Use of TILRR as target in prevention and treatment of kidney diseases

Abstract

By constructing a mouse model of the specific knockout TILRR, it is found that the specific knockout TILRR remarkably slows down the kidney inflammation signal flow of an object, inhibits kidney diseases of the object and reduces the kidney fibrosis degree and / or inflammatory response of the object. Thus, the present invention relates to the use of TILRR as a target in the prevention and / or treatment of kidney disease. Specifically, the invention provides application of the following reagents in preparation of drugs for slowing down kidney inflammation signal flow of an object, inhibiting kidney diseases of the object and reducing kidney fibrosis degree and / or inflammatory response of the object: (1) a reagent for reducing expression of TILRR protein of the object; and / or (2) an agent that reduces the activity of a TILRR protein expressed by a subject.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of TILRR as a target in the prevention and treatment of kidney disease. Background Art

[0002] The prevalence of chronic kidney disease (CKD) is increasing dramatically worldwide, with the rise in diabetes, hypertension, and obesity, as well as the aging of the population. This devastating disease is approaching epidemic proportions, and the incidence of CKD and its absolute contribution to cardiovascular disease and mortality increase with age, with more than 60% of people aged ≥80 years suffering from CKD. Regardless of its cause, CKD is characterized by progressive and irreversible nephron loss, reduced renal regeneration, microvascular damage, metabolic alterations, oxidative stress, and inflammation, ultimately leading to fibrosis. Fibrosis is part of the normal injury repair process, triggered by the body in response to injury, and occurs to preserve the structural and functional integrity of tissues. However, dysregulation of this process leads to excessive accumulation of extracellular matrix (ECM) proteins, primarily pathological accumulation of collagen. In CKD, podocytes are lost and replaced by ECM (known as glomerulosclerosis), and tubular cell damage and subsequent tubulointerstitial fibrosis lead to nephron loss. These processes lead to the replacement of parenchymal tissue with ECM, resulting in irreversible damage.

[0003] The current approach to solving the clinical problem of irreversible renal fibrosis is prevention, which should aim to prevent the transition from acute kidney injury to chronic kidney disease (AKI-to-CKD) and / or the progression of existing fibrosis. For example, the progression of the disease is mainly slowed down by controlling clinical indications such as hypertension drugs, diabetes drugs, angiotensin-converting enzyme inhibitors (ACEI) / angiotensin receptor blockers (ARB). These drugs take a long time to take, have limited efficacy, and cannot control the progression of the disease to the terminal stage. Therefore, the clinical use of drugs for CKD is in a dilemma, and there is no specific target drug that can reverse or cure CKD. Persistent inflammatory stimulation is also a key feature that causes CKD to develop into renal fibrosis. In mice with global knockout of the interleukin-1 type-1 receptor (IL1RI), the expansion of the interstitial matrix and the fibrotic phenotype were significantly reduced in the early period after acute kidney injury, indicating the role of IL-1β in the initiation of fibrosis. Recent studies have shown that inhibition of IL-1 receptor-associated kinase 4 (IRAK4) in mouse renal stromal cells can reduce the progression of interstitial fibrosis after renal ischemia, which directly proves that IL-1 receptor-mediated innate inflammatory signals can directly drive the occurrence and development of fibrosis. These studies have shown that by blocking the innate immune signaling pathway, anti-fibrotic effects can be successfully exerted and can be used as a potential therapeutic target for future clinical applications. However, Nowak et al. demonstrated that the use of antagonists through the IL-1R1 receptor can improve the inflammation and level and antioxidant properties of CKD in stages 3-5. Although the blockade of IL-1R1 has a certain effect on CKD, blocking IL-1R1 may also have side effects like atherosclerosis (reduced plaques, but poor plaque stability). Therefore, finding drugs that can reduce CDK inflammatory signals while controlling the side effects caused by inhibiting inflammation is of great significance for the treatment and prevention of CDK and CDK-induced fibrosis. Summary of the invention

[0004] In a first aspect, the present invention provides the use of the following agents in the preparation of a medicament for slowing the flow of inflammatory signals in the kidneys of a subject, inhibiting a kidney disease or symptom in a subject, or reducing the degree of kidney fibrosis and / or inflammatory response in a subject: (1) an agent that reduces the expression of a TILRR protein in a subject; and / or

[0005] (2) An agent that reduces the activity of a TILRR protein expressed in a subject.

[0006] In one or more embodiments, the kidney disease is chronic or acute kidney disease.

[0007] In one or more embodiments, the kidney disease is a disease associated with kidney fibrosis or inflammation.

[0008] In one or more embodiments, the renal fibrosis is tubulointerstitial fibrosis.

[0009] In one or more embodiments, the kidney disease is chronic nephritis, glomerular disease or renal injury. In one or more embodiments, the glomerular disease is IgA nephropathy and membranous nephropathy. In one or more embodiments, the renal injury is drug-induced renal injury, renal injury induced by other diseases (such as diabetes, liver disease), and renal injury induced by autoimmune disease.

[0010] In one or more embodiments, the medicament is for reducing the incidence of or treating the kidney disease or condition.

[0011] In one or more embodiments, the agent that reduces the expression of TILRR protein in a subject is an agent that inhibits the expression of the TILRR gene or reduces its expression level.

[0012] In one or more embodiments, the agent that inhibits TILRR gene expression or reduces its expression level is selected from:

[0013] (a) an agent that inhibits the transcriptional activity of the TILRR gene;

[0014] (b) an agent that inhibits the transcription level of TILRR mRNA;

[0015] (c) agents that promote TILRR mRNA degradation;

[0016] (d) siRNA targeting TILRR gene;

[0017] (e) an agent that inhibits translation of TILRR mRNA;

[0018] (f) an agent that specifically recognizes the guide nucleic acid of the TILRR gene and cleaves it to reduce its expression level; and

[0019] (g) an agent for partially or completely knocking out the TILRR gene; and

[0020] The agent for reducing the activity of the TILRR protein expressed by the subject is a specific antibody of the TILRR protein or a small molecule compound with inhibitory activity.

[0021] In one or more embodiments, the TILRR-specific antibody is an anti-TILRR antibody or an antigen-binding fragment thereof, wherein the anti-TILRR antibody has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and / or LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively. In one or more embodiments, the FR1 of the VH of the TILRR antibody is as shown in SEQ ID NO: 4.

[0022] In one or more embodiments, FR2 of VH of the TILRR antibody is as shown in SEQ ID NO:5.

[0023] In one or more embodiments, FR3 of VH of the TILRR antibody is as shown in SEQ ID NO:6.

[0024] In one or more embodiments, FR4 of VH of the TILRR antibody is as shown in SEQ ID NO:7.

[0025] In one or more embodiments, FR1 of the VL of the TILRR antibody is as shown in SEQ ID NO:12.

[0026] In one or more embodiments, FR2 of the VL of the TILRR antibody is as shown in SEQ ID NO:13.

[0027] In one or more embodiments, FR3 of the VL of the TILRR antibody is as shown in SEQ ID NO:14.

[0028] In one or more embodiments, FR4 of the VL of the TILRR antibody is as shown in SEQ ID NO:15.

[0029] In one or more embodiments, FR1, FR2, FR3 and FR4 of the VH of the TILRR antibody have the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively, and FR1, FR2, FR3 and FR4 of the VL of the TILRR antibody have the amino acid sequences shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, respectively.

[0030] In one or more embodiments, the VH of the TILRR antibody is shown as SEQ ID NO:8, and / or the VL of the TILRR antibody is shown as SEQ ID NO:16.

[0031] The second aspect of the present invention also provides an anti-TILRR antibody or an antigen-binding fragment thereof, wherein the anti-TILRR antibody has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and / or LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively. FR1 of VH of the TILRR antibody is shown in SEQ ID NO: 4.

[0032] In one or more embodiments, FR2 of VH of the TILRR antibody is as shown in SEQ ID NO:5.

[0033] In one or more embodiments, FR3 of VH of the TILRR antibody is as shown in SEQ ID NO:6.

[0034] In one or more embodiments, FR4 of VH of the TILRR antibody is as shown in SEQ ID NO:7.

[0035] In one or more embodiments, FR1 of the VL of the TILRR antibody is as shown in SEQ ID NO:12.

[0036] In one or more embodiments, FR2 of the VL of the TILRR antibody is as shown in SEQ ID NO:13.

[0037] In one or more embodiments, FR3 of the VL of the TILRR antibody is as shown in SEQ ID NO:14.

[0038] In one or more embodiments, FR4 of the VL of the TILRR antibody is as shown in SEQ ID NO:15.

[0039] In one or more embodiments, FR1, FR2, FR3 and FR4 of the VH of the TILRR antibody have the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively, and FR1, FR2, FR3 and FR4 of the VL of the TILRR antibody have the amino acid sequences shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, respectively.

[0040] In one or more embodiments, the VH of the TILRR antibody is shown as SEQ ID NO:8, and / or the VL of the TILRR antibody is shown as SEQ ID NO:16.

[0041] The third aspect of the present invention provides a nucleic acid molecule comprising a sequence selected from the following or its RNA counterpart:

[0042] (1) a nucleic acid sequence as shown in any one of SEQ ID NOs: 17-19;

[0043] (2) a sequence encoding an antibody or antigen-binding fragment thereof as described in any embodiment herein, and

[0044] (3) A complementary sequence of the sequence described in (1) or (2).

[0045] In one or more embodiments, (1) the nucleic acid sequence is SEQ ID NO:17 and 18, or SEQ ID NO:19.

[0046] In one or more embodiments, the nucleic acid molecule is DNA or RNA.

[0047] The fourth aspect of the present invention provides a nucleic acid construct comprising the nucleic acid molecule described herein.

[0048] In one or more embodiments, the nucleic acid construct is a vector, such as a cloning vector or an expression vector.

[0049] In one or more embodiments, the nucleic acid construct is mRNA.

[0050] The fifth aspect of the present invention also provides a genetically engineered host cell, wherein:

[0051] (1) expressing the antibody or antigen-binding fragment described in any embodiment of the present invention,

[0052] (2) comprising or having integrated into its genome a nucleic acid molecule or nucleic acid construct as described in any embodiment of the present invention.

[0053] In one or more embodiments, the host cell is a non-human mammalian cell.

[0054] In one or more embodiments, the host cell is a rodent cell.

[0055] In one or more embodiments, the host cell is an embryonic stem cell, a pluripotent stem cell, or a somatic cell of a human or non-human mammal.

[0056] In one or more embodiments, the host cell is a human embryonic stem cell or a pluripotent stem cell, which contains or has a nucleic acid molecule integrated into its genome or a nucleic acid construct containing the nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleic acid sequence shown in any one of SEQ ID NOs: 17-19 or its RNA counterpart. The human embryonic stem cell or pluripotent stem cell does not express TILRR protein, or the expression amount of TILRR protein is reduced compared with the control cell, or expresses an inactive TILRR protein or a TILRR protein with reduced activity.

[0057] The sixth aspect of the present invention also provides a method for constructing a transgenic mouse, the method comprising:

[0058] (1) providing a nucleic acid construct as described herein;

[0059] (2) introducing the nucleic acid construct in step (1) into a mouse fertilized egg;

[0060] (3) transplanting the mouse fertilized eggs in step (2) into the uterus of a pseudo-pregnant female mouse to obtain positive offspring mice;

[0061] (4) backcrossing the positive offspring mice obtained in step (3) to obtain homozygous positive mice, i.e., the transgenic mice.

[0062] In one or more embodiments, the method comprises:

[0063] (1) providing a nucleic acid construct as described herein;

[0064] (2) transcribing the nucleic acid construct in step (1) into mRNA in vitro and injecting it into mouse fertilized eggs;

[0065] (3) transplanting the mouse fertilized eggs in step (2) into the uterus of a pseudo-pregnant mouse, and identifying the offspring mice by PCR analysis after birth to obtain positive offspring mice;

[0066] (4) backcrossing the positive offspring mice obtained in step (3), and performing PCR identification to obtain homozygous positive mice, i.e., the mouse model.

[0067] In one or more embodiments, the nucleic acid construct is RNA.

[0068] In one or more embodiments, the transgenic mouse is characterized by not expressing TILRR protein in the whole body or kidney, or expressing a reduced amount of TILRR protein compared to control animals, or expressing inactive TILRR protein or TILRR protein with reduced activity.

[0069] The seventh aspect of the present invention also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment, nucleic acid molecule, nucleic acid construct and / or host cell described in any embodiment herein.

[0070] The eighth aspect of the present invention also provides the use of TILRR gene or protein as a target in screening drugs for preventing and / or treating kidney diseases or symptoms.

[0071] In one or more embodiments, the prevention and / or treatment of kidney disease or symptoms comprises one or more selected from the following: slowing down the flow of inflammatory signals in the subject's kidneys, inhibiting the subject's kidney disease, or reducing the degree of kidney fibrosis and / or inflammatory response in the subject.

[0072] In one or more embodiments, the kidney disease is chronic or acute kidney disease.

[0073] In one or more embodiments, the kidney disease is a disease associated with kidney fibrosis or inflammation.

[0074] In one or more embodiments, the renal fibrosis is tubulointerstitial fibrosis.

[0075] In one or more embodiments, the kidney disease is chronic nephritis, glomerular disease or renal injury. In one or more embodiments, the glomerular disease is IgA nephropathy and membranous nephropathy. In one or more embodiments, the renal injury is drug-induced renal injury, renal injury induced by other diseases (such as diabetes, liver disease), and renal injury induced by autoimmune disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1Identification of hiPSCs cell lines with TILRR gene knockout. (A) DNA sequencing results of TILRR knockout site. (B) Bright field imaging photos of hiPSCs and hiPSCs-TILRR- / - cell lines. (C) qRT-PCR analysis to identify TILRR knockout in hiPSCs-TILRR- / - cell lines, n=3, **p<0.01. (D) WB analysis to identify the expression level of TILRR protein in hiPSCs-TILRR- / - cell lines. (E) qRT-PCR detection of pluripotency of hiPSCs-TILRR- / - cell lines. (F) Immunofluorescence staining to detect pluripotency of hiPSCs-TILRR- / - cell lines. (G) H&E staining results of teratoma formation in hiPSCs-TILRR- / - cell lines. (H) Karyotype analysis of hiPSCs-TILRR- / - cell lines.

[0077] Figure 2 Identification of TILRR knockout mice. A. Identification of the genotype of TILRR knockout mice. B. Identification of the TILRR RNA expression level of TILRR knockout mice by RT-PCR. The three lanes of each mouse are brain, kidney, and spleen from left to right. C. Identification of the TILRR protein expression level of TILRR knockout mice by Western blotting.

[0078] Figure 3 TILRR knockout alleviates renal inflammation and fibrosis in the mouse UUO model. (A) Phenotype of mouse renal UUO model and H&E staining of tissue sections. (B) Comparison of Masson staining and quantitative analysis of collagen content after mouse renal UUO model. (C) qRT-PCR analysis to compare the mRNA expression of related proinflammatory cytokines, renal injury and fibrosis marker genes after renal UUO model in WT and TILRR knockout mice.

[0079] Figure 4 TILRR knockout reduces the inflammation and fibrosis levels of IL-1β-induced renal organoid fibrosis. (A) qRT-PCR analysis and comparison of the mRNA expression of related proinflammatory cytokines, renal injury and fibrosis marker genes in renal organoids under different conditions. (B) Immunofluorescence staining and quantitative comparison of the expression of fibrosis marker COL1A1 in renal organoids under different conditions.

[0080] Figure 5 TILRR blocking antibodies inhibit the inflammation and fibrosis levels of IL-1β-induced renal organoid fibrosis. (A) qRT-PCR detection of different concentrations of TILRR antibodies blocking the fibrosis effect of renal organoids stimulated by IL-1β. (B) Immunofluorescence staining to identify the effect of TILRR antibodies blocking the fibrosis of renal organoids stimulated by IL-1β. DETAILED DESCRIPTION

[0081] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a preferred technical solution.

[0082] In earlier studies, the inventors reported that overexpression of TILRR increases the binding ratio of MyD88 to IL-1RI and enhances IL-1RI receptor-associated kinase 4 (IRAK4) signal transduction. Blocking the interaction between TILRR and IL-1RI can reduce the response of cells to IL-1β stimulation, but it does not completely inhibit IL1R1-mediated signals. In this paper, TILRR was used as a therapeutic target, and a tubulointerstitial fibrosis model was constructed using the classic UUO modeling method to explore the role and biological function of TILRR in the kidney. This paper found that knocking out TILRR can reduce the degree of renal fibrosis, block the interaction between TILRR and IL-1RI, reduce the flow of inflammatory signals, inhibit the occurrence of CKD caused by IL-1β stimulation, prevent the development of renal fibrosis, and reduce the risk of complications.

[0083] Therefore, this article involves using TILRR as a target to slow down the flow of inflammatory signals in the subject's kidneys, inhibit the subject's kidney disease, or reduce the degree of kidney fibrosis and / or inflammatory response in the subject, so as to effectively reduce the incidence of primary or other disease-induced secondary nephritis such as chronic kidney disease, acute kidney injury, renal fibrosis, or treat these kidney diseases or their symptoms.

[0084] Herein, TILRR is a variant of FREM1 (Fras-related extracellular matrix 1), a regulator of many genes in NF-κB signaling and inflammatory responses. Various extracellular matrix proteins defined as TILRR in the art are included herein, such as TILRR from different species. The different species include, but are not limited to, mammals and rodents, such as mice. The coding gene for human TILRR is located on chromosome 9 (NC_000009.12 (14737152..14910995, complement)), and the accession numbers in NCBI are NM_001177704.3 and NP_001171175.1. The coding gene of mouse TILRR is located on chromosome 4 (NC_000070.7 (82816157..82970743, complement)), and the accession numbers of its amino acid sequence in NCBI are XM_036164192.1 and XP_036020085.1.

[0085] As used herein, "subject" refers to various animals in need including, but not limited to, mammals and rodents, such as humans and mice.

[0086] The above purpose is achieved by inhibiting the activity of TILRR protein. The inhibition of TILRR protein activity can be achieved by the following methods:

[0087] (1) reducing TILRR protein expression in a subject; and / or

[0088] (2) reducing the activity of TILRR protein expressed by the subject;

[0089] Reducing the TILRR protein expression level of the object includes but is not limited to inhibiting the expression of the coding gene of the TILRR protein or the expression level of the gene. For example, an agent that inhibits the expression of the TILRR gene or reduces its expression level may be given, including: an agent that inhibits the transcriptional activity of the TILRR gene, an agent that inhibits the transcriptional level of the TILRR mRNA, an agent that promotes the degradation of the TILRR mRNA, an siRNA for the TILRR gene, an agent that inhibits the translation of the TILRR mRNA, and an agent that specifically recognizes the guide nucleic acid of the TILRR gene and cuts it to reduce its expression level. In certain embodiments, the TILRR full gene can be knocked out by administering a targeting vector, thereby achieving inhibition or reduction of TILRR gene expression. In other embodiments, the TILRR gene expression can be inhibited or its expression level can be reduced by administering siRNA of the TILRR gene. In other embodiments, the TILRR protein expression of the object can be reduced by gene editing technology, such as administering an agent, such as sgRNA, that knocks out or knocks down the TILRR gene using ZFN, TALEN or CRISPR technology. As described above, the agent for reducing the activity of TILRR protein can be, for example, a specific antibody of TILRR or a small molecule compound with inhibitory activity. Therefore, the present invention also provides a TILRR-specific antibody.

[0090] Herein, the term "antibody" includes monoclonal antibodies (including full-length antibodies, which have an immunoglobulin Fc region), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies and single-chain molecules, as well as antibody fragments, especially antigen-binding fragments, such as Fab, Fab', F(ab')2, Fv and scFV). Herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably. Preferably, the antigen-binding fragment is composed of or contains a partial sequence of the heavy chain variable region or light chain variable region of the antibody from which it is derived, and the partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived. For the antigen-binding fragment of the TILRR blocking antibody, it is preferably at least equal to 1 / 100 of the affinity of the antibody from which it is derived, and in a more preferred manner at least equal to 1 / 10. Such an antibody fragment will contain a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which it is derived.

[0091] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the two heavy chains are connected to each other by one or more disulfide bonds, the number of which depends on the isotype of the heavy chain. Each heavy chain and light chain also has a regularly spaced intrachain disulfide bridge. Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (CH1, CH2 and CH3 for each α and γ chain) and four (CH1, CH2, CH3 and CH4 for μ and ε isotypes) constant domains (CH) and a hinge region (Hinge) between the CH1 domain and the CH2 domain. Each light chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at the other end. VL is arranged together with VH, and CL is arranged together with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are considered to form an interface between the light chain and the heavy chain variable domain. Paired VH and VL together form an antigen binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, the eighth edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw, ed., Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6. Light chains from any vertebrate species can be classified into one of two distinct types called κ and λ, according to their constant domain amino acid sequences. According to their heavy chain constant domain (CH) amino acid sequences, immunoglobulins can be classified into different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, each with a heavy chain called α, δ, ε, γ and μ. Based on relatively minor differences in CH sequence and function, the gamma and alpha classes can be further divided into subclasses, for example humans express the following subclasses: IgGl, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0092] The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen binding site. In one or more embodiments, the VH and VL of the antibodies described herein have the sequences shown in SEQ ID NO: 8 and SEQ ID NO: 16, or variants thereof having 90% sequence identity.

[0093] The term "variable" refers to the situation that some segments in the variable domains differ widely in the antibody sequence. The variable domain mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, the variability is not evenly distributed over all the amino acids spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (in both the light chain and heavy chain variable domains), namely, HCDR1, HCDR2, HCDR3 of the heavy chain variable region and LCDR1, LCDR2 and LCDR3 of the light chain variable region, respectively. The more highly conserved parts in the variable domains are called framework regions (FRs). The variable domains of natural heavy and light chains each include four FR regions (FR1, FR2, FR3 and FR4), most of which adopt a β-folded conformation, connected by three HVRs that form a loop connection and form a part of the β-folded structure in some cases. The HVR in each chain is held together in close proximity by the FR region and contributes to the formation of the antigen binding site of the antibody together with the HVR of the other chain (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD, 1991). Typically, the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as the participation of the antibody in antibody-dependent cell-mediated cytotoxicity. In a preferred embodiment, the CDRs of the TILRR-specific antibody of the present invention are as follows: HCDR1 comprises SYWIH (SEQ ID NO: 1), HCDR2 comprises MIDPSDSETRLNQRFKD (SEQ ID NO: 2), HCDR3 comprises SLYGGSCY (SEQ ID NO: 3), LCDR1 comprises KSSQSLLDSDGKTYLN (SEQ ID NO: 9), LCDR2 comprises LVSKLDS (SEQ ID NO: 10), and LCDR3 comprises WQGTHFPHT (SEQ ID NO: 11).In a preferred embodiment, the FR1 of the TILRR-specific antibody VH of the present invention comprises QVQLQQSGPQLVRPGASVEISCKASGYSFT (SEQ ID NO: 4), the FR2 of VH comprises WVKQRPGQGLEWIG (SEQ ID NO: 5), the FR3 of VH comprises KATLTVDKSSSTAYMQLSSPTSEDSAVYFCAR (SEQ ID NO: 6), the FR4 of VH comprises WGQGTTLTVSS (SEQ ID NO: 7), the FR1 of VL comprises DVVMTQTPLTLSVTIGQPASISC (SEQ ID NO: 12), the FR2 of VL comprises WLLQRPGQSPKRLIY (SEQ ID NO: 13), the FR3 of VL comprises GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC (SEQ ID NO: 14), and the FR4 of VL comprises FGSGTKLEIK (SEQ ID NO: 15). The CDRs described herein are obtained using the IMGT antibody numbering system. After knowing the sequence of the antibody or antigen-binding fragment thereof, those skilled in the art can easily obtain the corresponding CDR using other numbering systems.

[0094] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of an immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells), or binding to the first component (C1q) of the classical complement system. In IgG, IgA, and IgD antibody isotypes, the Fc region is composed of two identical protein fragments from the CH2 domain and CH3 domain of the two heavy chains of the antibody; the Fc region of IgM and IgE contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as the sequence segment from the amino acid residue at position C226 or P230 of the heavy chain to the carboxyl terminus, where the numbering is according to the EU index. As used herein, the Fc region may be a native sequence Fc or a variant Fc.

[0095] Herein, the "RNA counterpart" of a DNA sequence refers to an RNA sequence obtained by replacing T with U in the DNA sequence.

[0096] In certain embodiments, the activity of the TILRR protein can also be reduced by introducing a mutation in the TILRR protein. Herein, the activity of the TILRR protein refers in particular to the activity of the kidney disease or the symptoms described herein. Therefore, in certain embodiments, a mutation that causes the corresponding activity to be weakened or lost is introduced into the functional domain of the TILRR protein. The mutation can be the insertion, deletion or substitution of one or more (e.g., more than 10, more than 20, more than 30) amino acids. By administering an agent that acts on the TILRR gene, a mutation that causes the relevant biological activity to be weakened or lost can be present in the functional domain of the TILRR protein encoded by it. Such agents can change the sequence of the TILRR gene, resulting in the presence of corresponding mutations in the encoded TILRR protein, thereby having weakened activity, or loss of activity. For example, the TILRR gene in the wild-type cell can be replaced by a mutated TILRR gene by homologous recombination technology, thereby causing it to express a weakly active or inactive TILRR protein.

[0097] In the embodiment of reducing the expression of the subject TILRR protein by CRISPR gene editing technology, the knockout or knockdown of the TILRR gene is achieved by expressing a specific sgRNA in the cell. SEQ ID NO: 17-19 is the coding sequence of the sgRNA that can effectively reduce the expression of the TILRR protein screened by the inventors.

[0098] Therefore, the present invention includes the pharmaceutical use of the following agents: (1) an agent that reduces the expression of a subject's TILRR protein; and / or (2) an agent that reduces the activity of a subject's expressed TILRR protein. The agent that reduces the expression of a subject's TILRR protein includes the agent that inhibits TILRR gene expression or reduces its expression level as described above, and the agent that reduces the activity of a subject's expressed TILRR protein includes the agent that reduces the activity of a TILRR protein as described above.

[0099] Such agents can be used to prevent and / or treat kidney diseases or symptoms, particularly those mediated by IL-1β protein. Herein, kidney diseases or symptoms mediated by IL-1β protein include, but are not limited to, renal fibrosis or inflammation-related diseases, such as chronic nephritis, glomerular diseases such as IgA nephropathy and membranous nephropathy, drug-induced renal injury, renal injury induced by other diseases (such as diabetes, liver disease), and renal injury induced by autoimmune diseases. It should be understood that it is possible to use recognized standards in the art to determine whether the subject is a kidney disease or symptom mediated by IL-1β protein. Such agents can be used to prepare drugs for treating and / or preventing kidney disease or alleviating its symptoms, for treating and / or preventing kidney disease or alleviating its symptoms.

[0100] Herein, reducing the activity of TILRR protein includes reducing the activity of TILRR protein by at least 30% compared to its corresponding wild-type protein, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even completely inactive.

[0101] Also included herein are methods for treating and / or preventing a kidney disease or symptoms described herein, comprising administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of:

[0102] (1) an agent that reduces the expression of a subject's TILRR protein; and / or

[0103] (2) An agent that reduces the activity of a TILRR protein expressed in a subject.

[0104] An animal model is also provided herein, which is preferably a rodent model, especially a mouse. The animal model is characterized in that the TILRR protein is not expressed in the whole body or kidney, or the expression of the TILRR protein is reduced compared with the control animal, or an inactive TILRR protein or a TILRR protein with reduced activity is expressed. In certain embodiments, the animal model is a transgenic mouse that specifically knocks out TILRR. The transgenic mouse can be obtained using gene knockout and mouse hybridization techniques known in the art. In certain embodiments, the transgenic mouse is constructed as a tubulointerstitial fibrosis model using the UUO modeling method.

[0105] The mouse with specific TILRR knockout can be constructed by hybridization of TILRR knockout mice. Specifically, the mouse with specific TILRR knockout can be constructed as follows:

[0106] (1) providing a nucleic acid construct as described herein;

[0107] (2) introducing the nucleic acid construct in step (1) into a mouse fertilized egg;

[0108] (3) transplanting the mouse fertilized eggs in step (2) into the uterus of a pseudo-pregnant female mouse to obtain positive offspring mice;

[0109] (4) backcrossing the positive offspring mice obtained in step (3) to obtain homozygous positive mice, i.e., the transgenic mice.

[0110] In one or more embodiments, the method comprises:

[0111] (1) providing a nucleic acid construct as described herein;

[0112] (2) transcribing the nucleic acid construct in step (1) into mRNA in vitro and injecting it into mouse fertilized eggs;

[0113] (3) transplanting the mouse fertilized eggs in step (2) into the uterus of a pseudo-pregnant mouse, and identifying the offspring mice by PCR analysis after birth to obtain positive offspring mice;

[0114] (4) backcrossing the positive offspring mice obtained in step (3), and performing PCR identification to obtain homozygous positive mice, i.e., the mouse model.

[0115] In one or more embodiments, the nucleic acid construct is RNA.

[0116] Herein, positive mice refer to mice expressing the transgene or in which the target gene is knocked out.

[0117] Herein, "in vitro transcription" refers to the process of transcribing DNA into RNA outside an organism or cell. Conventional in vitro transcription methods are known to those skilled in the art.

[0118] As an illustrative example, genotype identification can be performed by PCR identification of the toes and tails of the mice, or RNA level identification can be performed by extracting RNA from mouse tissues. The PCR primers can be shown as SEQ ID NOs: 22-24; the qPCR primers for RNA identification can be shown as SEQ ID NOs: 39-40. It should be understood that the plasmid of step (1) of the above construction method can be constructed using techniques and sequences well known in the art. The plasmid can also contain other regulatory elements well known in the art to facilitate the integration of the TILRR gene into the genome of, for example, embryonic stem cells. In addition, the genetic mouse can be constructed using various experimental mice well known in the art.

[0119] Similar methods can be used to construct other animal models.

[0120] The present invention also provides nucleic acid molecules encoding the antibodies or sgRNA described herein. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. The present invention also includes degenerate variants of the polynucleotides, i.e., nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences. RNA may be mRNA that expresses antibodies in vivo and / or in vitro.

[0121] Therefore, the present invention also relates to polynucleotides that hybridize with the above-mentioned polynucleotide sequence and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0122] The nucleic acid molecules of the present invention or their fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0123] The present invention also provides nucleic acid constructs comprising the above-mentioned nucleic acid molecules for cloning or expressing genetic functions such as genes. Nucleic acid constructs can be expression cassettes or vectors (or plasmids). Therefore, in addition to the coding sequence, the nucleic acid constructs can also include other elements required for cloning or expression, such as promoters, multiple cloning sites and / or terminators (e.g., polyA signal sequences), which are operably connected to the coding sequence. For example, the nucleic acid construct can be an RNA construct or a DNA vector that can be transfected with cells and expressed. Herein, "nucleic acid molecules", "polynucleotides", and "nucleic acids" are used interchangeably.

[0124] Nucleic acid constructs can be constructed using methods well known to those skilled in the art, see, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory), Ausubel et al. (1989, Short Protocols in Molecular Biology, Wiley) or other standard textbooks. Alternatively, the nucleic acid construct can be reconstructed into a liposome for delivery to a target cell. The nucleic acid construct containing the present invention can be transferred to a host cell by a well-known method, which varies according to the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts, see Sambrook et al. (see above).

[0125] The nucleic acid sequence herein may be contained in a plasmid or vector. The vector may be a plasmid, a cosmid, a virus, or a viral vector. The vector includes a cloning vector, an integration vector, or an expression vector. The expression vector generally contains sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequence (collectively referred to as "flanking sequence" in certain embodiments) generally includes one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, a replication origin, a transcription termination sequence, a complete intron sequence containing donor and acceptor splicing sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a multiple cloning site for inserting a nucleic acid encoding a protein to be expressed, and a selectable marker element. Generally, the promoter is located at the 5' end of the multiple cloning site or the polypeptide coding sequence. Other regulatory sequences suitable for expressing DNA or RNA in vivo or in vitro are common knowledge in the art. In one or more embodiments, the plasmid or vector may be a plasmid or vector for expressing a protein or gene knockout. The plasmid or vector may also contain other elements that are conducive to expressing a protein or gene knockout in a host cell. The plasmids or vectors described herein can be constructed using methods known in the art. In one or more embodiments, the vector comprises a coding sequence for an antibody or antigen-binding fragment thereof described herein, and an element for expressing the antibody or antigen-binding fragment thereof. In one or more embodiments, the vector comprises a coding sequence for an sgRNA described herein and an element for expressing the sgRNA. In one or more embodiments, the nucleic acid construct is an mRNA encoding the sgRNA.

[0126] The present invention also includes host cells that contain or have integrated into their genome the nucleic acid molecules or nucleic acid constructs described herein. Host cells can be used as recipients of vectors. Host cells can be "transfected" or "transformed," which refers to the process of transfection or transduction of exogenous nucleic acids into host cells. Transformed cells include primary subject cells and their progeny. The terms "engineered" and "recombinant" cells or host cells used herein often refer to cells into which exogenous nucleic acid sequences, such as vectors, have been introduced. Therefore, recombinant cells can be distinguished from naturally occurring cells that do not contain the introduced recombinant nucleic acids.

[0127] The host cells suitable for introducing the nucleic acid constructs described herein can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc. Examples of mammalian cells include a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), and other human or non-human mammal embryonic stem cells, pluripotent stem cells or somatic cells.

[0128] In one or more embodiments, the mammalian cell is an embryonic stem cell, pluripotent stem cell or somatic cell of a rodent cell (e.g., mouse) or a primate (e.g., human). In certain embodiments, the host cell is an embryonic stem cell or pluripotent stem cell of a human, or a somatic cell of a mouse (e.g., C57 mouse). Herein, embryonic stem cells are or are derived from stem cells separated or obtained from a human or animal embryo within 14 days of fertilization without in vivo development. In one or more embodiments, embryonic stem cells are embryonic stem cell lines established in the art. Human pluripotent stem cells include hiPSCs, such as hES cell lines hiPSCs obtained after induction reprogramming of urine cells according to literature records (Xue Y, et.al., PLoS One. 2013Aug 5; 8(8): e70573. and Fan K, et.al., SciRep. 2017Oct 18; 7(1): 13496).

[0129] The method of transferring the nucleic acid construct of the present invention into the cell of interest is a conventional method in the art, including but not limited to: viral transduction, microinjection, particle bombardment, gene gun transformation and electroporation, etc. In certain embodiments, electroporation or microinjection is used. In one or more embodiments, when the host cell (e.g., Escherichia coli) is cultured under appropriate conditions, it is synthesized protein, and the protein can then be collected from the culture medium (if the host cell secretes it into the culture medium) or directly collected from the host cell that produces it (if it is not secreted). The recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties, and these methods are well known to those skilled in the art.

[0130] Also provided herein is a pharmaceutical composition comprising any one or more of the antibodies or antigen-binding fragments thereof, nucleic acid molecules, nucleic acid constructs and cells described herein, and a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient is non-toxic to the recipient at the dosage and concentration used, and may include at least one of a buffer (e.g., neutral buffered saline, sulfate buffered saline), an antioxidant, a preservative, an isotonic agent, a stabilizer, a chelating agent (e.g., EDTA or glutathione), an adjuvant (e.g., aluminum hydroxide) and a surfactant.

[0131] The present invention also provides a method of treatment, comprising administering a therapeutically effective amount of a pharmaceutical composition to a patient in need. As used herein, "effective amount" refers to an amount that can produce function or activity in humans and / or animals and can be accepted by humans and / or animals. The mode of administration, dosage and treatment regimen for a specific patient can be easily determined by a technician in the medical field by monitoring the patient's disease signs and adjusting the treatment accordingly. For example, gene therapy can be used. For example, an inhibitor of TILRR can be directly administered to a subject by methods such as injection; alternatively, an expression unit carrying an inhibitor of TILRR (such as an antibody expression vector or virus, or an sgRNA construct) can be delivered to a target (such as a tumor cell) through a certain route, and an active TILRR inhibitor can be expressed, depending on the type of inhibitor, which is well known to those skilled in the art.

[0132] This article also relates to the use of TILRR gene or protein as a target in screening drugs for preventing and / or treating kidney diseases or symptoms, including but not limited to screening for the prevention and / or treatment of renal fibrosis (e.g., tubulointerstitial fibrosis) or renal inflammation-related diseases, such as chronic nephritis, glomerular disease or renal injury. The glomerular disease is such as IgA nephropathy and membranous nephropathy. The renal injury can be drug-induced renal injury, renal injury induced by other diseases (such as diabetes, liver disease), and renal injury induced by autoimmune diseases. In one or more embodiments, renal diseases or symptoms may include tubular dilatation, tubulointerstitial scarring, ureteral obstruction, hydronephrosis, renal pelvic enlargement, renal atrophy, renal cortical thinning, renal medulla thinning, or renal collagen deposition. Specifically, TILRR protein or gene can be used as a molecular target to screen out molecules that can inhibit its activity or expression level, which can be new small molecule compounds or known compounds. The screening can be performed using cells or tissues expressing or not expressing TILRR, such as renal cells, renal tissues, or TILRR- / - kidney organoids or various tubes therein, such as epithelial cells, podocytes, endothelial cells, and fibroblasts of distal and / or proximal tubes of TILRR- / - mice. Such agents include, but are not limited to, small molecule compounds, siRNAs, and polypeptides. It should be understood that the renal cells of TILRR- / - mice can be derived from mice with systemic TILRR knockout as described herein.

[0133] An exemplary method for screening, preventing and / or treating kidney diseases or symptoms comprises the following steps:

[0134] (1) treating a system expressing TILRR with a candidate substance; and (2) detecting the expression or activity of TILRR in the system, or detecting whether the candidate substance targets cells in which TILRR expression is increased. If the candidate substance reduces the expression or activity of TILRR compared to a control group, or the candidate substance targets cells that highly express TILRR, it indicates that the candidate substance is a potential substance for preventing or treating kidney disease or symptoms, wherein the control group is a system expressing TILRR to which the candidate substance is not added. Generally, if the expression or activity of TILRR in the test group is statistically lower (preferably significantly lower, such as lower by more than 20%, preferably lower by more than 50%; more preferably lower by more than 80%) than that of the control group, it indicates that the candidate substance is a potential substance for preventing or treating kidney disease or symptoms. The system includes: a cell system (or a cell culture system), a subcellular system, a solution system, a tissue system, an organ system or an animal system.

[0135] For example, renal organoids with symptoms (such as inflammation or fibrosis, including but not limited to IL-1β-induced inflammation or fibrosis) are cultured separately with a culture medium containing a candidate substance, and the expression of pro-inflammatory factor genes (such as IL8), fibrosis marker genes (such as ACTA2 / FN1), renal injury marker genes (such as NGAL), and typical fibrosis markers (such as COL1A1) are detected. If the expression of one or more of these genes decreases or the increase is decreased compared to the control cultured without the candidate substance, the substance can be used as a candidate for preventing and / or treating kidney diseases or symptoms. Such a substance can be a small molecule, a polypeptide, a protein, or a nucleic acid.

[0136] The present invention will be further described below by way of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and reagents used in the examples are, unless otherwise stated, conventional methods and reagents in the art.

[0137] 1. Experimental Materials and Reagents

[0138] Table 1 Experimental reagents and manufacturers

[0139]

[0140]

[0141] Table 2 Experimental consumables and manufacturers

[0142]

[0143]

[0144] Table 3 Experimental instruments and equipment and manufacturers

[0145]

[0146]

[0147] 2. Experimental Methods

[0148] 2.1 Cell culture method

[0149] The subculture, cryopreservation and recovery of hiPSCs cells and other gene-edited human pluripotent stem cells all use the same operating steps (taking the single-well operation of a 6-well plate as an example, the reagent dosage of other culture vessels should be adjusted accordingly).

[0150] (1) Before subculturing, the wells should be coated with 1 mL of Matrigel (1:200 dilution in F12 medium) and incubated in a 37°C incubator for at least 30 min.

[0151] (2) When the cell confluence reaches about 80%, subculture. Aspirate the culture supernatant, add 1 mL of F12 culture medium to wash the cells, and aspirate F12.

[0152] (3) Add 1 mL of mild cell dissociation reagent GCDR to digest the cells and incubate at room temperature for 6-8 min. When the edges of the cell clones become brighter and gaps appear between the cells under a microscope, aspirate GCDR.

[0153] (4) Add 1 mL of mTeSR1 (or mTeSR1 plus) medium and gently blow with a 1 mL pipette tip to form a cell suspension (the cells should be uniform aggregates of 50-200 μm in size, and avoid blowing into single cells). Then, plate the cells at a ratio of 1:6 (adjust the subculture ratio as needed) into a new culture well coated with Matrigel and add mTeSR1 (or mTeSR1 plus) medium to 2 mL.

[0154] (5) Place the plate in a 37°C incubator and shake it back and forth to ensure that the cells are evenly distributed. Change the medium once a day, and generally subculture once every three days (depending on the cell subculture density).

[0155] 2.2 Kidney organoid differentiation construction and culture methods

[0156] 2.2.1 Preparation of culture medium

[0157] The BPEL (bovine serum albumin polyvinylalcoholessential lipids) and Stage II culture medium required for the differentiation and culture process of kidney organoids were prepared according to Tables 4 and 5 below, respectively, and stored in a refrigerator at 4°C after being sterilized by filtration through a 0.22 μm filter membrane.

[0158] Table 4 BPEL medium formula

[0159]

[0160] Table 5 Stage II culture medium formula

[0161]

[0162] 2.2.2 Static culture method on flat plate

[0163] The following is an example of a culture system where one 10 cm dish of cells corresponds to one ultra-low adhesion 6-well plate:

[0164] (1) Cell preparation: hiPSCs cells were cultured in a 10 cm dish, and subsequent operations were started after the cells reached a confluence of 80%.

[0165] (2) Preparation of BPEL complete medium. Add 14.4 μL of CHIR99021 (10 mM) to 18 mL of BPEL medium to a final concentration of 8 μM, add 6 μL of Y-27632 (10 mM) to a final concentration of 3.3 μM, and add 32.7 μL of β-mercaptoethanol (55 mM) to a final concentration of 100 μM. After the medium is mixed, add it to an ultra-low adhesion 6-well plate at a volume of 2 mL / well for later use.

[0166] (3) On day 0, wash the cells twice with 5 mL of DPBS each time. After aspirating the DPBS, add 3 mL of Dispase digestion enzyme and incubate in a 37°C incubator for 6 min. When the edges of the cell colonies are observed to curl up under a microscope, stop the digestion, aspirate Dispase, and wash the cells three more times with 5 mL of DPBS each time.

[0167] (4) Add the remaining 6 mL of BPEL complete medium to a 10 cm dish and scrape the cell clones from the bottom of the dish in the same direction with a cell scraper. Use a 10 mL pipette to blow the cell suspension 3-5 times. The cell clumps should be uniform and about 100 μm in size. Evenly plate the cell suspension into an ultra-low adhesion 6-well plate, 1 mL per well. Place the plate in a 37°C incubator, shake it up and down, and let it stand for 48 hours.

[0168] (5) On the second day, change the medium. First prepare 12 mL and add 9.6 μL of 10 mM CHIR99021 (final concentration is 8 μM). Then tilt the plate 45 degrees. After the formed embryoid bodies settle at the bottom of the wells, aspirate 2 mL from each well and retain 1 mL of the culture supernatant. Add 2 mL of the newly prepared BPEL culture medium to each well. Return the well to the 37°C incubator, shake again, and continue culturing.

[0169] (6) On day 3, the second stage of culture, the culture medium was replaced with Stage II medium. Use a 10 mL pipette to transfer the embryoid bodies and the culture medium to a 50 mL centrifuge tube. Wash the culture plate with 1 mL of DMEM medium per well and transfer all of it to a 50 mL centrifuge tube.

[0170] (7) After all the embryoid bodies have settled to the bottom of the centrifuge tube, aspirate the culture medium supernatant until about 5 mL remains, then add 10 mL of DMEM culture medium for washing. After the embryoid bodies have settled to the bottom of the centrifuge tube again, aspirate the supernatant.

[0171] (8) Add 2 mL / well of Stage II medium to the original ultra-low adhesion 6-well plate. Add 6 mL of Stage II medium to the 50 mL centrifuge tube containing embryoid bodies, and then use a pipette to transfer the mixture of embryoid bodies and medium and evenly distribute it to the original ultra-low adhesion 6-well plate at 1 mL / well.

[0172] (9) Return the plate to the 37°C incubator, shake it up and down, and continue culturing. Change the medium every other day. Tilt the plate 45 degrees when changing the medium. After the organoids have fully settled, aspirate 2 mL of supernatant from each well and add 2 mL of Stage II medium to each well. Continue culturing until the 14th day.

[0173] 2.2.3 Collection and processing of kidney organoid samples

[0174] According to the specific experimental needs, kidney organoid samples were collected at the corresponding time points.

[0175] For gene transcription level expression analysis: pipette an appropriate amount of kidney organoids (about 50 / sample) into a 1.5 mL centrifuge tube; aspirate the culture medium supernatant, wash with DPBS, and aspirate DPBS; add 0.75 mL of TRIzol reagent, transfer to a -80 °C refrigerator for storage until RNA extraction.

[0176] For immunofluorescence staining analysis: aspirate an appropriate amount of kidney organoids (about 15 / sample) into a 24-well plate; aspirate the supernatant of the culture medium, wash with DPBS, and aspirate DPBS; add 1 mL / well of 4% paraformaldehyde, and fix the kidney organoids at room temperature for about 20 minutes; aspirate 4% paraformaldehyde, wash several times with DPBS, and aspirate DPBS; add 1 mL / well of 30% sucrose solution, and dehydrate in a 4°C refrigerator for 24 hours; transfer the kidney organoids to an embedding mold, aspirate excess solution, embed with OCT embedding agent, and then transfer to a -20°C refrigerator for storage until frozen sections.

[0177] 2.3 Frozen sections

[0178] (1) Install the microtome and set the temperature of the cryostat to -20°C. Place the embedded sample in the microtome to equilibrate the temperature.

[0179] (2) Remove the sample block from the embedding mold and fix it on the sample holder after freezing with OCT.

[0180] (3) Fix the cooled sample onto the machine sample head, use the sample fast forward button to move the sample closer to the cutting edge, and use the slow forward button to start trimming and adjust the plane to be cut.

[0181] (4) Adjust the anti-roll plate and set the slice thickness to 16 μm. Turn the slicer wheel to start collecting slices. The slices should be complete and smooth.

[0182] (5) Place a glass slide close to the cut slices and adhere the tissue to the slide. Generally, 3-5 slices are attached to one slide.

[0183] (6) Place the slides in a section box and place them in a 4°C refrigerator for short-term storage for subsequent experiments, or place them in a -80°C refrigerator for long-term storage.

[0184] 2.4 Hematoxylin and eosin staining

[0185] Refer to the following instructions for the Hematoxylin-Eosin (H&E) staining kit.

[0186] For frozen section samples:

[0187] (1) Take the slides out of the refrigerator and warm them to room temperature.

[0188] (2) Add PBS dropwise onto the sample slices and wash several times until the OCT is completely dissolved and washed.

[0189] (3) Place the slide in hematoxylin solution for about 1 min, then soak and rinse in tap water.

[0190] (4) Place the slide in differentiation solution for 30 seconds.

[0191] (5) Soak the slide in tap water for 15 minutes.

[0192] (6) Place the slide in eosin stain for about 30 seconds and then soak and rinse in tap water.

[0193] (7) Soak in tap water for 2-5 minutes.

[0194] (8) Dehydration, transparency, and sealing: sequentially pass the slides through 30% ethanol for 2-3 seconds, 50% ethanol for 2-3 seconds, 70% ethanol for 2-3 seconds, 80% ethanol for 2-3 seconds, 90% ethanol for 2-3 seconds, anhydrous ethanol for 2-3 seconds, anhydrous ethanol for 1 minute, xylene (I) for 2 minutes, and xylene (II) for 2 minutes; seal with neutral resin, cover with a coverslip, and place in a fume hood overnight until the neutral resin solidifies.

[0195] (9) Use a panoramic slide scanner to scan and take photos.

[0196] For paraffin section samples: Dewaxing and hydration are required first. The slides are sequentially passed through xylene (Ⅰ) for 5 minutes, xylene (Ⅱ) for 5 minutes, anhydrous ethanol for 3 minutes, anhydrous ethanol for 3 minutes, 90% ethanol for 3 minutes, 80% ethanol for 3 minutes, 70% ethanol for 3 minutes, 50% ethanol for 3 minutes, 30% ethanol for 3 minutes, and distilled water for 3 minutes. The subsequent steps are the same as for frozen sections.

[0197] 2.5 RNA extraction, reverse transcription and quantitative PCR

[0198] 2.5.1 Cell lysis and RNA extraction

[0199] (1) Adherent cells: Add 0.75 mL of TRIzol reagent to one well of a 6-well plate, pipette thoroughly, transfer to a 1.5 mL centrifuge tube, and let stand at room temperature for 5 min;

[0200] Kidney organoids: After the samples collected in 2.2.2.4 are thawed, two 2 mm steel beads are added to each sample tube, and the samples are placed in a pre-cooled tissue grinder, ground at 40 Hz for 1 min, and allowed to stand at room temperature for 5 min;

[0201] Mouse kidney tissue: Take an appropriate amount of tissue block into a 1.5 mL centrifuge tube, add 0.75 mL TRIzol reagent, add 3 2 mm steel beads, put it into a pre-cooled tissue grinder, grind at 70 Hz for 5 min, and let it stand at room temperature for 5 min.

[0202] (2) Add 0.2 volume of 1,3-bromochloropropane, shake vigorously for 15 seconds, and place on ice for 15 minutes.

[0203] (3) Centrifuge at 12,000 × g for 12 min at 4°C.

[0204] (4) Aspirate the upper aqueous phase into a new 1.5 mL centrifuge tube, add an equal volume of isopropanol, mix by inversion for 15 seconds, and place at -20°C for 10 minutes.

[0205] (5) Centrifuge at 12,000 × g for 10 min at 4°C.

[0206] (6) Discard the supernatant, add 1 mL of 75% ethanol prepared with RNase-free H2O, invert, centrifuge at 7500 × g at 4°C for 5 min, aspirate the ethanol, and repeat this step twice.

[0207] (7) After absorbing the ethanol, dry it at room temperature for 5 min until the ethanol evaporates completely and the precipitate becomes transparent.

[0208] (8) Add 30-50 μL RNase-free H2O to dissolve the precipitate and place on ice.

[0209] (9) After measuring the concentration of the obtained RNA using SPECTRA MAX 190, reverse transcription is performed or the RNA is transferred to a -80°C freezer for storage.

[0210] 2.5.2 Reverse transcription

[0211] Using GoScript TM Reverse Transcription Mix, Oligo(dT) Kit:

[0212] (1) Take 1 μg RNA from each sample into a PCR tube and add Nuclease-Free Water to a total volume of 10 μL.

[0213] (2) Preheat the tube at 70°C for 5 min in a PCR instrument and place on ice for 5 min. Centrifuge gently for 5 s in a microcentrifuge and place on ice.

[0214] (3) On ice, prepare Reverse Transcription Mix (1 reaction system) according to the following composition:

[0215]

[0216] (4) Mix 10 μL of RNA sample and 10 μL of Reverse Transcription Mix, place in a PCR instrument, and perform reverse transcription according to the set program.

[0217] Reverse transcription procedure:

[0218]

[0219] (5) After the reaction is completed, the obtained cDNA sample is stored at -20°C.

[0220] 2.5.3 Real-time fluorescence quantitative PCR

[0221] Use SsoAdvanced Universal SYBR Green Supermix Kit for quantitative real-time PCR (qRT-PCR):

[0222] (1) The cDNA sample obtained by reverse transcription was diluted at 1:20 and used as a template for qRT-PCR. The template amount of cDNA for a single well was 10 ng.

[0223] (2) For each sample, three replicate wells were prepared for different genes, and the reaction system in each well was as follows:

[0224]

[0225] (3) The standard reaction program of Applied Biosystems StepOnePlus real-time fluorescence quantitative PCR instrument was used: 95°C, 10 min; (95°C, 15 s)*40 cycles; 60°C, 1 min.

[0226] Plate reading

[0227] Melting curve: 95℃15s, 60℃1min, 60℃-95℃+0.3℃ / s plate reading, 95℃15s plate reading

[0228] (4) When testing, GAPDH is generally used as the internal reference, and mouse is Gapdh. The ΔΔCt method is used to process the experimental results.

[0229] 2.6 Immunofluorescence staining

[0230] For adherent cells cultured in well plates:

[0231] (1) The cells were seeded into a 24-well plate. When the cells reached a density of 30-50%, the medium was discarded, the cells were washed twice with 500 μL PBS, and 500 μL 4% paraformaldehyde was added to fix the cells at room temperature for 20 min.

[0232] (2) Aspirate and discard 4% paraformaldehyde, and wash three times with PBS, 5 min each time.

[0233] (3) Permeabilization staining: Use primary antibody diluent (PBS + 10% serum from the corresponding secondary antibody species + 0.3% TritonX-100) to dilute the primary antibody in proportion, add 300 μL, and incubate at 4°C overnight.

[0234] (4) Aspirate and discard the antibody dilution solution and wash three times with PBS, 5 min each time.

[0235] (5) Dilute the secondary antibody (1:1000) and DAPI (1:5000) of the species corresponding to the primary antibody in PBS, add 300 μL of the secondary antibody mixture, and incubate at room temperature in the dark for 1-1.5 h.

[0236] (6) Aspirate and discard the antibody dilution solution and wash three times with PBS, 5 min each time.

[0237] (7) Add 300 μL PBS or anti-fluorescence quencher to prevent the cells from drying out, and use a laser confocal microscope Zeiss LSM 800 to detect and photograph.

[0238] For cryosection samples of renal organoids / mouse kidney tissue:

[0239] (1) Take the slides out of the refrigerator and warm them to room temperature.

[0240] (2) Carefully add PBS onto the sample slice from the edge using a Pasteur pipette and wash several times until the OCT is completely dissolved and washed.

[0241] (3) Blocking and permeabilization. Prepare blocking and permeabilization solution (PBS + 5% serum from the corresponding secondary antibody species + 0.3% TritonX-100). Use an immunohistochemical PAP pen to draw a circle around the slice sample on the slide, and add an appropriate amount of blocking and permeabilization solution into the circle. Place the slide in a humidified box at room temperature for 1-2 hours.

[0242] (4) Use primary antibody diluent (PBS + 1% BSA + 0.3% Triton X-100) to dilute the primary antibody in proportion. Use absorbent paper to absorb the blocking permeabilization solution from the edge of the sample circle, then add an appropriate amount of primary antibody diluent, place the slide in a humidified box, and incubate overnight at 4°C.

[0243] (5) Use absorbent paper to absorb the primary antibody dilution from the edge of the sample circle, and add PBS to wash three times, 5 minutes each time.

[0244] (6) Dilute the secondary antibody (1:1000) and DAPI (1:5000) of the species corresponding to the primary antibody in PBS, add an appropriate amount of the secondary antibody mixture, and incubate in a humidified box at room temperature in the dark for 1-1.5 h.

[0245] (7) Use absorbent paper to absorb the secondary antibody mixture from the edge of the sample circle, and add PBS to wash three times, 5 min each time.

[0246] (8) Use absorbent paper to absorb the liquid in the sample circle, and add an appropriate amount of anti-fluorescence quenching agent before the sample dries up. Then, add a cover glass to seal the sample, and use a two-photon laser confocal microscope Zeiss LSM 710 to detect and take pictures.

[0247] 2.7 Experimental study on TILRR blocking antibody in renal organoid fibrosis model

[0248] (1) Preparation of TILRR blocking antibodies. Antibodies were prepared based on TILRR using hybridoma technology. The prepared antibodies were validated for antibody specificity using Western Blot.

[0249] (2) Determination of the optimal concentration of TILRR blocking antibody. On the 9th day of kidney organoid culture, kidney organoids in good condition were selected and placed in two ultra-low adhesion 96-well plates, with one kidney organoid placed in each well, and 12 wells in a row as a group; 0, 500, 1000, 2000 ng / mL, a total of 4 concentrations were set, and the corresponding concentrations (500, 1000, 2000 ng / mL) of isotype control Normal Rabbit IgG (#2729, CST) antibody groups were set, and the IL-1β action groups corresponding to the above antibody action groups were set at the same time, so the samples of the two ultra-low adhesion 96-well plates can be divided into 14 groups in total; on the 9th day, Stage II culture medium containing the corresponding antibody concentrations was added to the 14 groups of samples in advance, 200 μL of culture medium per well, and the action was carried out for 24 hours; on the 10th day, half of the medium was replaced, and each group was added with Stage II culture medium containing the corresponding antibody concentration. II culture medium, and 100 ng / mL IL-1β was added to 7 IL-1β action groups to induce fibrosis; 48 hours later, that is, on the 12th day, 14 groups of samples were collected uniformly for RNA extraction and qRT-PCR detection to analyze the expression of specific marker genes.

[0250] (2) Batch experiment on the effect of optimal concentration of TILRR blocking antibody. On the 9th day of renal organoid culture, the medium of 4 wells of renal organoids cultured in ultra-low adhesion 6-well plates was completely replaced, and 2 wells were added with Stage II medium containing 1000 ng / mL TILRR blocking antibody, and the other 2 wells were added with Stage II medium containing 1000 ng / mL isotype control IgG antibody for 24 h; on the 10th day, the medium of 4 wells was completely replaced with Stage II medium containing the corresponding concentration of antibody, and 2 wells were added with 100 ng / mL IL-1β in the presence of two contrasting antibodies; 48 h later, on the 12th day, the samples of 4 wells were collected for subsequent qRT-PCR and immunofluorescence detection analysis.

[0251] 2.8 Experiment on renal fibrosis modeling in mice with unilateral ureteral obstruction

[0252] (1) The experimental mice were wild-type (WT) and TILRR knockout (TILRR- / -, constructed in the early laboratory) mice of the C57BL / 6 strain. Several 6-week-old male mice were selected for the experiment.

[0253] (2) According to the weight of the mouse, the anesthetic Avertin was injected intraperitoneally to anesthetize the mouse. After the mouse entered a deep anesthesia state, the abdominal hair was shaved with a shaver, the mouse skin was wiped with a cotton ball, and the mouse was placed on the operating table. The limbs were fixed with tape, and the abdominal skin was wiped with iodine.

[0254] (3) Surgical operation. Use ophthalmic scissors to cut about 2 cm long along the linea alba of the mouse abdomen; use a mouse spreader to spread the mouse abdominal skin from both sides. After the intestines and liver and other organs are exposed, use forceps to carefully push the intestines apart; use cotton balls to separate the kidneys from the liver and spleen; use curved forceps to pass through the left kidney ureter, tie both ends with 4-0 black silk thread, cut in the middle, and only perform ureteral stripping on the opposite right kidney without ligation; suture the muscle layer and epidermis layer in sequence; place the mouse under a warm light for 1 hour.

[0255] (4) Seven days after the experiment, the mice were dissected and the kidneys on both sides were removed. The kidney appearance was recorded by photographing, and each kidney was cut longitudinally from the left to the right. Half of the kidney was fixed with 4% paraformaldehyde at 4°C overnight, and then embedded in paraffin, sectioned, stained with H&E, and stained with Masson's staining; the other half of the kidney was partially placed in TRIzol reagent, and RNA was extracted and analyzed by qRT-PCR.

[0256] 2.9 Paraffin embedding and sectioning

[0257] (1) Sample pretreatment before embedding: 4% paraformaldehyde fixation at 4℃; PBS soaking at 4℃ for 0.5h, PBS soaking at room temperature for 0.5h, twice; 70% ethanol soaking at room temperature for 0.5h; 80% ethanol soaking at room temperature for 0.5h; 95% ethanol soaking at room temperature for 0.5h; anhydrous ethanol soaking at room temperature for 0.5h, twice; anhydrous ethanol + xylene (1:1) soaking at room temperature for 0.5h; xylene soaking at room temperature for 0.5h, twice.

[0258] (2) Embedding. Place the paraffin in a 58°C oven to fully dissolve. The following steps are all performed in a 58°C oven in a fume hood: soak overnight in xylene + paraffin (1:1); soak in paraffin for 1 hour, 3 times; wait until the paraffin completely permeates the tissue, place the tissue block in a pre-prepared mold, pour paraffin to cover and embed; place the entire mold on ice water to pre-cool, wait for the upper layer of wax to solidify and turn white, then place the entire wax block in ice water to cool overnight, and then proceed with subsequent sectioning the next day.

[0259] (3) Slicing. Use a blade to trim the embedded wax block into a neat quadrangular pyramid, fix it on the sample holder, and clamp it in the wax block clamp of the rotary slicer so that the wax block surface is parallel to the slice blade. The slice thickness is set to 6 μm. After the wax slice is cut, use small tweezers in the right hand to pick up the wax slice, and use a brush in the left hand to gently separate the wax slice along the blade. Place the slice with the cut surface facing down in 42°C water, flatten it, and gently separate the continuous wax slices with tweezers, then pick it up with a slide, dry it, put it in a slice box, and bake it at 42°C overnight.

[0260] 2.10 Immunohistochemical staining

[0261] Using GTVision TM III anti-mouse / anti-rabbit universal immunohistochemistry detection kit.

[0262] (1) Paraffin sections were routinely dewaxed and hydrated.

[0263] (2) Antigen retrieval. Place the slices in 0.1M sodium citrate buffer, pH 9.0, microwave on medium heat until bubbling but not boiling. After 5 minutes, microwave on medium heat again until bubbling but not boiling. Repeat 3 times and cool to room temperature. After cooling, wash the slices 3 times with PBS, 3 minutes each time.

[0264] (3) Incubate the sections in 3% hydrogen peroxide solution in the dark for 20 min, and wash with PBS three times, each time for 3 min.

[0265] (4) After drying the slices, circle the samples with an immunohistochemistry pen and block with 3% BSA + 0.3% Triton X-100 + PBS for 1 hour.

[0266] (5) After drying the blocking solution, add the primary antibody (Anti-TILRR, Rabbit Polyclonal antibody, GenScript custom antibody, 1:150 dilution) to the sample and incubate at 4°C overnight.

[0267] (6) Dry the antibody diluent and wash with PBS three times, 3 min each time.

[0268] (7) Add solution A from the immunohistochemistry test kit onto the sample and incubate at room temperature for 1 hour. After incubation, wash with PBS three times, 3 minutes each time.

[0269] (8) Add the prepared DAB working solution (add 1 drop of 20 μL of solution B per mL of solution C) and incubate at room temperature for 1-3 min (observe under a microscope to control the color development time) until the brown-yellow color no longer deepens. Rinse with distilled water to terminate the color development.

[0270] (9) Counterstain with hematoxylin for 30 seconds and then rinse with tap water.

[0271] (10) Dehydrate with different levels of alcohol (30%-100%) for 1 min each level, then place in xylene for 2 min, repeat twice.

[0272] (11) Cover with a coverslip, seal with neutral resin, and place in a fume hood overnight until the neutral resin solidifies.

[0273] (12) Use a panoramic slide scanner to scan and take photos.

[0274] 2.11 Masson staining

[0275] Staining was performed using Masson's trichrome staining kit.

[0276] (1) Paraffin sections were routinely dewaxed and hydrated.

[0277] (2) Stain with prepared Weigert iron hematoxylin staining solution for 5 min-10 min; differentiate with acidic ethanol differentiation solution for 5-15 s, wash with water; return to blue with Masson blueing solution for 3-5 min, wash with water; wash with distilled water for 1 min.

[0278] (3) Stain with Ponceau fuchsin solution for 5-10 min and wash with weak acid working solution for 1 min.

[0279] (4) Wash with phosphomolybdic acid solution for 1-2 minutes and with weak acid working solution for 1 minute.

[0280] (5) Stain in aniline blue staining solution for 1-2 minutes and wash in weak acid working solution for 1 minute.

[0281] (6) Dehydrate the sections in 95% ethanol for rapid dehydration; dehydrate in anhydrous ethanol three times, each time for 5-10 seconds; and clear in xylene three times, each time for 1-2 minutes.

[0282] (7) Cover with a coverslip, seal with neutral resin, and place in a fume hood overnight until the neutral resin solidifies.

[0283] (8) Use a panoramic slide scanner to scan and take photos.

[0284] Table 6. List of qRT-PCR primer sequences

[0285]

[0286] Table 7. List of antibodies used in immunofluorescence staining

[0287]

[0288]

[0289] Example 1: Construction of TILRR knockout mice and human stem cell lines

[0290] 1. Construction and identification of TILRR gene knockout human stem cell lines

[0291] (1) According to the literature records Xue Y, et al., PLoS One. 2013Aug 5; 8(8): e70573. and Fan K, et al., Sci Rep. 2017Oct 18; 7(1): 13496. hiPSCs stem cell line hiPSCs was obtained by inducing reprogramming of urine cells and then purifying. hiPSCs were seeded in a 6-well plate. When the cells grew to 80% confluence, the cells were first washed with F12 medium, and then 1.0 mL of digestive enzyme Accutase was added at 37°C for 4-6 minutes to digest into single cells; 3 volumes of F12 were added to terminate the digestion, and the cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 300×g for 3 minutes, and the supernatant was discarded to obtain a cell pellet.

[0292] (2) Using Basic Nucleofector TM Kit for Primary Mammalian Epithelial Cells, pX459-TILRR-sgRNA (sgRNA sequence: Forward (5'-3'): TGAGATCACACTGGAGACTG (SEQ ID NO: 17); Reverse (5'-3'): ACTCTAGTGTGACCTCTGAC (SEQ ID NO: 18)) plasmid was electroporated, and the electroporation program was B-016, and electroporation was performed:

[0293] (3) 24 h after electroporation, replace with new mTeSR1 medium (containing 10 mM Y-27632) and continue culturing.

[0294] (4) 48 h after electroporation, replace the mTeSR1 medium with fresh one and add Puromycin (0.5 μg / mL) to screen positive clones.

[0295] (5) After 24 h of Puromycin treatment, the mTeSR1 medium was replaced with new medium and the cell clones were cultured and observed for growth.

[0296] (6) Generally, continue culturing for about one week and pick single clones. Replace mTeSR1 medium (containing 100× ampicillin-streptomycin). After all clones are picked, place them in a 37°C cell culture incubator for continued culture.

[0297] (7) When the cell clones reach a certain size, they can be subcultured.

[0298] Cell clone identification

[0299] (1) DNA sequencing identification of knockout sites. PCR identification primers are as follows:

[0300] F:TTAAGCACTGATGAGCCTGAGAAATATGATTG (SEQ ID NO: 20);

[0301] R:GTGTGGCTTTTGCTCCTTTCTGTGCTAC(SEQ ID NO:21)

[0302] (2) The knockout cell clones were expanded and cultured in accordance with the cells that were retained in 2.1, and the cells were frozen. At the same time, RNA was extracted and qRT-PCR analysis was performed to identify the transcriptional expression of TILRR and pluripotency-related marker genes;

[0303] (3) Protein was extracted and the expression level of TILRR protein was identified by western blot. The cells were plated into new 24-well plates and the expression of pluripotency-related marker proteins was identified by immunofluorescence staining to further confirm the knockout of TILRR gene and the maintenance of pluripotency of pluripotent stem cells.

[0304] Results and Analysis

[0305] A gene mutation clone cell line (hiPSCs-TILRR- / -) was obtained through knockout screening. DNA sequencing results showed that this cell line had an 8-base pair deletion in the front double-stranded DNA of exon 26 (E26) of the TILRR (FREM1 isoform2) genomic sequence ( Figure 1 (A)). The deletion of this base pair caused the translation of the 715-amino acid TILRR to change at the 36th amino acid and terminate the translation prematurely at the 61st amino acid. The cell morphology of hiPSCs-TILRR- / - cells was not significantly different from that of wild-type hiPSCs by bright-field imaging under an inverted microscope ( Figure 1 (B)). Then, qRT-PCR and WB were used to detect the expression of TILRR at the transcriptional and protein levels in the knockout cells. Figure 1 (C) and Figure 1 The results of (D) show that TILRR in hiPSCs-TILRR- / - cells is almost not expressed at the transcriptional and protein levels. At the same time, the expression of cell pluripotency marker genes was detected by qRT-PCR. Figure 1 The results in (E) showed that the expression levels of pluripotency-related genes OCT4 / SOX2 / NANOG were the same between hiPSCs-TILRR- / - cells and wild-type hiPSCs. Figure 1(F) Immunofluorescence staining analysis of the pluripotency markers OCT4 / SSEA4 / TRA-1-60 / TRA-1-81 protein levels also confirmed that the pluripotency of hiPSCs-TILRR- / - cells was no different from that of wild-type hiPSCs. In addition, the H&E staining results of the teratoma formation experiment showed that hiPSCs-TILRR- / - cells had multipotency after being transplanted into mice and could differentiate into cells of the three germ layers ( Figure 1 (G)). In addition, karyotype analysis showed that hiPSCs-TILRR- / - cells had normal 23 pairs (or 22 pairs +XY) of chromosomes, with no obvious abnormalities, such as Figure 1 (H) shown.

[0306] The above results indicate that this example successfully obtains a human stem cell TILRR- / - knockout cell line with normal pluripotency maintenance and self-renewal capabilities.

[0307] 2. Construction of TILRR knockout mice

[0308] (1) GGACACAAGAGCCCATGCTGA (SEQ ID NO: 19) is used as the sgRNA sequence. The AGG after this sequence is the NGG site where Cas9 theoretically acts.

[0309] (2) The constructed Cas9 vector was transcribed into mRNA in vitro and microinjected into the fertilized eggs of C57 wild-type mice.

[0310] (3) The embryos are then transplanted into the uterus of pseudopregnant mice for culture, and the offspring are observed and identified after birth.

[0311] The genomic DNA of multiple knockout embryos was identified after birth. The identification primers are as follows:

[0312] mTILRR F1 CGTAGGAGGGGTATCAGTGATGCT (SEQ ID NO: 22);

[0313] mTILRR R1 AGCGCTGTCCACAGTCTGCAGTG (SEQ ID NO:23)

[0314] The PCR products were sequenced and analyzed, and the positive edited offspring mice were retained.

[0315] (4) The successfully edited offspring mice are backcrossed, and the resulting mice are subjected to tail DNA extraction and PCR identification (identification primers are the same as above).

[0316] Characterization of TILRR knockout mice

[0317] (1) When the mice were genotyped, the toes of the newborn mice were clipped, numbered, and tails were cut when they reached two weeks of age. About 5 mm of the mouse tail tip was taken, and a proteinase K lysis solution was prepared using a mouse genotype identification kit (Vazyme, #PD101). The tail tip was immersed in the lysis solution and placed in a 55°C water bath overnight to lyse the tissue. The genotype of the TILRR mice was identified by sequencing the PCR product. PCR primers, the product fragment was about 600 bp. After agarose gel electrophoresis, the PCR product was cut and sent for sequencing; the sequencing primers used (m105: CGTAGGAGGGGTATCAGTGATGCT (SEQ ID NO: 22); m106: CACAAACTACGTGCCTGCCATAC (SEQ ID NO: 24)) After obtaining the sequencing results, the sequencing results were compared with the mouse TILRR knockout sequence by BLASTx to determine whether the sample was a knockout sequence and whether it was homozygous.

[0318] (2) RNA level identification was performed by extracting RNA from mouse tissues. Homozygous mice of appropriate age were selected for identification and killed by cervical dislocation. Brain, kidney, and spleen tissues were dissected and ground into powder in a pre-cooled mortar with liquid nitrogen. RNA was extracted using the Trizol method. The mTILRR primers qm001-F: CCTTGCCTCTCTTTGCCAGAT (SEQ ID NO: 39) and qm001-R: GCCAGAAAGAAGGTCAGGTTCTC (SEQ ID NO: 40) were used for qPCR. The internal reference gene was mGapdh; cDNA from the corresponding tissue of wild-type C57 mice was used as a control. After PCR, the PCR products were subjected to agarose gel electrophoresis and photographed to observe whether TILRR bands were present.

[0319] (3) Mouse tissue protein extraction was obtained by similar liquid nitrogen grinding. Homozygous genotype mice of appropriate age were selected for identification and killed by cervical dislocation. Brain, kidney and spleen tissues were obtained by dissection and ground into powder in a pre-cooled mortar with liquid nitrogen. Lysis Buffer was prepared according to the instructions of the protein extraction kit (KeyGEN BioTECH, #KGP2100). Phosphatase inhibitors, protease inhibitors and PMSF were added to the lysis buffer. The tissue powder in the mortar was washed and recovered with an appropriate volume of lysis buffer. Lysis was carried out on ice for 50 minutes. During this period, a 1 mL syringe needle was used to aspirate several times every ten minutes to complete the lysis. After lysis, centrifugation was performed at 17,000×g and 4℃ for 10 minutes. The supernatant was taken into a new tube. After sampling and determining the protein concentration (quantitatively using the BCA method as described above), 4× protein loading buffer was added at a ratio of 1:3 and mixed. The mixture was denatured in boiling water for 10 minutes and stored at -20℃. The knockout of TILRR protein was characterized by Western blotting.

[0320] Results and Analysis

[0321] The final genotype obtained was Figure 2 (A) TILRR knockout heterozygous mice (F1 generation) from mouse #1. The F1 generation mice were self-bred with the F2 generation mice, and the homozygous TILRR- / - mice were obtained by sequencing and identification; the homozygous TILRR knockout mice were used for the next experiment. TILRR knockout homozygous mice of about 8 weeks old and wild-type mice of similar age were taken, and the whole brain tissue, unilateral kidney and spleen of the mice were extracted for total RNA, reverse transcription was performed, and PCR and agarose gel electrophoresis were performed on the cDNA. Figure 2(B) With GAPDH as the internal reference, the knockout mice and wild-type tissue RNA can obtain GAPDH bands; the knockout mice tissue RNA cannot obtain the corresponding TILRR bands, while the wild-type mice in the control group have obvious TILRR bands, indicating that the TILRR knockout mice do not have normal TILRR gene mRNA expression. TILRR knockout homozygous mice of about 8 weeks of age and wild-type mice of similar age were taken, and the whole protein of the unilateral kidney and spleen of the mice was extracted, and after lysis, SDS-PAGE polyacrylamide gel electrophoresis and immunoblotting (Western blotting) were used. The immunoblotting experiment used GAPDH as the internal reference, and the expression of TILRR protein in the tissues of TILRR knockout mice and C57 wild-type mice was identified by TILRR monoclonal antibody. After ECL staining and imaging, a clear TILRR band of about 55 kDa was visible in the protein lane of wild-type mice; in the protein of TILRR knockout mice, a fuzzy immunoblot band of similar size also appeared, but the intensity was significantly lower than that of the wild type, which largely indicated that the expression of TILRR protein in TILRR knockout mice was knocked out or strongly knocked down ( Figure 2 (C)).

[0322] Example 2: TILRR knockout can reduce inflammation and fibrosis levels in the renal UUO model

[0323] In order to verify that the expression of TILRR can regulate the occurrence and development of renal fibrosis, a tubulointerstitial fibrosis model was constructed based on the TILRR knockout mice previously constructed in the laboratory, and the classic UUO modeling method was used to examine the role of TILRR in renal fibrosis in the mouse model. Wild-type (WT, 9 mice) and TILRR knockout (TILRR- / -, 10 mice) mice of the same age (6 weeks old) and gender (male) were selected. Each mouse underwent ureteral ligation surgery on the left kidney, while the contralateral (contralat) kidney was used as a control, with only ureteral stripping without ligation; 7 days after modeling, anatomical sampling was performed (due to unexpected factors such as surgical risks and infection-related deaths, 1 mouse died in the WT group and 5 mice died in the TILRR- / - group during the modeling period). When sampling, the renal appearance phenotype was observed, such as Figure 3 As shown in (A), the appearance of the kidneys with ureteral obstruction in both the WT and TILRR- / - groups changed significantly, becoming pale and enlarged due to hydronephrosis, but the degree of enlargement in the TILRR- / - group was less than that in the WT group. Further H&E staining of tissue sections showed that the obstructed kidneys atrophied, with obvious thinning of the renal cortex and medulla, and the degree of atrophy in the TILRR- / - group was less than that in the WT group. The renal coronal section is shown in Figure 2. Figure 3(A) As shown in the large image below. Through the observation of magnified H&E tissue sections, the histology of the normal untreated contralateral kidney showed that the glomeruli were surrounded by tight tubulointerstitial and renal tubules, and the renal tubules had clearly visible lumens; in the kidney after ureteral obstruction, although the glomeruli appeared normal, the tubules were significantly dilated and scars appeared in the tubulointerstitial. These fibrosis phenomena were generally weaker in the TILRR- / - group than in the WT group.

[0324] In order to determine the actual collagen deposition in renal fibrosis after UUO modeling in mice, the tissue sections were subjected to classic Mssson staining. Figure 3 As shown in (B), the staining results showed that muscle fibers were red and collagen fibers were blue. The kidneys with ureteral obstruction in the WT group and the TILRR- / - group had obvious collagen deposition compared with the contralateral kidneys, and the proportion of collagen in the WT group was slightly higher than that in the TILRR- / - group. Then, the blue collagen fibers in all tissue sections were quantitatively analyzed. The larger the area density (ArealDensity) value, the higher the positive expression level. It can be seen that the content of collagen in the kidneys with ureteral obstruction in the WT group is indeed higher than that in the TILRR- / - group.

[0325] After pathological analysis of the kidneys of UUO mice, traditional molecular biological methods were further used to analyze the expression of related marker genes. Figure 3 (C) qRT-PCR analysis showed that the expression of the proinflammatory cytokine IL-1β gene and the fibrosis marker gene Col1a1 / Fn1 / Acta2 in the ureteral obstruction kidney of the WT group was significantly upregulated and significantly higher than that in the ureteral obstruction kidney of the TILRR- / - group; in addition, although there was no significant statistical difference in the expression of the proinflammatory cytokine IL8 and the kidney injury marker gene Ngal, there was also a similar expression difference trend. It can be seen that the TILRR knockout mice in the UUO model have a lower degree of fibrosis at the gene expression level compared with WT mice.

[0326] Example 3: TILRR knockout reduces IL-1beta-induced inflammation and fibrosis in renal organoids

[0327] After constructing the hiPSCs-TILRR- / - cell line, the organoids were cultured in ultra-low adhesion six-well plates using the kidney organoid construction method. The previously reported method of adding IL-1β to induce kidney organoid fibrosis modeling (flat static culture method) was used to investigate the role of TILRR as an IL-1RI co-receptor. HiPSCs and hiPSCs-TILRR- / -derived kidney organoids were cultured at the same time. At the D10 period when the kidney organoids were basically mature, the experimental group replaced the culture medium containing 100 ng / mL IL-1β, and the control group replaced the normal culture medium. After continuing to culture for 48 hours until the D12 period, samples were taken for observation and analysis.

[0328] qRT-PCR was performed to analyze the overall transcriptional expression of related marker genes in four groups of samples (hiPSCs-OGD12, hiPSCs-OGD12+IL-1β48h, hiPSCs-TILRR- / --OGD12, hiPSCs-TILRR- / --OGD12+IL-1β48h). Figure 4 The results of (A) show that the expression levels of TILRR in the two groups of hiPSCs-TILRR- / --OGD12 samples were significantly lower than those in the two groups of hiPSCs-OGD12 samples, and the effectiveness of TILRR knockout was confirmed in kidney organoids again. At the same time, under the condition of IL-1β stimulation for 48h, the expression levels of proinflammatory factor gene IL-8, kidney injury marker gene NGAL, and fibrosis marker gene COL1A1 / ACTA2 / FN1 in hiPSCs-derived kidney organoids were significantly increased compared with their respective control groups, while there was no significant difference in hiPSCs-TILRR- / -derived kidney organoids (NGAL was only detected in hiPSCs-OGD12+IL-1β48h samples).

[0329] At the same time, immunofluorescence staining analysis was performed on the above four groups of samples to investigate the expression and distribution of the typical fibrosis marker COL1A1 in kidney organoids. The results are as follows Figure 4 (B) As shown. Under the condition of IL-1β stimulation for 48h, the expression of COL1A1 in hiPSCs-derived kidney organoids was enhanced and more abundantly distributed compared with the respective control groups, mainly expressed in the periphery of the renal tubules, that is, in the tubulointerstitium, while there was no significant difference in the expression of COL1A1 in hiPSCs-TILRR- / -derived kidney organoids. The quantitative statistical analysis of the mean fluorescence intensity of COL1A1 also confirmed these differences. The expression of COL1A1 can represent that under the condition of IL-1β stimulation for 48h, the degree of fibrosis in hiPSCs-TILRR- / -derived kidney organoids is lower than that in hiPSCs-derived kidney organoids. Combined with the results of the above qRT-PCR and immunofluorescence staining analysis, it is shown that the knockout of TILRR will lead to the restricted development of renal fibrosis mediated by IL-1RI, a receptor of IL-1β, which is mainly manifested in the reduction of tubulointerstitial fibrosis.

[0330] Example 4: TILRR blocking antibodies can significantly reduce IL-1β-induced renal organoid inflammation and fibrosis

[0331] Kidney organoids were cultured separately with culture medium containing blocking antibodies with different concentration gradients, and the optimal concentration of the antibodies was determined while judging the effect of the antibodies. This experiment examined a total of four concentrations of 0, 500, 1000 and 2000 ng / mL, and an isotype control IgG antibody group of TILRR blocking antibodies with the same concentration gradient was set up. Briefly, when some structures of kidney organoids had appeared on D9, a single kidney organoid was placed in a single 96-well plate well, with a total of 12 kidney organoids in each group, and then antibodies of corresponding concentrations were added, and the antibodies were allowed to act on the kidney organoids for 24 hours in advance. At D10, the culture medium containing antibodies of corresponding concentrations was replaced, and 100 ng / mL of IL-1β was added to the experimental group. qRT-PCR detection and analysis was performed after 48 hours of action. Figure 5 As shown in (A), the qRT-PCR test results showed that at antibody concentrations of 1000 and 2000 ng / mL, under the same IL-1β treatment conditions, the expression of the pro-inflammatory gene IL8 and the fibrosis marker gene ACTA2 / FN1 in the sample group treated with TILRR blocking antibodies were significantly lower than those in the IgG antibody control group. Although there was no statistical difference in the expression of the renal injury marker gene NGAL, it also showed the same trend. This preliminarily confirmed the role of TILRR blocking antibodies in inhibiting fibrosis at the transcription level of typical marker genes.

[0332] Based on the comprehensive judgment of the qRT-PCR test results, a TILRR blocking antibody with a concentration of 1000ng / mL was selected for batch experiments. Four groups of samples were set up, namely the 1000ng / mL IgG isotype antibody group, the 1000ng / mL TILRR blocking antibody group, and the corresponding IL-1β group. The experimental strategy was the same as before. Immunofluorescence staining analysis was used to identify the effect of TILRR blocking antibody in inhibiting renal organoid fibrosis caused by IL-1β stimulation. Figure 5 The results of (B) show that compared with the IgG isotype antibody control group, the expression of COL1A1, a typical marker of fibrosis, did not increase significantly in the TILRR blocking antibody group after IL-1β stimulation and induction. This contrast difference was confirmed by quantitative statistical analysis of the mean fluorescence intensity of COL1A1. In the normal WT group, IL-1β stimulation induced an increase in the expression of COL1A1, which led to fibrosis. However, after TILRR blocking treatment, IL-1β stimulation did not lead to COL1A1. From the above results, it can be seen that the occurrence and development of fibrosis in the IL-1β-induced renal organoid fibrosis disease model can be alleviated by TILRR blocking antibodies.

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[0342] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0343] <h2 style=";text-align:left;direction:ltr"> FR2:WVKQRPGQGLEWIG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0344] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0345] <h2 style=";text-align:left;direction:ltr"> FR3:KATLTVDKSSSTAYMQLSSPTSEDSAVYFCAR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0346] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0347] <h2 style=";text-align:left;direction:ltr"> FR4:WGQGTTLTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0348] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0349] <h2 style=";text-align:left;direction:ltr"> VH:MGWSCIILLLVSTATGVHSQVQLQQSGPQLVRPGASVEISCKASGYSFTSYWIHWVKQRPGQGLEWIGMIDPSDSETRLNQRFKDKATLVDKSSSTAYMQLSSPTSEDSAVYFCARSLYGGSCYWGQGTTLTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0350] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0351] <h2 style=";text-align:left;direction:ltr"> LCDR1:KSSQSLLDSDGKTYLN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0352] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0353] <h2 style=";text-align:left;direction:ltr"> LCDR2:LVSKLDS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0354] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:11<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0355] <h2 style=";text-align:left;direction:ltr"> LCDR3:WQGTHFPHT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0356] <h2 style=";text-align:left;direction:ltr">SEQ ID NO:12<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0357] <h2 style=";text-align:left;direction:ltr"> FR1:DVVMTQTPLTLSVTIGQPASISC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0358] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:13<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0359] <h2 style=";text-align:left;direction:ltr"> FR2:WLLQRPGQSPKRLIY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0360] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:14<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0361] <h2 style=";text-align:left;direction:ltr"> FR3:GVPDRFTGSGSGTDFTLKISRVEEAEDLGVYYC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0362] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0363] <h2 style=";text-align:left;direction:ltr"> FR4:FGSGTKLEIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0364] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:16<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0365] <h2 style=";text-align:left;direction:ltr"> VL:MSPAQFLFLLVLWIRETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPHTFGSGTKLEIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0366] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:17<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0367] <h2 style=";text-align:left;direction:ltr"> TGAGATCACACTGGAGACTG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0368] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:18<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0369] <h2 style=";text-align:left;direction:ltr"> ACTCTAGTGTGACCTCTGAC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0370] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:19<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0371] <h2 style=";text-align:left;direction:ltr"> GGACACAAGAGCCCATGCTGA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0372] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:20<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0373] <h2 style=";text-align:left;direction:ltr"> TTAAGCACTGATGAGCCTGAGAAATATGATTG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0374] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:21<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0375] <h2 style=";text-align:left;direction:ltr"> GTGTGGCTTTTGCTCCTTTCTGTGCTAC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0376] SEQ ID NO:22

[0377] CGTAGGAGGGGTATCAGTGATGCT

[0378] SEQ ID NO:23

[0379] AGGCGCTGTCCACAGTCTGCAGTG

[0380] SEQ ID NO:24

[0381] CACAAACTACGTGCCTGCCATAC

[0382] SEQ ID NO:25

[0383] GGCATGGACTGTGGTCATGAG

[0384] SEQ ID NO:26

[0385] TGCACCACCAACTGCTTAGC

[0386] SEQ ID NO:27

[0387] AGAGCCCTGCCTGTGGTAAC

[0388] SEQ ID NO:28

[0389] GAAGGGGAATGCAAGAGTGTGATA

[0390] SEQ ID NO:29

[0391] ACTGAGAGTGATTGAGAGTGGAC

[0392] SEQ ID NO:30

[0393] AACCCTCTGCACCCAGTTTTC

[0394] SEQ ID NO:31

[0395] GACAACCAATTCCAGGGGAAG

[0396] SEQ ID NO:32

[0397] GCATACATCTTTTGCGGGTCT

[0398] SEQ ID NO:33

[0399] GAGGGCCAAGACGAAGACATC

[0400] SEQ ID NO:34

[0401] CAGATCACGTCATCGCACAAC

[0402] SEQ ID NO:35

[0403] CTATGAGGGCTATGCCTTGCC

[0404] SEQ ID NO:36

[0405] GCTCAGCAGTAGTAACGAAGGA

[0406] SEQ ID NO:37

[0407] AACAAACACTAATGTTAATTGCCCA

[0408] SEQ ID NO:38

[0409] TCGGGAATCTTCTCTGTCAGC

[0410] SEQ ID NO:39

[0411] CCTTGCCTCTCTTTGCCAGAT SEQ ID NO:40GCCAGAAAGAAGGTCAGGTTCTC SEQ ID NO:41GAAATGCCACCTTTTGACAGTG

[0412] SEQ ID NO:42

[0413] TGGATGCTCTCATCAGGACAG

[0414] SEQ ID NO:43

[0415] CAAGGCTGGTCCATGCTCC

[0416] SEQ ID NO:44

[0417] TGCTATCACTTCCTTTCTGTTGC

[0418] SEQ ID NO:45

[0419] GGGAAATATGCACAGGTATCCTC

[0420] SEQ ID NO:46

[0421] CATGGCGAACTGGTTGTAGTC

[0422] SEQ ID NO:47

[0423] GCTCCTCTTAGGGGCCACT

[0424] SEQ ID NO:48

[0425] CCACGTCTCACCATTGGGG

[0426] SEQ ID NO:49

[0427] TTCAAGTGTGATCCCCATGAAG

[0428] SEQ ID NO:50

[0429] CAGGTCTACGGCAGTTGTCA

[0430] SEQ ID NO:51

[0431] GTCCCAGACATCAGGGAGTAA

[0432] SEQ ID NO:52

[0433] TCGGATACTTCAGCGTCAGGA

Claims

1. Use of the following agents in the preparation of a drug for slowing down the flow of inflammatory signals in the kidneys of a subject, inhibiting kidney disease in a subject, or reducing the degree of kidney fibrosis and / or inflammatory response in a subject: (1) an agent that reduces the expression of a subject's TILRR protein; and / or (2) An agent that reduces the activity of a TILRR protein expressed in a subject. Preferably, The kidney disease is a disease related to kidney fibrosis or inflammation.

2. The use according to claim 1, characterized in that The agent for reducing the expression of the subject TILRR protein is an agent that inhibits the expression of the TILRR gene or reduces its expression level, and is preferably selected from: (a) an agent that inhibits the transcriptional activity of the TILRR gene; (b) an agent that inhibits the transcription level of TILRR mRNA; (c) agents that promote TILRR mRNA degradation; (d) siRNA targeting TILRR gene; (e) an agent that inhibits translation of TILRR mRNA; (f) an agent that specifically recognizes the guide nucleic acid of the TILRR gene and cleaves it to reduce its expression level; and (g) Reagents for partial or complete knockout of the TILRR gene.

3. The use according to claim 1, characterized in that The agent for reducing the activity of the TILRR protein expressed by the subject is a specific antibody of the TILRR protein or a small molecule compound with inhibitory activity, Preferably, The TILRR-specific antibody is an anti-TILRR antibody or an antigen-binding fragment thereof, wherein the anti-TILRR antibody has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and / or LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively, More preferably, The FR1 of the VH of the TILRR antibody is shown in SEQ ID NO: 4, The FR2 of the VH of the TILRR antibody is shown in SEQ ID NO: 5, The FR3 of the VH of the TILRR antibody is shown in SEQ ID NO: 6, The FR4 of the VH of the TILRR antibody is shown in SEQ ID NO: 7, The FR1 of the VL of the TILRR antibody is shown in SEQ ID NO: 12, The FR2 of the VL of the TILRR antibody is shown in SEQ ID NO: 13, The FR3 of the VL of the TILRR antibody is shown in SEQ ID NO: 14, The FR4 of the VL of the TILRR antibody is shown in SEQ ID NO:

15.

4. An anti-TILRR antibody or an antigen-binding fragment thereof, characterized in that: The TILRR antibody has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and / or LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively, Preferably, The FR1 of the VH of the TILRR antibody is shown in SEQ ID NO: 4, The FR2 of the VH of the TILRR antibody is shown in SEQ ID NO: 5, The FR3 of the VH of the TILRR antibody is shown in SEQ ID NO: 6, The FR4 of the VH of the TILRR antibody is shown in SEQ ID NO: 7, The FR1 of the VL of the TILRR antibody is shown in SEQ ID NO: 12, The FR2 of the VL of the TILRR antibody is shown in SEQ ID NO: 13, The FR3 of the VL of the TILRR antibody is shown in SEQ ID NO: 14, The FR4 of the VL of the TILRR antibody is shown in SEQ ID NO: 15, More preferably, FR1, FR2, FR3 and FR4 of the VH of the TILRR antibody have the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively, and FR1, FR2, FR3 and FR4 of the VL of the TILRR antibody have the amino acid sequences shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, respectively, The VH of the TILRR antibody is shown in SEQ ID NO:8, and / or the VL of the TILRR antibody is shown in SEQ ID NO:

16.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a sequence selected from the group consisting of: (1) a nucleic acid sequence as shown in any one of SEQ ID NOs: 17-19; (2) a sequence encoding the antibody or antigen-binding fragment thereof according to claim 4, and (3) A complementary sequence of the sequence described in (1) or (2). Preferably, (1) the nucleic acid sequence is SEQ ID NO: 17 and 18, or SEQ ID NO: 19, The nucleic acid molecule is DNA or RNA.

6. A nucleic acid construct, characterized in that The nucleic acid construct comprises the nucleic acid molecule according to claim 5, Preferably, The nucleic acid construct is a vector, such as a cloning vector or an expression vector. The nucleic acid construct is mRNA.

7. A genetically engineered host cell, wherein: (1) expressing the antibody or antigen-binding fragment thereof according to claim 4, (2) comprising or having integrated into its genome the nucleic acid molecule according to claim 5 or the nucleic acid construct according to claim 6, Preferably, The host cell is a non-human mammalian cell, The host cell is a rodent cell, The host cell is an embryonic stem cell, a pluripotent stem cell or a somatic cell of a human or non-human mammal, The host cell is a human embryonic stem cell or a pluripotent stem cell, which contains or has integrated into its genome the nucleic acid molecule described in claim 5 or a nucleic acid construct comprising the nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleic acid sequence shown in any one of SEQ ID NOs: 17-19 or its RNA counterpart, and / or the human embryonic stem cell or pluripotent stem cell does not express TILRR protein, or the expression level of TILRR protein is reduced compared to control cells, or an inactive TILRR protein or a TILRR protein with reduced activity is expressed.

8. A method for constructing a transgenic mouse, characterized in that: The method comprises: (1) Providing the nucleic acid construct according to claim 6; (2) introducing the nucleic acid construct in step (1) into a mouse fertilized egg; (3) transplanting the mouse fertilized eggs in step (2) into the uterus of a pseudo-pregnant female mouse to obtain positive offspring mice; (4) backcrossing the positive offspring mice obtained in step (3) to obtain homozygous positive mice, i.e., the transgenic mice, Preferably, the method comprises: (1) Providing the nucleic acid construct according to claim 6; (2) transcribing the nucleic acid construct in step (1) into mRNA in vitro and injecting it into mouse fertilized eggs; (3) transplanting the mouse fertilized eggs in step (2) into the uterus of a pseudo-pregnant mouse, and identifying the offspring mice by PCR analysis after birth to obtain positive offspring mice; (4) backcrossing the positive offspring mice obtained in step (3), and performing PCR identification to obtain homozygous positive mice, i.e., the transgenic mice; Preferably, the nucleic acid construct is RNA, Preferably, the transgenic mouse is characterized by not expressing TILRR protein in the whole body or kidney, or the expression level of TILRR protein is reduced compared with control animals, or inactive TILRR protein or TILRR protein with reduced activity is expressed.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to claim 4, the nucleic acid molecule according to claim 5, the nucleic acid construct according to claim 6 and / or the host cell according to claim 7.

10. Use of TILRR gene or protein as a target in screening drugs for preventing and / or treating kidney diseases or symptoms. Preferably, the prevention and / or treatment of kidney diseases or symptoms includes one or more selected from the following: slowing down the flow of inflammatory signals in the subject's kidneys, inhibiting the subject's kidney disease, or reducing the degree of kidney fibrosis and / or inflammatory response in the subject.

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