Application of BMP9 (bone morphogenetic protein 9) as intervention target in preparation of medicine for treating diabetic nephropathy

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of BMP9 as an intervention target in preparation of a medicine for treating diabetic nephropathy. More specifically, the invention provides application of a BMP9 accelerant in preparation of a medicine for preventing and / or treating diabetic nephropathy. The BMP9 accelerant is a substance shown in the following (i) or (ii): (i) a substance for increasing the content of BMP9 in a subject; (ii) a substance that increases BMP9 activity in a subject. According to the invention, a DKD mouse model is constructed, and liver RNA-seq sequencing is carried out, such that a differential expression gene BMP9 exists between a normal mouse and a DKD mouse. It is found that BMP9 can be used as a new target for prevention and treatment of DKD and has important theoretical significance and potential clinical application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of BMP9 as an intervention target in preparation of a drug for treating diabetic nephropathy. BACKGROUND

[0002] Diabetic kidney disease (DKD, for short, diabetic nephropathy) is one of the main microvascular complications of diabetes and is the main cause of chronic kidney disease and end-stage renal disease.

[0003] At present, the research on DKD mainly focuses on glomerular [1] However, the renal tubule and renal tubular interstitium account for 90% of the mass of the renal parenchyma, suggesting that they are crucial for the occurrence and development of DKD. The renal tubule of an early-stage diabetic patient undergoes a series of structural changes, such as atrophy of the renal tubule, interstitial fibrosis, and sparseness of the peritubular capillary, each of which is closely related to the decline in renal function [2] . The degree of damage to the renal tubular interstitium is closely related to long-term renal function. The protective effect of sodium-glucose cotransporter-2 inhibitors on renal tubular epithelial cells verifies that they can be used as a therapeutic target for DKD [3] . Due to its basic function of reabsorbing and secreting solutes, the renal tubular epithelial cell is a high-energy-consumption cell, and thus depends on the production of ATP, which can only be ensured when mitochondrial energy metabolism is maintained [4] In summary, renal tubular lesions are the main and important factors affecting the progression of DKD, and the damage to the renal tubule cannot be ignored.

[0004] It has been found that tissue-tissue interactions caused by endocrine factors (including secreted proteins) are an important mechanism for maintaining normal physiological homeostasis. Organs such as the liver and the kidney exhibit a wide range of interactions, and in clinical studies, it is usually observed that diseases of other organs affect the liver, and vice versa [5] .

[0005] Bone morphogenetic protein 9 (BMP9) is a protein mainly secreted by the liver and belongs to the BMP family. Many members of the BMP family have been reported to play an important role in chronic metabolic diseases. For example, BMP2 / BMP4 / BMP7 / BMP8b molecules have been confirmed to affect the occurrence of chronic metabolic diseases such as diabetes and obesity by regulating brown fat thermogenesis and improving insulin resistance. Studies have shown that the expression of liver BMP9 is down-regulated in insulin-resistant mice and diabetic patients, and overexpression of liver BMP9 in mice fed with a high-fat diet can improve glucose tolerance and insulin resistance [6]However, the specific function and mechanism of BMP9 in chronic metabolic disease complications such as DKD are rarely studied.

[0006] References:

[0007] [1]. DeFronzo R; Reeves W; Awad A. Pathophysiology of Diabetic Kidney Disease: Impact of SGLT2 Inhibitors. Nat Rev Nephrol 2021, 17, 319-334.

[0008] [2]. Xu F; Jiang H; Li X; Pan J; Li H; Wang L; Zhang P; Chen J; Qiu S; Xie Y; et al. Discovery of PRDM16-Mediated TRPA1 Induction as the Mechanism for Low Tubulo-Interstitial Fibrosis in Diabetic Kidney Disease. Adv Sci (Weinh) 2024, 11, e2306704.

[0009] [3]. Mora-Fernández C; M; Donate-Correa J; E; Pérez-Delgado N; L; Fernández-Fernández B; Ortiz A; Navarro-González J. Sodium-Glucose Co-Transporter-2 Inhibitors Increase Klotho in Patients with Diabetic Kidney Disease: A Clinical and Experimental Study. Biomed Pharmacother 2022, 154, 113677.

[0010] [4]. Chen H; Zhang H; Liu Y; Liu Y; Zhang W; Yang C; Song N; Zhan M; Yang S. VDR Regulates Mitochondrial Function as a Protective Mechanism against Renal Tubular Cell Injury in Diabetic Rats. Redox Biol 2024, 70, 103062.

[0011] [5]. Moller S, Bernardi M. Interactions of the heart and the liver [J]. Eur Heart J, 2013, 34(36): 2804-2811.

[0012] [6]. Yang M, Liang Z, Yang M, Jia Y, Yang G, He Y, Li X, Gu HF, Zheng H, Zhu Z, Li L. Role of bone morphogenetic protein-9 in the regulation of glucose and lipid metabolism. FASEB J. 2019 Sep;33(9): 10077-10088. SUMMARY

[0013] Problem to be solved by the invention

[0014] At present, it is found that some drugs with kidney protection can delay the progression of diabetic nephropathy, such as sodium-glucose cotransporter-2 inhibitor drugs, angiotensin converting enzyme inhibitors / angiotensin receptor antagonists, and mineralocorticoid receptor antagonists. However, for diabetic nephropathy as a serious complication of chronic metabolic diseases, such drug research is still not sufficient, and the treatment target of diabetic nephropathy still needs to be developed.

[0015] Although, for example, the above reference 6 and other studies have found that the expression of BMP9 is related to glucose tolerance and insulin resistance, but there is no research report on the relationship between BMP9 and diabetic nephropathy, a complication of diabetes.

[0016] However, in the process of in-depth study of diabetic nephropathy, it is accidentally found that the content of BMP9 in diabetic nephropathy patients is significantly reduced, and gradually reduces with the progress of pathological staging. Based on this, the present application aims to study the correlation between BMP9 and diabetic nephropathy, and to provide a new target for the prevention and treatment of diabetic nephropathy.

[0017] Solution to solve the problem

[0018] [1]. Use of a BMP9 promoter in the preparation of a drug for preventing and / or treating diabetic nephropathy; the BMP9 promoter is a substance as shown in (i) or (ii) below:

[0019] (i) a substance that increases the content of BMP9 in the body of a subject;

[0020] (ii) a substance that increases the activity of BMP9 in the body of a subject.

[0021] [2]. The use according to [1], wherein the BMP9 promoter is selected from recombinant BMP9, a polynucleotide encoding recombinant BMP9, a vector carrying recombinant BMP9, and a vector carrying a polynucleotide encoding recombinant BMP9; the vector includes any one or more of plasmid, lentivirus, adenovirus, adeno-associated virus, retrovirus, liposome, lipid nanoparticle, N-acetylgalactosamine, and extracellular vesicle.

[0022] [3]. The use according to [1] or [2], wherein the BMP9 promoter is recombinant BMP9; the amino acid sequence of the recombinant BMP9 comprises a sequence as shown in (i) or (ii) below:

[0023] (i) a sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3;

[0024] (ii) a sequence that substitutes, deletes or adds one or more amino acids in the sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3 and has the protein activity of the sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

[0025] [4]. The use according to [1] or [2], wherein the BMP9 promoter is an adeno-associated virus carrying a polynucleotide encoding recombinant BMP9; the amino acid sequence of the recombinant BMP9 comprises a sequence as shown in (i) or (ii) below:

[0026] (i) a sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3;

[0027] (ii) a sequence in which one or more amino acids are substituted, deleted or added in the sequence shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3 and has the protein activity of the sequence shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3;

[0028] Preferably, the polynucleotide encoding the recombinant BMP9 comprises a nucleic acid sequence shown in (a) or (b) below:

[0029] (a) a sequence shown as SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6;

[0030] (b) a sequence which hybridizes to a sequence shown as SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6 under stringent conditions and encodes a protein having the protein activity of the sequence shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

[0031] [5]. The use according to [4], wherein, in the genome of the adeno-associated virus carrying the polynucleotide encoding the recombinant BMP9, the polynucleotide encoding the recombinant BMP9 is driven by a transthyretin promoter.

[0032] [6]. The use according to any one of [1] to [5], wherein the prevention and / or treatment of diabetic nephropathy comprises improving kidney function, liver function and blood lipids of the subject.

[0033] [7]. The use according to [6], wherein the improvement of kidney function of the subject comprises at least one of reducing urinary albumin / creatinine ratio, reducing blood urea nitrogen level and reducing uric acid level.

[0034] [8]. The use according to [6], wherein the improvement of liver function of the subject comprises reducing glutamic-pyruvic transaminase level.

[0035] [9]. The use according to [6], wherein the improvement of blood lipids of the subject comprises at least one of reducing total cholesterol level, reducing triglyceride level and reducing low-density lipoprotein level.

[0036]

[10] . The use according to any one of [1] to [9], wherein the subject comprises a mammal.

[0037] Preferably, the subject is a human.

[0038] Effects of the invention

[0039] The present application finds that there is a differentially expressed gene BMP9 between normal mice and DKD mice by constructing a DKD mouse model and performing liver RNA-seq sequencing. Further, the present application finds that the plasma BMP9 level is correlated with the occurrence and severity of DKD in a cohort of diabetic patients. Through in-depth research, the present application finds that overexpression of BMP9 can improve the liver function, blood lipid and renal function of DKD patients, and can effectively reduce the renal tubular injury.

[0040] Through the above research, the present application finds that BMP9 can be used as a new target for the prevention and treatment of DKD, which has important theoretical significance and potential clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 : Flow chart of example experimental scheme.

[0042] Figure 2A : Schematic diagram of DKD mouse model construction method.

[0043] Figure 2B : DKD mouse weight detection results.

[0044] Figure 2C : DKD mouse blood glucose detection results.

[0045] Figure 2D : DKD mouse IPGTT detection results.

[0046] Figure 2E : DKD mouse liver and kidney tissue morphological staining results.

[0047] Figure 2F : DKD mouse liver lipid droplet positive area statistics results.

[0048] Figure 2G : DKD mouse kidney lipid droplet positive area statistics results.

[0049] Figure 2H : DKD mouse urine albumin / creatinine ratio (UACR) detection results.

[0050] Figure 3A : Schematic diagram of DKD mouse model liver tissue RNA-seq sequencing.

[0051] Figure 3B : DKD mouse model liver tissue RNA-seq sequencing results.

[0052] Figure 3C : Top 30 results of GO signaling pathways enriched by DKD mouse model liver tissue RNA-seq sequencing.

[0053] Figure 3D VENN results of RNA-seq sequencing of liver tissues of DKD mouse models.

[0054] Figure 3E RT-qPCR detection results of BMP9 expression levels of liver tissues of DKD mouse models.

[0055] Figure 3F Western blot detection results of BMP9 expression levels of liver tissues of DKD mouse models.

[0056] Figure 3G Immunohistochemical staining detection results of BMP9 expression levels of liver tissues of DKD mouse models.

[0057] Figure 4A DKD patient grouping schematic diagram.

[0058] Figure 4B DKD patient plasma BMP9 level detection results.

[0059] Figure 4C Linear correlation calculation results of DKD patient plasma BMP9 level and UACR.

[0060] Figure 5A Schematic diagram of overexpression BMP9 mouse construction method.

[0061] Figure 5B Overexpression BMP9 mouse blood alanine aminotransferase (ALT) level detection results.

[0062] Figure 5C Overexpression BMP9 mouse blood aspartate aminotransferase (AST) level detection results.

[0063] Figure 5D Overexpression BMP9 mouse blood total cholesterol (CHO) level detection results.

[0064] Figure 5E Overexpression BMP9 mouse blood triglyceride (TG) level detection results.

[0065] Figure 5F Overexpression BMP9 mouse blood high-density lipoprotein (HDL) level detection results.

[0066] Figure 5G Overexpression BMP9 mouse blood low-density lipoprotein (LDL) level detection results.

[0067] Figure 5H Overexpression BMP9 mouse blood blood urea nitrogen (BUN) level detection results.

[0068] Figure 5I: The results of detecting the blood uric acid (UA) level of the overexpression BMP9 mice.

[0069] Figure 5J : The results of detecting the blood creatinine (CREA) level of the overexpression BMP9 mice.

[0070] Figure 5K : The results of detecting the urine albumin / creatinine ratio (UACR) of the overexpression BMP9 mice.

[0071] Figure 5L : The results of detecting the blood BMP9 level of the overexpression BMP9 mice.

[0072] Figure 5M : The results of detecting the urine kidney injury molecule 1 (KIM-1) level of the overexpression BMP9 mice.

[0073] Figure 5N : The results of detecting the mRNA level of the liver BMP9 of the overexpression BMP9 mice.

[0074] Figure 5O : The results of detecting the kidney triglyceride (TG) content of the overexpression BMP9 mice.

[0075] Figure 5P : The results of kidney morphological staining of the overexpression BMP9 mice.

[0076] Figure 5Q : The results of detecting the level of other genes of the BMP family in the liver of the overexpression BMP9 mice.

[0077] Figure 6A : The results of identifying the tail genotypes of the BMP9 knockout mice; wherein, 1#, 2#, 3#, 5#, 6# lanes represent the wild type BMP9 mice, and 4# lane represents the whole body knockout BMP9 mice.

[0078] Figure 6B : The results of detecting the urine albumin / creatinine ratio (UACR) of the BMP9 knockout mice.

[0079] Figure 7A : The results of oil red O staining of the HK2 cells treated by rhBMP9.

[0080] Figure 7B : The results of RT-qPCR detection of the expression levels of the lipid metabolism related proteins of the HK2 cells treated by rhBMP9.

[0081] Figure 7C : The results of Western blot detection of the expression levels of the lipid metabolism related proteins of the HK2 cells treated by rhBMP9. DETAILED DESCRIPTION

[0082] Embodiments of the present application will be described below, but the present application is not limited thereto. Various modifications can be made within the scope of the present application, and embodiments obtained by appropriately combining the technical means disclosed in each of the different embodiments and examples are also included in the technical scope of the present application.

[0083] The present application has found that the plasma BMP9 level is correlated with the occurrence and severity of diabetic nephropathy, and BMP9 can be used as a new target for the prevention and treatment of diabetic nephropathy. Based on this, the present application provides the use of a BMP9 promoter in the preparation of a drug for preventing and / or treating diabetic nephropathy.

[0084] The "BMP9 promoter" described in the present application has the function of increasing the content and / or activity level of BMP9 in the body of a subject.

[0085] The "diabetic nephropathy" described in the present application refers to chronic kidney disease caused by diabetes. The early symptoms are mild and difficult to detect, and the first symptom is usually an increase in urinary foam. Progressive increase in urinary protein and decline in kidney function are the main clinical features. In some embodiments, the diabetic nephropathy described in the present application includes clinical stages I-V diabetic nephropathy.

[0086] The "prevention" described in the present application means that before a disease (e.g., diabetic nephropathy) occurs, an individual is contacted (e.g., injected) with an appropriate drug (e.g., the BMP9 promoter described in the present application) or the like, so that the symptoms after the disease are alleviated compared with when no contact is made, and it does not mean that the disease must be completely inhibited. The occurrence of a disease means that the body has symptoms of the disease.

[0087] The "treatment" described in the present application means that after a disease (e.g., diabetic nephropathy) occurs, an individual is contacted (e.g., injected) with an appropriate drug (e.g., the BMP9 promoter described in the present application) or the like, so that the symptoms of the disease are alleviated compared with when no contact is made, and it does not mean that the symptoms of the disease must be completely inhibited.

[0088] The "individual", "subject", "patient" or "subject" described in the present application includes a mammal. The mammal includes, but is not limited to, a domestic animal (e.g., a cow, a sheep, a cat, a dog, or a horse, etc.), a primate (e.g., a human, a non-human primate such as a monkey or a chimpanzee, etc.), a rabbit, and a rodent (e.g., a mouse, a rat, or a guinea pig, etc.). In some preferred embodiments, the "individual", "subject", "patient" or "subject" described in the present application is a human.

[0089] In some embodiments, the prevention and / or treatment of diabetic nephropathy described in the present application includes simultaneously improving the kidney function, liver function, and blood lipids of the subject.

[0090] In some specific embodiments, the improvement of the renal function in the subject comprises at least one of decreasing urinary albumin / creatinine ratio, decreasing blood urea nitrogen level, and decreasing uric acid level. In some preferred embodiments, the improvement of the renal function in the subject comprises decreasing blood urea nitrogen level, decreasing uric acid level, and decreasing urinary albumin / creatinine ratio simultaneously.

[0091] In some specific embodiments, the improvement of the liver function in the subject comprises decreasing glutamic-pyruvic transaminase level.

[0092] In some specific embodiments, the improvement of the blood lipid in the subject comprises at least one of decreasing total cholesterol level, decreasing triglyceride level, and decreasing low-density lipoprotein level. In some preferred embodiments, the improvement of the blood lipid in the subject comprises decreasing total cholesterol level, decreasing triglyceride level, and decreasing low-density lipoprotein level simultaneously.

[0093] In some embodiments, the BMP9 promoter of the present application is a substance as shown in (i) or (ii) below: (i) a substance that increases the content of BMP9 in the subject; (ii) a substance that increases the activity of BMP9 in the subject. The "increasing the content of BMP9 in the subject" of the present application is not limited to increasing the content of natural BMP9 protein, but also includes increasing the content of a fragment of natural BMP9 protein, and increasing the content of a protein that is modified based on natural BMP9 protein (e.g., substitution, addition, or deletion in the amino acid sequence). In some preferred embodiments, the "increasing the content of BMP9 in the subject" of the present application is increasing the content of a protein having the same amino acid sequence as natural BMP9 protein in the subject or increasing the content of a fragment of natural BMP9 protein in the subject.

[0094] Further, in some embodiments, the BMP9 promoter of the present application is selected from any one or more of recombinant BMP9, a polynucleotide encoding recombinant BMP9, a vector carrying recombinant BMP9, and a vector carrying a polynucleotide encoding recombinant BMP9; the vector includes any one or more of plasmid, lentivirus, adenovirus, adeno-associated virus, retrovirus, liposome, lipid nanoparticle, N-acetylgalactosamine, and extracellular vesicle.

[0095] The "recombinant BMP9" according to the present application refers to a non-naturally occurring BMP9 protein. Exemplarily, the recombinant BMP9 according to the present application includes, but is not limited to, a protein having the same amino acid sequence as a natural (e.g., human-derived) BMP9 protein prepared by biosynthetic means, a protein having an amino acid sequence obtained by substituting, deleting and / or adding one or more amino acids in the amino acid sequence of a natural (e.g., human-derived) BMP9 protein prepared by biosynthetic means, a protein having a functional active amino acid sequence fragment of a natural (e.g., human-derived) BMP9 protein prepared by biosynthetic means, and the like.

[0096] In some embodiments, the BMP9 promoter according to the present application is a recombinant BMP9 comprising an amino acid sequence as shown in (i) or (ii) below: (i) a sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3; (ii) a sequence obtained by substituting, deleting or adding one or more amino acids in the sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3 and having the activity of the sequence as shown in SEQ ID NO. 1 (including the activity of improving the renal function, liver function and / or blood lipid of a subject); preferably, the amino acid sequence of the recombinant BMP9 is as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

[0097] SEQ ID NO. 1 (ab94350, Abeam): SAGAGSHCQKTSLRVNFEDIGWDSWIIAPKEYEAYECKGGCFFPLADDVTPTKHAIVQTLVHLKFPTKVGKACCVPTKLSPISVLYKDDMGVPTLKYHYEGMSVAECGCR.

[0098] SEQ ID NO. 2 (NP_057288.1): MCPGALWVALPLLSLLAGSLQGKPLQSWGRGSAGGNAHSP LGVPGGGLPEHTFNLKMFLENVKVDFLRSLNLSGVPSQDKTRVEPPQYMIDLYNRYTSDKSTTPASNIVRSFSMEDAISITATEDFPFQKHILLFNISIPRHEQITRAELRLYVSCQNHVDPSHDLKGSVVIYDVLDGTDAWDSATETKTFLVSQDIQDEGWETLEVSSAVKRWVRSDSTKSKNKLEVTVESHRKGCDTLDISVPPGSRNLPFFVVFSNDHSSGTKETRLELREMISHEQESVLKKLSKDGSTEAGESSHEEDTDGHVAAGSTLARRKRSAGAGSHCQKTSLRVNFEDIGWDSWIIAPKEYEAYECKGGCFFPLADDVTPTKHAIVQTLVHLKFPTKVGKACCVPTKLSPISVLYKDDMGVPTLKYHYEGMSVAECGCR.

[0099] SEQ ID NO. 3 (NP_062379.3): MSPGAFRVALLPLFLLVCVTQQKPLQNWEQASPGENAHSSL GLSGAGEEGVFDLQMFLENMKVDFLRSLNLSGIPSQDKTRAEPPQYMIDLYNRYTTDKSSTPASNIVRSFSVEDAISTAATEDFPFQKHILIFNISIPRHEQITRAELRLYVSCQNDVDSTHGLEGSMVVYDVLEDSETWDQATGTKTFLVSQDIRDEGWETLEVSSAVKRWVRADSTTNKNKLEVTVQSHRESCDTLDISVPPGSKNLPFFVVFSNDRSNGTKETRLELKEMIGHEQETMLVKTAKNAYQVAGESQEEEGLDGYTAVGPLLARRKRSTGASSHCQKTSLRVNFEDIGWDSWIIAPKEYDAYECKGGCFFPLADDVTPTKHAIVQTLVHLKFPTKVGKACCVPTKLSPISILYKDDMGVPTLKYHYEGMSVAECGCR.

[0100] The amino acid deletion according to the present application can mean deletion of 1, 2, or more than 3 amino acids from the amino acid sequence, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence.

[0101] The amino acid addition according to the present application can mean addition of 1, 2, or more than 3 amino acids at any position of the C-terminus, N-terminus, or between the C-terminus and N-terminus of the amino acid sequence, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence.

[0102] The amino acid substitution according to the present application can mean substitution of an amino acid at a certain position of the amino acid sequence with another amino acid, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence. The amino acid substitution can be a conservative amino acid substitution, which means that several amino acids are substituted with amino acids having similar or nearly similar properties to form a peptide (conservatively modified peptide) compared to the original amino acid sequence. Exemplarily, these conservatively modified peptides can be generated by substitution of Val, Leu, or He for Ala, Lys, Gin, Asn, or His for Arg, Gin, His, Lys, or Arg for Asn, Glu or Asn for Asp, Ser or Ala for Cys, Asn or Glu for Gin, Asp or Gin for Glu, Ala for Gly, Asn, Lys, Gin, or Arg for His, Leu, Met, Ala, Val, or Phe for He, He, Met, Ala, Val, or Phe for Leu, Asn, Gin, or Arg for Lys, He, Leu, or Phe for Met, Leu, Val, He, Ala, or Tyr for Phe, Ala for Pro, Thr for Ser, Ser or Val for Thr, Phe or Tyr for Trp, Trp, Phe, Thr, or Ser for Tyr, and Phe, Ala, Met, He, or Leu for Val. The amino acid substitution can also be a non-conservative amino acid substitution.

[0103] The "polynucleotide" according to the present application means a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or their analogs. Exemplarily, the polynucleotide according to the present application includes, but is not limited to, DNA, cDNA, and mRNA. The polynucleotide according to the present application can be linear or circular, and can have any three-dimensional structure.

[0104] In some embodiments, the BMP9 promoter of the present application is an adeno-associated virus carrying a polynucleotide encoding a recombinant BMP9 comprising an amino acid sequence as shown in (i) or (ii) below: (i) a sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3; (ii) a sequence with substitution, deletion, or addition of one or more amino acids in a sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3 and having the protein activity (including the activity of improving the kidney function, liver function and / or blood lipid of a subject) of the sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3; preferably, the polynucleotide encoding the recombinant BMP9 comprises a nucleic acid sequence as shown in (a) or (b) below: (a) a sequence as shown in SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6; (b) a sequence hybridizing to a sequence as shown in SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6 under stringent conditions and encoding a protein having the protein activity (including the activity of improving the kidney function, liver function and / or blood lipid of a subject) of the sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3; more preferably, the nucleic acid sequence of the polynucleotide encoding the recombinant BMP9 is as shown in SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6.

[0105] SEQ ID NO. 4: AGCGCCGGGGCTGGCAGCCACTGTCAAAAGACCTCCCTGCGGGTAAACTT CGAGGACATCGGCTGGGACAGCTGGATCATTGCACCCAAGGAGTATGAAGCCTACGAGTGTAAGGGCGGCTGCTTCTTCCCCTTGGCTGACGATGTGACGCCGACGAAACACGCTATCGTGCAGACCCTGGTGCATCTCAAGTTCCCCACAAAGGTGGGCAAGGCCTGCTGTGTGCCCACCAAACTGAGCCCCATCTCCGTCCTCTACAAGGATGACATGGGGGTGCCCACCCTCAAGTACCATTACGAGGGCATGAGCGTGGCAGAGTGTGGGTGCAGG.

[0106]

[0107]

[0108] "Hybridization" according to the application means the ability of a polynucleotide or oligonucleotide to bind to a substantially complementary sequence under stringent conditions, without non-specific binding to non-complementary sequences under these conditions. Preferably, the sequences are 90 to 100% complementary. According to the application, hybridization occurs under stringent conditions. Such hybridization conditions are described in Sambrook, J., Fritsch, E.F., Maniatis, T., in: Molecular Cloning (A Laboratory Manual), 2nded., Cold Spring Harbor Laboratory Press, 1989, pp. 9.31-9.57, or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Stringent hybridization conditions mean, in particular, incubation in a solution consisting of 50% formamide, 5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g / ml denatured, sheared salmon sperm DNA, overnight at 42°C, followed by washing with 0.1x SSC at 65°C.

[0109] In some embodiments, in the genome of the adeno-associated virus carrying the polynucleotide encoding the recombinant BMP9, the polynucleotide encoding the recombinant BMP9 is driven by a transthyretin promoter.

[0110] Examples

[0111] Embodiments of the application will be described in detail in the following with reference to examples, but a person skilled in the art will understand that the following examples are only for illustration of the application and should not be considered as limiting the scope of the application. In the examples, specific conditions not mentioned are carried out under conventional conditions or under conditions recommended by the manufacturer. The materials or instruments used are, unless otherwise specified, commercially available conventional products that can be used.

[0112] To explore the effect of liver secreted proteins on DKD, a DKD mouse model was first constructed in this embodiment. RNA-seq was performed on the fresh liver tissue of the DKD mouse model, and the candidate gene growth differentiation factor 2 (GDF2), also known as BMP9, was screened. Further, by collecting DKD patients at different pathological stages, it was found that the content of BMP9 in DKD patients was significantly reduced, and gradually decreased with the progression of the pathological stage. At the same time, it was found by using an animal model that tail vein injection of liver-specific overexpression of BMP9 adeno-associated virus significantly improved the kidney function of the DKD mouse model, while systemic knockdown of BMP9 aggravated the damage of kidney function. Finally, the mechanism of action of BMP9 in DKD was found by cell experiments. The specific experimental scheme is shown in Figure 1 , and the experimental results are as follows.

[0113] (1) Construction of DKD mouse model

[0114] 6-week-old C57BL / 6J male mice were fed with high-fat diet (HFD) (60% fat, 20% carbohydrate, and 1% cholesterol) for 4 weeks, and 80 mg / kg streptozotocin (STZ) was injected intraperitoneally at the 5th week of HFD feeding, with a total of three injections every other day. HFD+STZ was induced for 24 weeks Figure 2A ). It was found that, compared with the control group of mice without any treatment (referred to as the ND group), the body weight and blood glucose of the HFD+STZ group of mice (referred to as the HFD / STZ group) were significantly increased Figure 2B and Figure 2C ), and the glucose tolerance of the mice was tested by intraperitoneal glucose tolerance test (IPGTT), and the results showed that the glucose tolerance of the HFD+STZ group of mice was significantly impaired Figure 2D ). Morphological staining was performed on the liver and kidney tissues of the mice, and the results of HE and oil red O staining showed that the lipid accumulation in the liver and kidney of the HFD+STZ group of mice was significantly increased compared with the control group, especially in the renal tubular epithelial cells; the results of kidney PAS staining showed that the glycogen deposition was increased Figures 2E-2G ). The results of urine white UACR detection showed that the urinary protein of the kidney of the HFD+STZ group of mice was significantly increased Figure 2H ), and the above results indicated that the DKD modeling of the HFD+STZ induced C57BL / 6J mice was successful.

[0115] (2) BMP9 expression in liver tissue of DKD mouse model is significantly reduced

[0116] RNA-seq sequencing was performed on liver tissues of DKD mouse model and control group mice Figure 3A ), and the differential gene BMP9 Figures 3B-3D ) was screened. The expression level of BMP9 in liver tissues of DKD mouse model and control group mice was further detected by Western blot, RT-qPCR and immunohistochemical staining, and the results showed that the expression of BMP9 in liver tissues of DKD mouse model was significantly lower than that in control group mice Figures 3E-3G ).

[0117] (3) The expression of BMP9 in serum of DKD patients at different stages was significantly reduced

[0118] 220 cases of non-diabetic controls and 533 cases of DKD patients with abnormal kidney function were collected, and according to the eGFR (glomerular filtration rate) and UACR level, the 533 cases of DKD patients with abnormal kidney function were divided into four groups according to the severity of DKD Figure 4A ), and the plasma BMP9 level of 220 cases of non-diabetic controls and 533 cases of DKD patients with abnormal kidney function was further detected by ELISA method, and it was found that compared with non-diabetic controls, the plasma BMP9 level gradually decreased with the severity of DKD Figure 4B and Figure 4C ).

[0119] (4) Overexpression of BMP9 improves HFD combined with STZ induced renal tubular injury

[0120] AAV8-Vector and liver-specific AAV8-BMP9 (carrying BMP9 gene (SEQ ID NO. 6) of AAV8) were constructed and injected into HFD+STZ induced DKD mouse model through tail vein respectively (marked as HFD / STZ+Vector group and HFD / STZ+BMP9 group respectively), and the samples were taken after 8 weeks Figure 5A ). The liver function, blood lipid and kidney function of each group of mice were detected. The results showed that after overexpression of BMP9, the liver function index alanine aminotransferase (ALT) level of mice was significantly improved, and the aspartate aminotransferase (AST) level had no significant change Figure 5B and Figure 5C ). Blood lipid and kidney function were significantly improved Figures 5D-5K ). ELISA results showed that BMP9 overexpression in HFD / STZ+BMP9 group of mice was successful Figure 5L ), and the renal tubular injury index KIM-1 also had a slight change Figure 5M ), and the liver RT-qPCR results showed that BMP9 mRNA overexpression in HFD / STZ+BMP9 group of mice was successful Figure 5N). The kidney lipid content of BMP9 overexpression mice was significantly improved Figure 5O ), and morphological staining results showed that tubular injury and lipid accumulation were reduced in the HFD / STZ+BMP9 group of mice after BMP9 overexpression Figure 5P ).

[0121] At the same time, the levels of other BMPs family genes in the livers of mice in each group were detected, and according to Figure 5Q It can be seen that after overexpression of BMP9 in the liver, the levels of other genes in the BMPs family did not change significantly.

[0122] (5) Knockout of BMP9 expression aggravates HFD combined with STZ induced kidney injury

[0123] BMP9 knockout mice (denoted as BMP9 KO) were constructed and induced by HFD+STZ together with wild type mice (denoted as WT), and the results of mouse tail genotype identification are shown in Figure 6A After knockout of BMP9, the urine UACR level was significantly increased Figure 6B , aggravating HFD combined with STZ induced kidney injury.

[0124] (6) Explore the specific molecular mechanism of BMP9 acting on renal tubules

[0125] Bovine serum albumin (BSA), high glucose and palmitic acid (HGPA), recombinant protein rhBMP9 (ab94350, Abeam, USA) or their combination were used to treat human renal proximal tubular epithelial cell line HK2 cells for 24 hours, and the results of oil red O staining showed that recombinant protein rhBMP9 could reduce the lipid accumulation of HK2 cells induced by high glucose and palmitic acid Figure 7A ); RT-qPCR and Western blot were used to detect the expression levels of lipid metabolism related proteins, and the results showed that at the mRNA and protein levels, recombinant protein rhBMP9 could significantly increase the expression of perixisome proliferation-activated receptor alpha (PPARα) Figure 7B and Figure 7C ).

Claims

1. Use of a BMP9 promoter in the manufacture of a medicament for preventing and / or treating diabetic nephropathy; the BMP9 promoter is a substance indicated in (i) or (ii) below: (i) a substance that increases the amount of BMP9 in a subject; (ii) a substance that increases the activity of BMP9 in a subject.

2. Use according to claim 1, characterized in that, The BMP9 promoter is selected from the group consisting of recombinant BMP9, a polynucleotide encoding recombinant BMP9, a vector carrying recombinant BMP9, and a vector carrying a polynucleotide encoding recombinant BMP9; the vector includes any one or more of a plasmid, a lentivirus, an adenovirus, an adeno-associated virus, a retrovirus, a liposome, a lipid nanoparticle, N-acetylgalactosamine, and an extracellular vesicle.

3. Use according to claim 1 or 2, characterized in that, The BMP9 promoter is recombinant BMP9; the amino acid sequence of the recombinant BMP9 comprises a sequence indicated in (i) or (ii) below: (i) a sequence as indicated in SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3; (ii) a sequence in which one or more amino acids are substituted, deleted, or added in a sequence as indicated in SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3, and has the protein activity of the sequence as indicated in SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO.

3.

4. Use according to claim 1 or 2, characterized in that, The BMP9 promoter is an adeno-associated virus carrying a polynucleotide encoding recombinant BMP9; the amino acid sequence of the recombinant BMP9 comprises a sequence indicated in (i) or (ii) below: (i) a sequence as indicated in SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3; (ii) a sequence in which one or more amino acids are substituted, deleted, or added in a sequence as indicated in SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3, and has the protein activity of the sequence as indicated in SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO.

3. Preferably, the polynucleotide encoding recombinant BMP9 comprises a nucleic acid sequence indicated in (a) or (b) below: (a) a sequence as indicated in SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6; (b) a sequence that hybridizes to a sequence as indicated in SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6 under stringent conditions, and encodes a protein having the protein activity of the sequence as indicated in SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO.

3.

5. Use according to claim 4, characterized in that, In the genome of the adeno-associated virus carrying the polynucleotide encoding recombinant BMP9, the polynucleotide encoding recombinant BMP9 is driven by a transthyretin promoter.

6. Use according to any one of claims 1 to 5, characterized in that, The prevention and / or treatment of diabetic nephropathy includes improving the renal function, liver function, and blood lipids of a subject.

7. Use according to claim 6, characterized in that, The improvement of the renal function of a subject includes at least one of reducing the urine albumin / creatinine ratio, reducing the blood urea nitrogen level, and reducing the uric acid level.

8. Use according to claim 6, characterized in that, The improvement in liver function in the subject includes a reduction in alanine aminotransferase levels.

9. Use according to claim 6, characterized in that, The improvement in blood lipids in the subject includes at least one of a reduction in total cholesterol levels, a reduction in triglyceride levels, and a reduction in low-density lipoprotein levels.

10. Use according to any one of claims 1 to 9, characterized in that, The subject includes a mammal. Preferably, the subject is a human.

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