overall condition. For the purposes of this disclosure, prognostic prediction can be interpreted as predicting the disease-free survival rate of gout patients by predicting the course of the disease and their post-treatment condition.

[0053] As used herein, the terms “effective amount” or “therapeutic effective amount” are used interchangeably and refer to the amount of a compound, formulation, substance or composition that effectively achieves a particular biological outcome or provides a therapeutic or preventive benefit as described herein.

[0054] Table 1. Abbreviations

[0055] II. Detailed Implementation Plan

[0056] In one aspect, this disclosure provides a use of the RINL gene or its encoded protein, wherein the RINL gene or its encoded protein is used as a biomarker for the diagnosis, detection or prognosis of hyperuricemia; (2) for the preparation of reagents or kits for the diagnosis or detection of hyperuricemia; or (3) for the preparation of drugs for the prevention and treatment of hyperuricemia.

[0057] On the other hand, this disclosure provides an RINL antagonist for the prevention and / or treatment of hyperuricemia, 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:

[0058] (1) Inhibit RINL gene expression;

[0059] (2) Cause the RINL gene to be deleted or silenced;

[0060] (3) Antagonize RINL protein.

[0061] In some embodiments, the RINL antagonist is selected from shRNA, siRNA, miRNA, dsRNA, stRNA, small molecule compounds, aptamers, or antibodies or their antigen-binding fragments.

[0062] In some embodiments, the antibody or its antigen-binding fragment is selected from whole antibodies, F(ab)-fragments, F(ab)2-fragments, single-chain antibodies, chimeric antibodies, bivalent antibody-constructions, synthetic antibodies, bispecific single-chain antibodies, or cross-clonal antibodies.

[0063] In some implementations, the RINL antagonist is an anti-RINL antibody.

[0064] In some embodiments, the RINL antagonist is siRNA, which comprises a sense strand and an antisense strand, each 15-30 base pairs in length, wherein the antisense strand contains a complementary region that is complementary to the mRNA encoding RINL.

[0065] In some embodiments, the nucleotide sequence of the siRNA is selected from any combination of the following sense and antisense strands:

[0066] (1) A positive chain having the sequence shown in SEQ ID NO.1, and an antisense chain having the sequence shown in SEQ ID NO.2; or

[0067] (2) A sense strand having the sequence shown in SEQ ID NO.3, and an antisense strand having the sequence shown in SEQ ID NO.4; or

[0068] (3) A sense chain having the sequence shown in SEQ ID NO.5 and an antisense chain having the sequence shown in SEQ ID NO.6.

[0069] In some embodiments, the siRNA comprises the sense strand shown in SEQ ID NO.3 and the antisense strand shown in SEQ ID NO.4.

[0070] In some embodiments, the sense and antisense strands of the siRNA are further modified, the modifications being selected from one or more of the following: 2'-methoxyethyl modification, 2'-methoxy modification, 2'-deoxy-2'-fluorine 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 mimicry modification.

[0071] In some implementations, at least one nucleotide of the siRNA is conjugated to one or more target ligands.

[0072] In some implementations, the targeting ligand is conjugated to the sense or antisense strand of the siRNA.

[0073] In some implementations, the targeting ligand is conjugated to any nucleotide on either the sense or antisense strand of the siRNA.

[0074] In some implementations, the targeting ligand is conjugated to the 5' end of the sense or antisense strand of the siRNA.

[0075] In some implementations, the targeting ligand is conjugated to the 3' end of the sense or antisense strand of the siRNA.

[0076] In some implementations, the targeting ligand comprises a protein, carbohydrate, or lipid.

[0077] In some implementations, the protein is selected from natural proteins, synthetic polyamino acids, and antibodies.

[0078] In some embodiments, the natural protein is selected from human serum albumin, low-density lipoprotein, or globulin.

[0079] 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, diethylene ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer, polyethylene glycol, polyvinyl alcohol, polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphoric acid.

[0080] In some embodiments, the carbohydrate is selected from dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, amino sugars, or hyaluronic acid.

[0081] In some embodiments, the lipid is selected from fatty acids, sterols, or phospholipids.

[0082] 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.

[0083] In some embodiments, the sterol is selected from cholesterol, cholesterol group, cholesterol alcohol, stigmasterol, cholesterol acid, or ergosterol.

[0084] In some embodiments, the phospholipid is selected from di-hexadecyl-racemic glycerol or triethylamine 1,2-di-O-hexadecyl-racemic glycerol-3-hydrophosphonate.

[0085] In some implementations, the targeting ligand comprises an amino sugar.

[0086] In some embodiments, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

[0087] In some implementations, the GalNac portion is a monovalent GalNAc portion, a divalent GalNAc portion, a trivalent GalNAc portion, or a tetravalent GalNAc portion.

[0088] In some implementations, the targeting ligand has the following structure:

[0089] In some embodiments, the targeting ligand is conjugated to the 3'-end of the positive strand of the siRNA via a linker, forming a conjugate as follows:

[0090] X can be selected from S or O.

[0091] In some implementations, the RINL antagonist can promote the expression of OAT3 and ABCG2 proteins, thereby increasing uric acid excretion.

[0092] On the other hand, this disclosure provides nucleic acids encoding the aforementioned RINL antagonist.

[0093] On the other hand, this disclosure provides an expression vector containing the aforementioned nucleic acid.

[0094] On the other hand, this disclosure provides a cell comprising the aforementioned nucleic acid and expression vector.

[0095] On the other hand, this disclosure provides a composition comprising the aforementioned RINL antagonist, and optionally a pharmaceutically acceptable carrier or excipient.

[0096] In some embodiments, the composition contains 1% to 99% of the RINL antagonist.

[0097] In some embodiments, the composition further comprises a second pharmaceutical ingredient selected from one or more of allopurinol, febuxostat, benzbromarone, probenecid, sodium bicarbonate, citrate preparations, and combinations thereof.

[0098] In some embodiments, the composition is formulated for administration via ocular, vaginal, rectal, nasal, transdermal, subcutaneous, intravenous infusion, intra-arterial, intralymphatic, intrabronchial, intrapleural, intraperitoneal, cerebrospinal, or intramuscular injection, or for administration via the lungs, intrathecal, or intracardiac. In some embodiments, subcutaneous administration is preferred.

[0099] On the other hand, this disclosure provides a method for inhibiting RINL expression in cells, the method comprising the following steps:

[0100] The aforementioned RINL antagonist or the aforementioned composition is delivered to cells to inhibit the expression of the RINL gene in the cells.

[0101] On the other hand, this disclosure provides the use of the aforementioned method, the aforementioned RINL antagonist, and / or the aforementioned composition in the preparation of a medicament for the prevention and / or treatment of individual hyperuricemia.

[0102] On the other hand, this 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.

[0103] On the other hand, this disclosure provides a prognostic method for hyperuricemia by detecting the expression and mutation status of RINL in subject samples.

[0104] Experimental Materials and Methods

[0105] Cell culture and transfection

[0106] HK2 (human renal cortical proximal tubular epithelial cells) were purchased from Procell Biotechnology Co., Ltd. (Wuhan, China). They were cultured in DMEM / F12 medium (Pricella, catalog number PM150312) with 10% fetal bovine serum (VivaCell, catalog number C04001) and 1% penicillin / streptomycin (Solarbio, catalog number P8420 / S8290) added, and cultured in an incubator containing 5% CO2.

[0107] Transfection was performed using Lipofectamine 2000 (Thermo, catalog number 11668-019).

[0108] HK2 cells were transiently transfected after co-incubation with Lipofectamine 2000 and si-NC and si-RINL. The si-RINL sequence is shown in Table 2 below.

[0109] Table 2. siRNA sequence information

[0110] Construction of conditional knockout mice

[0111] Mice were placed in a standard environment characterized by a 12-hour light / dark cycle, a temperature of 22–25°C, and 40–60% humidity, with free access to food and water. All animals were housed in a pathogen-specific sterile facility. 12-week-old male DB / M and DB / DB mice were obtained from Cavens Biotechnology (China). C57BL / 6J(B6) mice were obtained from Witter River Laboratories (China).

[0112] A large fragment of the RINL gene in mice was knocked out using the high-performance Crisper strategy (Figure 7a). These mice were then mated with B6 animals to obtain RINL heterozygous mice, which were then mated again to generate RIN homozygous mice. For RINL KO genotyping, 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 showed no band. PCR was also performed using the following primers: F: 5'-ATGAGGGAATGGATGAACGG-3' (SEQ ID NO: 11), R: 5'-TCTGGAGGTCAGTGAGGAGC-3' (SEQ ID NO: 12). RINL KO mice showed no band, while wild-type mice showed a 434 bp band.

[0113] To generate RINL loxP / loxP mice, a targeting vector was first constructed, inserting two loxP sites at both ends of the fourth exon of RINL. These were then mated with B6 animals to obtain RINL loxP / + mice, which were subsequently mated again to generate RINL loxP / loxP mice. For RINL loxP genotyping, 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 wild-type mice showed a 300 bp band.

[0114] RINL loxP / loxP mice and Pax8-Cre-ER T2 Mice were mated to generate renal tubule-specific RINL knockout mice, Pax8-Cre-ER. T2 + / RINL loxP / loxP(RINL cKO). Interbreeding with Pax8-Cre-ER T2 Positive siblings served as controls. Pax8-Cre-ER was detected by PCR amplification. T2The transgenic primer sequences are 5'-ATGCCCACCAAAGTCATCAGTGTAG--3' (SEQ ID NO:17) and 5'-CCAGTCCCAGGTACTGACAGAG-3' (SEQ ID NO:18).

[0115] Construction of stable cell lines by knockout and overexpression

[0116] LentiV2-RINL gRNA and pLV-RINL plasmid 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 a viral suspension. HK2 cells were then infected with the viral suspension for 8 hours, and finally, after puro selection, HK2 cell lines with RINL knockout and overexpression were obtained.

[0117] Immunohistochemical staining

[0118] Paraffin-embedded kidney sections were dewaxed and dehydrated using 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). Following this, sections were treated with 3% hydrogen peroxide for 10 min to inhibit endogenous catalase activity. Sections were then pre-incubated with 5% goat serum for 1 h, followed by overnight incubation at 4°C with anti-RINL antibody (1:200, Abcam, catalog number ab243527). Sections were then incubated with horseradish peroxidase-conjugated secondary antibody AffiniPure Goat Anti-Rabbit IgG (H+L) (Bioss) at 37°C for 1 h, followed by treatment with streptavidin HRP AffiniPure goat anti-rabbit IgG (H+L) (Boster, catalog number BA1039). RINL expression in the kidneys was analyzed using a Zeiss microscope (Oberkochen, Germany).

[0119] Immunofluorescence staining

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

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

[0124] Western blot analysis

[0125] Kidney tissue or cells were homogenized in RIPA buffer containing 0.1% benzyl sulfonyl fluoride (Solarbio, catalog number R0010) and then centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatant was collected, and protein concentration was determined using a BCA protein assay kit (Yamei, catalog number ZJ101). The protein sample was mixed with 5× loading buffer at a ratio of 4:1 and then boiled for 10 min. Subsequently, the mixture was separated by 8%–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride membrane (Merck, catalog number IPVH00010) for 2 hours.

[0126] After transfer, the membrane was incubated in 5% skim milk dissolved in TBST for 1 hour. Then, the membrane was incubated at 4°C for 12 hours using RINL-targeting antibodies (1:200, Abcam, catalog number ab243527), ABCG2 (CST, catalog number 42078), and OAT3 (Proteintech, catalog number A3119) at a dilution of 1:1000.

[0127] After three TBST washes, the membrane was incubated with the secondary antibodies AffiniPure Goat Anti-Mouse IgG (H+L) (Boster, catalog number BA1038) and AffiniPure Goat Anti-Rabbit IgG (H+L) (Boster, catalog number BA1039) at a 1:10000 dilution for 12 hours at room temperature. Subsequently, the membrane was imaged using an enhanced chemiluminescence assay kit (Advansta, catalog number K-12045-D50), and grayscale analysis was performed using ImageJ software.

[0128] Example

[0129] A further understanding of this disclosure can be obtained by referring to the specific embodiments given herein, which are for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. It is obvious that various modifications and variations can be made to this disclosure without departing from its spirit, and therefore such modifications and variations are also within the scope of protection claimed in this application. All clinical trial data used in this disclosure have been obtained with the informed consent of the subjects.

[0130] Example 1: RINL is a gene related to HUA.

[0131] The applicant screened a family with human invasive disease (HUA) in the Minnan region and collected whole blood samples from both HUA patients and healthy individuals within the family. Whole-exome sequencing was performed on the whole blood samples of all family members. Unexpectedly, the applicant discovered a heterozygous mutation in the RINL gene in all affected members, while this mutation was absent in healthy members (see Figures 1A and 1B). Furthermore, by comparing the RINL amino acid sequences of different species, it was found that the amino acid site corresponding to this mutation is conserved across multiple species (see Figure 1C).

[0132] Meanwhile, since the kidneys are the main organs for uric acid excretion, and the renal tubules are the main sites for uric acid excretion and reabsorption, the applicant obtained paraffin-embedded kidney samples from healthy humans at the Fifth Hospital of Xiamen. After sectioning and immunohistochemical staining, RINL was found to be expressed in the renal tubules, as shown in Figure 1D. Therefore, this also suggests that RINL may affect uric acid excretion in the renal tubules.

[0133] Based on the results of whole-exome sequencing, protein sequence alignment, and immunohistochemical detection of RINL expression location, it is indicated that RINL may be a potential HUA-related gene and may affect uric acid excretion in the renal tubules. In other words, RINL may be a novel target related to uric acid excretion.

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

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

[0136] To further investigate the specific effects of the RINL gene on uric acid excretion, the following cell experiments were conducted in this embodiment.

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

[0138] The above results indicate that RINL is an effective target for promoting uric acid excretion from renal tubular epithelial cells. Knocking down RINL significantly increases the expression of uric acid excretion protein ABCG2, thereby reducing intracellular uric acid content by promoting uric acid excretion to the extracellular space. This demonstrates that RINL plays an important physiological role in the physiological process of uric acid excretion.

[0139] Example 3: RINL knockout promotes uric acid secretion in HK2 cells

[0140] This embodiment further explores the role of RINL in uric acid excretion by constructing an HK2 cell line with RINL knockout and combining it with a HUA cell model.

[0141] A RINL knockout cell model was constructed using the human renal proximal tubular epithelial cell line (HK2) as the model cell. Cell viability was tested using the MTT assay, and the results are shown in Figure 3A. The results indicate that RINL knockout does not significantly affect cell viability. HUA cell models were constructed using normal and RINL knockout HK2 cell lines (cells were treated with 2.5 mM adenosine for 24 h, followed by 0.005 U / L XOD for 8 h). Cell culture medium was collected, and uric acid levels were measured using a uric acid assay kit (Nanjing Jiancheng). Simultaneously, cells were collected, resuspended in PBS, and after turbulence and centrifugation at 12000 rpm for 15 min, the supernatant was collected. Uric acid levels in the supernatant were measured using the same kit, and protein levels were also measured and corrected using protein levels. The results are shown in Figures 3B and 3C. Compared with the control group, RINL knockout significantly increased extracellular uric acid levels and significantly decreased intracellular uric acid levels.

[0142] HUA cell models were constructed using normal and RINL knockout HK2 cell lines, respectively. Intracellular proteins were collected by cell disruption, and the levels of intracellular uric acid excretion proteins OAT3 and ABCG2 were detected by Western blotting. As shown in Figures 3D and 3E, the levels of uric acid excretion proteins in RINL knockout HK2 cells were significantly increased, which also indicates that RINL knockout promotes the excretion of uric acid from cells.

[0143] In addition, immunofluorescence assays were performed on normal and RINL-knockout HK2 cells to detect intracellular RINL and ABCG2 levels, as shown in Figures 3F and 3G. The results showed that the intracellular uric acid excretion protein ABCG2 level was significantly increased after RINL knockout.

[0144] The results in summary indicate that RINL knockout can promote uric acid excretion from proximal tubular epithelial cells in the human kidney.

[0145] Example 4: RINL overexpression inhibits uric acid secretion in HK2 cells

[0146] In this embodiment, the effect of RINL on uric acid excretion was further explored by constructing an HK2 cell line overexpressing RINL and combining it with a HUA cell model.

[0147] A cell model stably overexpressing RINL was constructed, and its cell viability was tested by the MTT assay. The results are shown in Figure 4A, indicating that RINL overexpression did not significantly affect cell viability. HUA cell models were constructed using normal and RINL-overexpressing HK2 cell lines (cells were treated with 2.5 mM adenosine for 24 h, followed by 0.005 U / L XOD for 8 h). Cell culture medium was collected, and uric acid levels were detected using a uric acid assay kit (Nanjing Jiancheng). Simultaneously, cells were collected, resuspended in PBS, and after turbulence and centrifugation at 12000 rpm for 15 min, the supernatant was collected. Uric acid levels in the supernatant were detected using a uric acid assay kit (Nanjing Jiancheng), and protein levels were also measured and corrected using protein levels. The results are shown in Figures 4B and 4C. Compared with the control group, RINL overexpression significantly increased extracellular uric acid levels and significantly decreased intracellular uric acid levels.

[0148] HUA cell models were constructed using normal and RINL-overexpressing HK2 cell lines, respectively. Intracellular proteins were collected by cell lysis, and the levels of intracellular uric acid excretion proteins OAT3 and ABCG2 were detected by Western blotting. As shown in Figures 4D and 4E, the levels of uric acid excretion proteins in HK2 cells overexpressing RINL were significantly increased, which also indicates that RINL overexpression inhibits cellular uric acid excretion.

[0149] Immunofluorescence assays were performed on normal and RINL-overexpressing HK2 cells to detect intracellular RINL and ABCG2 levels, as shown in Figures 4F and 4G. The results showed that RINL overexpression significantly increased the intracellular uric acid excretion protein ABCG2 level.

[0150] In summary, the experimental results indicate that RINL overexpression inhibits uric acid excretion from proximal tubular epithelial cells in human kidneys.

[0151] Example 5: RINL (P139S) point mutation inhibits uric acid secretion in a HUA cell model

[0152] In this embodiment, the effect of RINL point mutation on uric acid excretion in renal proximal tubular epithelial cells was investigated by constructing a stable RINL(P139S) point mutation overexpressing HK2 cell line.

[0153] HK2 cell lines stably overexpressing the Flag-RINL and Flag-RINL(P139S) point mutations were constructed using lentiviral packaging and infection. As shown in Figure 5A, mRNA level detection revealed a significant increase in Flag-RINL(P139S) mRNA levels after overexpression, but no significant difference compared to Rinl mRNA in Flag-RINL overexpressing cells. This suggests that the RINL(P139S) point mutation has no effect on the transcription of this gene.

[0154] HUA cell models were constructed using HK2 cell lines with normal expression, RINL overexpression, and RINL (P139S) point mutation overexpression, respectively. Cells were treated with 2.5 mM adenosine for 24 h, followed by 0.005 U / L XOD for 8 h. Cell culture medium was collected, and uric acid levels were measured using a uric acid assay kit (Nanjing Jiancheng). Simultaneously, cells were collected, resuspended in PBS, and after vortexing to disrupt the cells, they were centrifuged at 12000 rpm for 15 min. The supernatant was collected, and uric acid levels in the supernatant were measured using the same kit. Protein levels were also measured and corrected using protein level analysis. As shown in Figures 5B-E, compared to HK2 cells with RINL overexpression, the intracellular uric acid excretion protein level was significantly reduced in cells with the RINL (P139S) point mutation, while the extracellular uric acid level showed a more significant decrease, and the intracellular uric acid level was significantly increased.

[0155] HK2 cell lines with normal expression, RINL overexpression, and RINL (P139S) point mutation overexpression were used. Intracellular proteins were collected by cell disruption, and the levels of RINL and uric acid excretion protein ABCG2 were detected by Western blotting. As shown in Figure 5F, compared with HK2 cells overexpressing RINL, the intracellular RINL level was significantly increased and the uric acid excretion protein ABCG2 level was significantly decreased in cells with the RINL (P139S) point mutation. This also indicates that the RINL (P139S) point mutation makes the protein less susceptible to degradation, allowing it to remain more stably within the cell and exert its inhibitory effect on uric acid excretion.

[0156] The results above show that the RINL (P139S) point mutation inhibits the degradation of the RINL protein in cells, leading to an increase in the intracellular level of the protein, which further inhibits uric acid excretion from HK2 cells.

[0157] Example 6: Complete knockout of RINL promotes uric acid excretion by the kidneys in mice and reduces uric acid levels in mice.

[0158] In this embodiment, the effect of RINL on uric acid excretion was investigated in vivo by constructing RINL knockout mice.

[0159] Mice with a large-scale systemic knockout of the RINL fragment were constructed using the CRISPR-Cas9 method, as shown in Figure 7A. After obtaining a sufficient number of pups, postnatal developmental indicators were examined, as shown in Figures 6A-6H. No significant differences were found between wild-type and RINL knockout mice in indicators such as ear separation, body hair growth, lower tooth eruption, upper tooth eruption, eye opening, ear opening, crawling, and righting. This indicates that RINL knockout has no significant impact on mouse development. Since newborn mice do not yet exhibit sex characteristics, developmental indicators were tested on all offspring of both wild-type and knockout mice. As shown in Figure 7B, genotyping was performed by clipping the toes of mice to obtain wild-type and homozygous knockout male mice. Kidney tissues from 8-month-old wild-type and RINL knockout male mice were analyzed using qRCR, Western blotting, immunofluorescence, and immunohistochemistry, as shown in Figures 7C-F. No RINL expression was observed in the kidneys of RINL knockout mice. The applicant used a uric acid detection kit (Nanjing Jiancheng) to detect serum uric acid levels in male and female mice aged 2, 4, 8, 12, and 16 months. The results showed that, compared to wild-type mice, RINL knockout mice exhibited significantly lower serum uric acid levels with increasing age. As shown in Figures 7G and 7H, this also indicates that RINL knockout can reduce uric acid levels in mice. The applicant collected blood and kidney samples from 8-month-old mice and measured mouse body weight, kidney-to-body weight ratio, serum uric acid, serum creatinine, and serum urea nitrogen levels, as shown in Figures 8A to 8F. There were no significant differences in body weight, kidney-to-body weight ratio, and serum creatinine (detected using a Nanjing Jiancheng creatinine detection kit) levels between wild-type and knockout mice. However, compared to wild-type mice, RINL knockout mice showed significantly lower serum urea nitrogen (detected using a Nanjing Jiancheng creatinine detection kit) and uric acid (detected using a Nanjing Jiancheng creatinine detection kit). Consistent with previous results, this indicates that RINL knockout can significantly reduce uric acid levels in mice without affecting their development. Kidney damage in 8-month-old wild-type and RINL knockout male mice was detected by sectioning and HE staining, as shown in Figure 8G. No obvious damage was observed in the kidneys of knockout mice. Interleukin-1β levels in the kidneys of 8-month-old wild-type and RINL knockout male mice were detected by ELISA, as shown in Figure 8H. No obvious inflammation was observed in the kidneys of RINL knockout mice. The levels of uric acid excretion proteins in the kidneys of 8-month-old wild-type and RINL knockout male mice were detected by Western blotting, as shown in Figure 8I. Compared with wild-type mice, the levels of uric acid excretion proteins OAT3 / ABCG2 in the kidneys of RINL knockout mice were significantly increased, indicating that RINL knockout can promote uric acid excretion in mice.

[0160] The results above show that complete knockout of RINL can promote uric acid excretion in the kidneys of mice without affecting their normal development.

[0161] Example 7: RINL knockout promotes uric acid excretion in the kidneys of HUA mice

[0162] In this embodiment, a 2-month-old wild-type (RINL) was constructed. + / + ) and RINL knockout (RINL - / - We used male and female HUA mouse models to examine the effect of RINL on uric acid excretion in HUA mice.

[0163] Developing human uric acid (HUA) was induced in mice by intraperitoneal injection of potassium oxychloride (PO) and free access to 10% fructose water. PO was dissolved in physiological saline and administered intraperitoneally to mice at a dose of 350 mg / kg daily for 28 days. A control group of mice used physiological saline as the solvent. Serum uric acid levels were measured on days 14 and 28, as shown in Figures 9A-9D. Regardless of sex, RINL knockout significantly reduced serum uric acid levels in HUA mice. Kidneys were harvested on day 29, and Western blot analysis was performed to detect the levels of the uric acid excretion proteins OAT3 / OAT1 in the kidneys of male mice, as shown in Figure 9E. Consistent with serum results, RINL knockout significantly increased the levels of OAT3 / OAT1 in the kidneys of mice.

[0164] The results above indicate that RINL knockout can rescue drug-induced increases in serum uric acid levels, making mice less susceptible to HUA.

[0165] Example 8: RINL tubule-specific knockout promotes uric acid excretion in mouse kidneys

[0166] In this embodiment, RINL renal tubule-specific knockout mice were constructed to further investigate the effect of RINL on renal tubular uric acid excretion.

[0167] A C57 / BL6 mouse model specifically knocking out renal tubule RINL (RINL Cko) was constructed, as shown in Figure 10A. The applicant inserted loxp sites at both ends of exon 4 of the RINL gene, through interaction with Pax8 CreER. T2 Mice were hybridized and induced with tamoxifen to obtain RINL tubule-specific knockout mice. As shown in Figure 10B, the applicant induced RINL gene tubule-specific knockout in male mice at 4 months of age by daily injection of 75 mg / kg tamoxifen (for 7 consecutive days). As shown in Figure 10C, kidney samples were collected after induction, and Rinl mRNA levels were detected. The results showed that the Rinl mRNA level in the kidneys of RINL tubule-specific knockout mice was significantly reduced, indicating that the RINL gene in the renal tubules was knocked out. As shown in Figures 10D-10F, at 6 months of age, the mice were weighed, and serum uric acid and creatinine levels were detected by submandibular blood sampling. It was found that RINL tubule-specific knockout mice also showed significantly reduced uric acid and creatinine levels.

[0168] Mice were sacrificed at 8 months of age, and blood and kidney tissue were collected for analysis. Similar to 6-month-old mice (Figures 10G-10J), compared with control mice (RINL not knocked out in the renal tubules), RINL cKO mice showed no significant difference in body weight and kidney-to-body ratio, but serum uric acid and creatinine levels were significantly reduced. As shown in Figures 10K and 10L, urine analysis of control and RINL cKO mice showed a significant increase in urinary uric acid and creatinine levels after RINL renal tubule-specific knockout. Based on the results, fractional uric acid excretion (FEUA = (urinary uric acid * serum creatinine) / (serum uric acid * urinary creatinine) * 100%) and urinary uric acid excretion rate (UUE = urinary uric acid / urinary creatinine) were calculated. As shown in Figures 10M and 10N, FEUA and UUE levels were significantly increased in RINL cKO mice. The serum and urine results suggest that RINL renal tubule-specific knockout promotes the excretion of uric acid in urine. The levels of uric acid excretion protein in the kidneys of 8-month-old control and RINL cKO mice were detected by immunoblotting, as shown in Figure 10O. Compared with control mice, the levels of uric acid excretion protein OAT3 / ABCG2 in the kidneys of RINL cKO mice were significantly increased, which also indicates that RINL tubule-specific knockout can promote uric acid excretion in the kidneys of mice.

[0169] The results above show that RINL can regulate renal tubular uric acid excretion by modulating the level of renal tubular uric acid excretion protein, and that RINL tubular-specific knockout promotes renal uric acid excretion.

[0170] In summary, the experimental results indicate that RINL is a HUA-related gene that significantly affects uric acid excretion in the renal tubules. Both RINL knockdown and knockout promote uric acid secretion from HK2 cells. In mice, complete RINL knockout promotes renal uric acid excretion and reduces uric acid levels. Specifically, in HUA mice, RINL knockout promotes renal uric acid excretion.

[0171] The above description is merely an embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

[0172] Table 3. Sequence Information

Claims

1. Use of the RINL gene or its encoded protein, wherein the RINL gene or its encoded protein is used as: (1) a biomarker for the diagnosis, detection or prognosis of hyperuricemia; (2) for the preparation of reagents or kits for the diagnosis or detection of hyperuricemia; or (3) for the preparation of drugs for the prevention and / or treatment of hyperuricemia.

2. A RINL antagonist for the prevention and / or treatment of hyperuricemia, 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) Cause the RINL gene to be deleted or silenced; (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 or antibodies or their antigen-binding fragments; Preferably, the antibody or its antigen-binding fragment is selected from whole antibodies, F(ab)-fragments, F(ab)2-fragments, single-chain antibodies, chimeric antibodies, bivalent antibody-constructs, synthetic antibodies, bispecific single-chain antibodies, or cross-clonal antibodies; 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 containing a sense strand and an antisense strand, each 15-30 base pairs in length, wherein the antisense strand contains a complementary region that is complementary to the mRNA encoding RINL. Preferably, the nucleotide sequence of the siRNA is selected from any combination of the following sense and antisense strands: (1) A positive chain having the sequence shown in SEQ ID NO.1, and an antisense chain having the sequence shown in SEQ ID NO.2; or (2) A sense strand having the sequence shown in SEQ ID NO.3, and an antisense strand having the sequence shown in SEQ ID NO.4; or (3) A sense chain having the sequence shown in SEQ ID NO.5 and an antisense chain having the sequence shown in SEQ ID NO.

6.

5. The RINL antagonist according to claim 4, wherein, The sense and antisense strands of the siRNA are further modified, and the modifications are selected from one or more of the following: 2'-methoxyethyl modification, 2'-methoxy modification, 2'-deoxy-2'-fluorine 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 mimicry 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 target ligands; Preferably, the targeting ligand is conjugated to the sense strand of the siRNA; Preferably, the targeting ligand is conjugated to any nucleotide position on 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, carbohydrate, or lipid.

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

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

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

10. Use of the RINL antagonist of any one of claims 2-6 and / or the composition of claim 7 or 8 in the preparation of a medicament for the prevention and / or treatment of individual hyperuricemia.

11. A method for treating hyperuricemia, wherein, The method includes the following steps: By administering to the subject a therapeutically effective amount of the RINL antagonist of any one of claims 2-6 and / or the composition of claim 7 or 8.

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