CN118599000A - Application of LRP5 as a targeting molecule in the treatment of diabetic nephropathy - Google Patents

Abstract

The present invention discloses a targeting molecule LRP5, whose amino acid sequence is shown in SEQ ID NO: 1. The present invention also discloses the use of the targeting molecule in the preparation of a drug for treating diabetic nephropathy. The targeting molecule LRP5 of the present invention regulates the expression level and transcriptional activity of the PPAR pathway, not only reduces the body's blood lipids, but also inhibits the kidney's reabsorption of glucose, enhances the fatty acid oxidation capacity of the proximal tubules of the kidney, improves the lipid metabolism function of the proximal tubules, improves the proximal tubule lesions and renal fibrosis, and achieves the purpose of better treating diabetic nephropathy.

Description

Application of LRP5 as a targeting molecule in the treatment of diabetic nephropathy

Technical Field

The present invention belongs to the field of biomedicine technology, and in particular, relates to the application of LRP5 as a targeting molecule in the treatment of diabetic nephropathy.

Background Art

Diabetic nephropathy (DKD) is a serious microvascular complication of diabetes with a long course of disease. Early DKD has no obvious symptoms and signs and is easily ignored. As the disease progresses, urine protein continues to be positive and the glomerular filtration rate (GFR) gradually decreases, eventually leading to late-stage renal failure. The pathological damage to the kidneys is irreversible.

Diabetic nephropathy is the main cause of end-stage renal disease. The mechanism of diabetic nephropathy is relatively complex. Tubulointerstitial fibrosis is a common pathway for most renal injuries and is one of the main pathological features of chronic kidney disease. Renal fibroblasts play an important role in maintaining the homeostasis of the interstitial matrix and adjacent tissues under physiological conditions. Many cells are involved in the process of renal fibrosis. The main cause of tubulointerstitial fibrosis comes from the activation and expansion of fibroblasts, the production and deposition of a large number of extracellular matrix components, and changes in renal tubules and microvessels. Continuous exposure to a high-sugar environment induces epithelial-mesenchymal transition (EMT) in renal tubular epithelial cells, leading to interstitial fibrosis and accumulation of extracellular matrix proteins in the mesangial interstitium, such as tubulointerstitial fibrosis caused by the accumulation of collagen and fibronectin.

According to statistics, 60% of diabetic patients have hyperlipidemia, and 20% to 40% of diabetic patients have DKD with insidious onset and rapid progression, which has become the leading cause of end-stage renal disease (ESRD), cardiovascular events and death in middle-aged and elderly people. DKD patients with decreased renal function are often accompanied by dyslipidemia, so the clinical incidence of DKD combined with hyperlipidemia is very high. For patients with advanced DKD, how to reduce blood lipids without causing kidney damage is a difficult clinical problem. At present, the control of the development of diabetic nephropathy includes preventing, alleviating and even reversing DKD by solving the interaction between hemodynamics and metabolic pathways, controlling blood sugar, blood pressure and blood lipids, using major drugs that have a blocking effect on the renin-angiotensin (ACE)-aldosterone system, and treating dyslipidemia, which are also effective strategies for preventing diabetic nephropathy. However, despite the control of blood sugar, blood lipids and blood pressure, many patients still develop diabetic nephropathy. The first-line medications recommended by current international guidelines are metformin, ACEI/ARB, SGLT-2i, GLP-1 agonists, and the recently approved third-generation aldosterone receptor antagonists, which can effectively reduce the risk of composite endpoint events of renal disease such as doubling of creatinine and ESRD. However, many DKD patients still experience progressive renal damage.

It is estimated that by 2040, the prevalence of diabetes is expected to exceed 600 million people, of which more than 20 million will develop DKD. Therefore, there is an urgent need to explore potential treatments for DKD to slow down the occurrence and development of DKD.

CN115960020B discloses a caffeic acid nitrone compound, a preparation method thereof and its use in preparing a drug for treating diabetic nephropathy. Animal test results show that the invented compound can reduce the urine albumin/creatinine ratio and the level of urea nitrogen, and has a certain effect on treating diabetic nephropathy. However, the therapeutic effect of the caffeic acid nitrone compound is limited, mainly playing a preventive role, and it is difficult to use for therapeutic purposes.

CN117959346A discloses the use of a pharmaceutical composition in the preparation of a drug for the prevention and/or treatment of diabetic nephropathy, wherein the active ingredients in the pharmaceutical composition at least include curcumin and yew. This invention first proposed that curcumin and yew can be used in combination to prepare drugs for the prevention and/or treatment of diabetic nephropathy. The established experimental diabetic nephropathy rats were given medication for treatment, and it was found that after curcumin + yew combined treatment, the blood sugar level, serum creatinine, and urea nitrogen level of the rats were significantly reduced. However, the use of this drug combination has limited efficacy, a long cycle, and the possibility of side effects.

CN117959336A discloses the application of exosomes derived from human umbilical cord mesenchymal stem cells in diabetic nephropathy treatment drugs, which specifically involves the extraction of HucMSC-Exo and its significant effect on the treatment of DKD mice. The invention proves that hucMSC-Exo has the effect of improving DKD in vivo, and introduces a new method for the clinical treatment of DKD, but its steps are cumbersome, the success rate is low and the cost is high, which makes it difficult to promote it on a large scale for patients.

EP4351617A1 discloses a compound for treating diabetic nephropathy, wherein the compound is a peptide derived from an IL-1R antagonist (ILIRA) protein selected from a specific sequence SEQ ID NO: 2-5, or a variant thereof is at least 70% identical to the peptide derived from I LIRA. The peptide derived from ILIRA disclosed in the patent can reduce creatinine-corrected urinary albumin and creatinine-corrected renal injury molecule (KIM-1/CR), and urinary albumin and KIM-1 are both biomarkers of innate inflammation of the kidney including diabetic nephropathy, so the compound can be used for the treatment of diabetic nephropathy. However, the peptide treatment effect described in the patent is limited and the cost is high.

CN117752673A discloses the application of miR-378c targeting GSK3β in regulating podocyte injury in diabetic nephropathy. It first discovered that the expression level of miRNA-378c in the kidney tissue of patients with diabetic nephropathy was significantly downregulated, and further verified that miR-378c was downregulated in podocytes under high glucose environment, and at the same time verified that miR-378c combined with the 3'UTR segment of GSK3β to target downregulated the expression level of GSK3β, and then upregulated the expression level of Nrf2, achieving the repair and inhibition of podocyte injury and apoptosis in high glucose environment, and providing a new treatment idea and new target for the treatment of diabetic nephropathy. However, this target can only repair and inhibit podocyte injury and apoptosis in high glucose environment, and the effect is not obvious.

CN117683881 discloses the application of AcircMRP4 as a new target in the diagnosis and treatment of diabetic nephropathy. Its research confirms that circMRP4 in renal podocytes is significantly upregulated in diabetic nephropathy, and it is found that it can promote podocyte apoptosis, thereby exacerbating the occurrence and development of diabetic nephropathy. Knocking down circMRP4 has the effect of inhibiting podocyte apoptosis and delaying the progression of diabetic nephropathy. Therefore, circMRP4 can be used as a new diagnostic indicator and therapeutic target in the treatment of diabetic nephropathy. However, the therapeutic effect of this target is limited, mainly playing a preventive role, and it is difficult to use for therapeutic purposes.

In addition, existing literature reports that oxidative stress is a key factor in the initiation, occurrence, and development of DKD (AntioxidRedox Sign 2016; 25: 639-641). Podocytes are renal cells that are highly susceptible to oxidative stress and are easily damaged, thereby causing proteinuria. A large number of studies have shown that oxidative stress-induced podocyte damage, especially podocyte apoptosis, is a key factor in the development of DKD proteinuria and glomerulosclerosis (Diabetologia 2016; 59: 379-389). As an oxidative stress response transcription factor, the forkhead protein FOXO3a is highly expressed in the podocytes of the renal tissue of DKD patients and can promote podocyte apoptosis by mediating the transcription of downstream pro-apoptotic target genes such as Bim and FasL (FASEB J, 2020, 34: 13300-13316). The mutual binding of FOXO3a and P53 to regulate the transcription of pro-apoptotic genes and activate the mitochondrial apoptosis pathway is a potential mechanism for FOXO3a to regulate apoptosis, and therefore can be used as a potential treatment for DKD.

Human neuroblastoma tumorigenicity suppressor 1 (NBL1) is a 165-amino acid long secreted protein that was originally identified as a tumor suppressor in neuroblastoma cell lines. Subsequently, NBL1 was shown to have bone morphogenetic protein (BMP) inhibitory activity, especially BMP-2 and BMP-7 inhibitory activity. A recent study showed that NBL1 is closely associated with the high risk of progression of DKD patients to ESKD, and the NBL1 content in the blood and urine of DKD patients is significantly increased. In addition, NBL1 can induce apoptosis of podocytes in vitro. Therefore, NBL1 is related to the occurrence, development and prognosis of DKD, and may become a new drug treatment target with great potential to curb the transformation of DKD to ESKD.

Although the prior art has disclosed a variety of potential treatments for DKD, the therapeutic effects of these treatments are not satisfactory, and new targets and treatment ideas are still needed.

Summary of the invention

In view of the deficiencies of the prior art and actual needs, the present invention provides a targeting molecule for treating diabetic nephropathy.

To achieve the above object, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides a targeting molecule LRP5, the amino acid sequence of the targeting molecule is shown in SEQ ID NO: 1:

MEAAPPGPPWPLLLLLLLLLALCGCPAPAAASPPLLLFANRRDVRLVDAGGVKLESTIVVSGLEDAAAVDFQFSKGAVYWTDVSEEAIKQTYLNQTGAAVQNVVISGLVSPDGLACDWVGKKLYWTDSETNRIEVANLNGTSRKVLFWQDLDQPRAIALDPAHGYMYWTDWGETPRIERAGMDGSTRKIIVDSDIYWPNGLTIDLEEQKLYWADA KLSFIHRANLDGSFRQKVVEGSLTHPFALTLSGDTLYWTDWQTRSIHACNKRTGGKRKEILSALYSPMDIQVLSQERQPFFHTRCEEDNGGCSHLCLLSPSEPFYTCACPTGVQLQDNGRTCKAGAEEVLLLARRTDLRRISLDTPDFTDIVLQVDDIRHAIAIDYDPLEGYVYWTDDEVRAIRRAYLD GSGAQTLVNTEINDPDGIAVDWVARNLYWTDTGTDRIEVTRLNGTSRKILVSEDLDEPRAIALHPVMGLMYWTDWGENPKIECANLDGQERRVLVNASLGWPNGLALDLQEGKLYWGDAKTDKIEVINVDGTKRRTLLEDKLPHIFGFTLLGDFIYWTDWQRRSIERVHKVKASRDVIIDQLPDLMGLKAVNVAKVVGTNPCADRNGGCS HLCFFTPHATRCGCPIGLELLSDMKTCIVPEAFLVFTSRAAIHRISLETNNNDVAIPLTGVKEASALDFDVSNNHIYWTDVSLKTISRAFMNGSSVEHVVEFGLDYPEGMAVDWMGKNLYWADTGTNRIEVARLDGQFRQVLVWRDLDNPRSLALDPTKGYIYWTEWGGKPRIVRAFMDGTNCMTLVDKVGRAN DLTIDYADQRLYWTDLDTNMIESSNMLGQERVVIADDLPHPFGLTQYSDYIYWTDWNLHSIERADKTSGRRNRTLIQGHLDFVMDILVFHSSRQDGLNDCMHNNGQCGQLCLAIPGGHRCGCASHYTLDPSSRNCSPPTTFLLFSQKSAISRMIPDDQHSPDLILPLHGLRNVKAIDYDPLDKFIYWVDGRQNIKRAKDDGTQPFV LTSLSQGQNPDRQPHDLSIDIYSRTLFWTCEATNTINVHRLSGEAMGVVLRGDRDKPRAIVVNAERGYLYFTNMQDRAAKIERAALDGTEREVLFTTGLIRPVALVVDNTLGKLFWVDADLKRIESCDLSGANRLTLEDANIVQPLGLTILGKHLYWIDRQQQMIERVEKTTGDKRTRIQGRVAHLTGIHAVEEVSLEE FSAHPCARDNGGCSHICIAKGDGTPRCSCPVHLVLLQNLLTCGEPPTCSPDQFACATGEIDCIPGAWRCDGFPECDDQSDEEGCPVCSAAQFPCARGQCVDLRLRCDGEADCQDRSDEADCDAICLPNQFRCASGQCVLIKQQCDSFPDCIDGSDELMCEITKPPSDDSPAHSSAIGPVIGIILSLFVMGGVYFVCQRVVCQRYAGANGPFPHEYVSGTPHV PLNFIAPGGSQHGPFTGIACGKSMMSSVSLMGGRGGVPLYDRNHVTGASSSSSSSTKATLYPPILNPPPSPATDPSLYNMDMFYSSNIPATARPYRPYIIRGMAPPTTPCSTDVCDSDYSASRWKASKYYLDLNSDSDPYPPPPTPHSQYLSAEDSCPPSPATERSYFHLFPPPPSPCTDSS

The DNA sequence of the targeting molecule is shown in SEQ ID NO: 2:

atggag gcagcgccgc ccgggccgcc gtggccgctg ctgctgctgc tgctgctgctgctggcgctg tgcggctgcccggcccccgc cgcggcctcg ccgctcctgc tatttgccaa ccgccgggacgtacggctgg tggacgccggcggagtcaag ctggagtcca ccatc gtggt cagcggcctg gaggatgcggccgcagtgga cttccagttttccaagggag ccgtgtactg gacagacgtg agcgaggagg ccatcaagcagacctacctg aaccagacgggggccgccgt gcagaacgtg gtcatctccg gcctggtctc tcccgacggcctcgcctgcg actgggt gggcaagaagctg tactggacgg actcagagac caaccgcatc gaggtggccaacctcaatgg cacatcccggaaggtgctct tctggcagga ccttgaccag ccgagggcca tcgccttggaccccgctcac gggtacatgtactggacaga ctggggtgag acgccccgga ttgagcgggc agggatggatggcagcaccc gga agatcattgtggactcg gacatttact ggcccaatgg actgaccatc gacctggaggagcagaagct ctactgggctgacgccaagc tcagcttcat ccaccgtgcc aacctggacg gctcgttccggcagaaggtg gtggagggcagcctgacgca ccccttcgcc ctgacgctct ccggggacac tctg tactggacagactggc agacccgctccatccatgcc tgcaacaagc gcactggggg gaagaggaag gagatcctgagtgccctcta ctcacccatggacatccagg tgctgagcca ggagcggcag cctttcttcc acactcgctgtgaggaggac aatggcggctgctcccacct gtgcctgctg tccccaagcg agcct ttcta cacatgcgcctgccccacgg gtgtgcagctgcaggacaac ggcaggacgt gtaaggcagg agccgaggag gtgctgctgctggcccggcg gacggaccta cggaggatct cgctggacac gccggacttc accgacatcg tgctgcaggtggacgacatc cggcacgcca ttgccatcga ctac gacccg ctagagggct atgtctactg gacagatgacgaggtgcggg ccatccgcag ggcgtacctg gacgggtctg gggcgcagac gctggtcaac accgagatcaacgaccccga tggcatcgcg gtcgactggg tggcccgaaa cctctactgg accgaccgg gcacggaccgcatcgaggtg acgcgcc tca acggcacctc ccgcaagatc ctggtgtcgg aggacctgga cgagccccgagccatcgcac tgcaccccgt gatgggcctc atgtactgga cagactgggg agagaaccct aaaatcgagtgtgccaactt ggatgggcag gagcggcgtg tgctggtcaa tgcctccctc gggtgg ccca acggcctggccctggacctg caggagggga agctctactg gggagacgcc aagacagaca agatcgaggt gatcaatgttgatgggacga agaggcggac cctcctggag gacaagctcc cgcacatttt tgggttcacg ctgctgggggacttcatcta ctggactgac tggcagcgcc gcagcatcga gc gggtgcac aaggtcaagg ccagccgggacgtcatcatt gaccagctgc ccgacctgat ggggctcaaa gctgtgaatg tggccaaggt cgtcggaaccaacccgtgtg cggacaggaa cggggggtgc agccacctgt gcttcttcac accccacgca acccggtgtggctgccccat cgg cctggag ctgctgagtg acatgaagac ctgcatcgtg cctgaggcct tcttggtcttcaccagcaga gccgccatcc acaggatctc cctcgagacc aataacaacg acgtggccat cccgctcacgggcgtcaagg aggcctcagc cctggacttt gatgtgtcca acaaccacat ctactggaca gacgtcagcctgaagac cat cagccgcgcc ttcatgaacg ggagctcggt ggagcacgtg gtggagtttg gccttgactaccccgagggc atggccgttg actggatggg caagaacctc tactgggccg acactgggac caacagaatcgaagtggcgc ggctggacgg gcagttccgg caagtcctcg tgtggaggga ctt ggacaac ccgaggtcgctggccctgga tcccaccaag ggctacatct actggaccga gtggggcggc aagccgagga tcgtgcgggccttcatggac gggaccaact gcatgacgct ggtggacaag gtgggccggg ccaacgacct caccattgactacgctgacc agcgcctcta ctggaccgac ctggacacca acat gatcga gtcgtccaac atgctgggtcaggagcgggt cgtgattgcc gacgatctcc cgcacccgtt cggtctgacg cagtacagcg attatatctactggacagac tggaatctgc acagcattga gcgggccgac aagactagcg gccggaaccg caccctcatccagggccacc tggacttcgt gat ggacatc ctggtgttcc actcctcccg ccaggatggc ctcaatgactgtatgcacaa caacgggcag tgtgggcagc tgtgccttgc catccccggc ggccaccgct gcggctgcgcctcacactac accctggacc ccagcagccg caactgcagc ccgcccacca ccttcttgct gttcagccagaaatctgcca tcagtcggat gatcccggac gaccagcaca gcccggatct catcctgccc ctgcatggactgaggaacgt caaagccatc gactatgacc cactggacaa gttcatctac tgggtggatg ggcgccagaacatcaagcga gccaaggacg acgggaccca gccctttgtt ttgacctctc tgagccaagg c caaaacccagacaggcagc cccacgacct cagcatcgac atctacagcc ggacactgtt ctggacgtgc gaggccaccaataccatcaa cgtccacagg ctgagcgggg aagccatggg ggtggtgctg cgtggggacc gcgacaagcccagggccatc gtcgtcaacg cggagcgagg gtacctgtac ttcaccaaca tgcagg accg ggcagccaagatcgaacgcg cagccctgga cggcaccgag cgcgaggtcc tcttcaccac cggcctcatc cgccctgtggccctggtggt ggacaacaca ctgggcaagc tgttctgggt ggacgcggac ctgaagcgca ttgagagctgtgacctgtca ggggccaacc gcctga ccct ggaggacgcc aacatcgtgc agcctctggg cctgaccatccttggcaagc atctctactg gatcgaccgc cagcagcaga tgatcgagcg tgtggagaag accaccggggacaagcggac tcgcatccag ggccgtgtcg cccacctcac tggcatccat gcagtggagg aagtcagcctggaggagttc tcagcccacc gtgcccg tgacaatggt ggctgctccc acatctgtat tgccaagggtgatgggacac cacggtgctc atgcccagtc cacctcgtgc tcctgcagaa cctgctgacc tgtggagagccgcccacctg ctccccggac cagtttgcat gtgccacagg ggagatcgac tgtatcc ccg gggcctggcgctgtgacggc tttcccgagt gcgatgacca gagcgacgag gagggctgcc ccgtgtgctc cgccgcccagttcccctgcg cgcggggtca gtgtgtggac ctgcgcctgc gctgcgacgg cgaggcagac tgtcaggaccgctcagacga ggcggactgt gacgccatct gcctgcccaa ccagttccgg tgtgcgagcg gccagtgtgtcctcatcaaa cagcagtgcg actccttccc cgactgtatc gacggctccg acgagctcat gtgtgaaatcaccaagccgc cctcagacga cagcccggcc cacagcagtg ccatcgggcc cgtcattggc atcatcctctct catctcttcgtg ggtggt gtctattttg tgtgccagcg cgtggtgtgc cagcgctatg cgggggccaacgggcccttc ccgcacgagt atgtcagcgg gaccccgcac gtgcccctca atttcatagc cccgggcggttcccagcatg gccccttcac aggcatcgca tgcggaaagt ccatgatgag ctccgtgagc ctgatggggggccgggg cgg ggtgcccctc tacgaccgga accacgtcac aggggcctcg tccagcagct cgtccagcacgaaggccacg ctgtacccgc cgatcctgaa cccgccgccc tccccggcca cggacccctc cctgtacaacatggacatgt tctactcttc aaacattccg gccactgcga gaccgtac agcccctacatc attcgaggaatggcgccccc gacgacgccc tgcagcaccg acgtgtgtga cagcgactac agcgccagcc gctggaaggccagcaagtac tacctggatt tgaactcgga ctcagacccc tatccacccc cacccacgcc ccacagccagtacctgtcgg cggaggacag ctgcccgccc t cgcccgcca ccgagaggag ctacttccat ctcttcccgccccctccgtc cccctgcacg gactcatcct ga

In a second aspect, the present invention provides the use of the targeting molecule LRP5 in the preparation of drugs for treating DKD and hyperlipidemia.

In one or more embodiments, the DKD is kidney disease caused by diabetes, and the diabetes is one or more of type 1 diabetes, type 2 diabetes, a special type of diabetes, and gestational diabetes.

In the present invention, the targeting molecule LRP5 treats diabetes and kidney disease by regulating the expression level and transcriptional activity of the PPAR pathway.

In the present invention, the targeting molecule LRP5 can reduce the body's blood lipid level, enhance the fatty acid oxidation capacity of the renal proximal tubules, improve the lipid metabolism function of the proximal tubules, inhibit proximal tubule lesions and renal fibrosis, and treat DKD and hyperlipidemia at the same time.

In a third aspect, the present invention provides a drug for treating DKD that can simultaneously reduce blood lipid levels and inhibit glucose reabsorption by the kidneys, wherein the drug comprises a targeting molecule LRP5 and a pharmaceutically acceptable carrier.

In one or more embodiments, the drug is administered by one or more of subcutaneous injection, intravenous injection, intramuscular injection or nasal administration.

In one or more embodiments, the pharmaceutically acceptable carrier is a delivery carrier.

In one or more embodiments, the delivery vector comprises at least one of a plasmid vector, a liposome, a viral vector, a nanovesicle, a membrane-penetrating peptide modification, or electroporation.

In one or more embodiments, the medicament may further comprise other drugs for treating diabetes and/or kidney disease.

Compared with the prior art, the present invention has the following beneficial effects:

The targeted molecule LRP5 described in the present invention not only reduces the body's blood lipid level and inhibits the kidney's reabsorption of glucose by regulating the expression level and transcriptional activity of the PPAR pathway, but also enhances the fatty acid oxidation capacity of the proximal tubules of the kidney, improves the lipid metabolism function of the proximal tubules, inhibits proximal tubule lesions and renal fibrosis, thereby treating DKD.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the changes in renal function caused by Lrp5 knockout, where (a) shows the timetable of high-fat feeding and testing of mice; (b) detects triglycerides (TG), cholesterol (TC), low-density lipoprotein (LDL-C) and high-density lipoprotein (HDL-C) in mouse serum; (c, d) detects creatinine levels in mouse serum and urine; (e) calculates creatinine clearance (CCR); (f) detects the total amount of albumin in 24-hour urine; (g) calculates the urine albumin-creatinine ratio (UACR).

Figure 2 shows that blood lipid levels are negatively correlated with renal function in the case of diabetes, including (a-d) the correlation between serum creatinine levels and TG, TC, LDL-C and HDL-C; (e-h) the correlation between CCR and TG, TC, LDL-C and HDL-C.

Figure 3 shows that Lrp5 knockout causes the renal proximal tubules to prefer glucose utilization, where (a, b) line graphs show the body weight and random blood glucose values of mice every two weeks after the start of HFD feeding; (c, d) fluorescent staining detection and statistics of the expression levels of glucose reuptake protein SGLT2 and glucose transporter GLUT2 in the renal proximal tubules; (e) real-time PCR detection of the expression levels of glycolytic genes in the kidney.

Figure 4 shows that Lrp5 knockout aggravates diabetic renal fibrosis, where (a) the bar graph shows the top 10 GO cellular component pathways of differentially enriched genes between the Lrp5- ^-/- HFD and WT-HFD groups, and the red arrows indicate extracellular matrix-related components; (b, c) Western blot and grayscale statistical analysis of the expression levels of extracellular matrix collagen I, vimentin, PAI-1 and α-SMA in the kidney lysates of each group; (df) H&E staining, Sirius red staining and vimentin staining detected and counted the tissue morphology, collagen deposition and fibrosis-specific marker molecule vimentin level of kidney sections in each group.

Figure 5 shows that LRP5 is an important factor in protecting the function of renal proximal tubule epithelium, where (a-d) GSEA showed that compared with WT-HFD, Lrp5- ^-/- HFD had significantly downregulated epithelial cell apoptosis, positive epithelial cell proliferation, positive epithelial cell migration and epithelial-epithelial adhesion pathways; (e, f) Western blot and grayscale statistical analysis of the expression levels of epithelial cell-specific marker molecule E-cadherin and proximal tubule epithelial-specific marker molecule SGLT2 in the kidney lysate of each group; (g, h) Fluorescence staining was used to detect the distribution and expression of E-cadherin in the kidney sections of each group.

Figure 6 shows that LRP5 overexpression protects the function of proximal tubule epithelial cells, where (a, b) Western blot and grayscale statistical analysis of the expression levels of E-cadherin, collagen I, α-SMA and vimentin in HK2 cells infected with LRP5 adenovirus for 24 hours and treated with PA for 24 hours; (c, d) Fluorescence staining detection and statistical analysis of the expression distribution of E-cadherin and vimentin in HK2 cells infected with LRP5 adenovirus for 24 hours and treated with PA for 24 hours; (e, f) Western blot and grayscale statistical analysis of the expression level of vimentin in primary WT PTECs infected with LRP5 adenovirus for 24 hours and treated with PA for 24 hours.

Figure 7 shows that LRP5 regulates fatty acid transport and synthesis in diabetic kidneys, where (a) Real-time PCR was used to detect the transcription levels of fatty acid transporters Fabp1, Fabp2, Fabp3, Fabp4 and Cd36 in the kidneys of each group; (b) Real-time PCR was used to detect the transcription levels of key enzymes for fatty acid synthesis Acaca, Fasn and Srebf1 in the kidneys of each group; (c, d) Western blot and grayscale statistical analysis were used to analyze the expression levels of p-ACC, ACC, SREBP1 and SREBP1c in the kidneys of each group; (e, f) Western blot and grayscale statistical analysis were used to analyze the protein levels of p-ACC and ACC in WT PTECs infected with LRP5 adenovirus for 24 hours and then treated with PA for 24 hours.

Figure 8 shows that LRP5 regulates fatty acid oxidation (FAO) in the proximal tubules of diabetic kidneys, where (a) GSEA shows that the lipid metabolism-related GO pathways are significantly changed in Lrp5- ^-/- HFD kidneys compared with WT-HFD; (b) the heat map shows the expression of FAO pathway components in the kidneys of each group; (c, d) fluorescent staining was used to detect the distribution of ACOX1 in the kidneys of each group; (e, f) Western blot and grayscale statistical analysis were used to analyze the expression levels of CPT1A, CPT2 and ACOX1 in HK2 cells after they were infected with LRP5 adenovirus for 24 hours and then treated with PA for 24 hours.

Figure 9 shows that Lrp5 knockout inhibits the PPAR/PGC-1 signaling pathway in diabetic kidneys, where (a) the bubble chart shows the top 10 most significantly changed KEGG pathways in Lrp5- ^-/- HFD kidneys compared with WT-HFD, and the PPAR pathway is the third most significantly changed pathway; (b) the heat map shows the transcriptional levels of each component in the PPAR signaling pathway in the kidneys of each group; (c, d) Western blot and grayscale statistical analysis of the expression levels of PGC-1β, PPARγ and PPARα in the kidneys of each group; (e, f) fluorescent staining to detect the expression and distribution of PGC-1α in the kidneys of each group.

Figure 10 shows the regulatory effect of LRP5 on renal FAO mediated by the PPAR/PGC-1 signaling pathway, where (a, b) Real-time PCR detected the transcription levels of PPARA, PPARG, PPARGC1A and PPARGC1G in HK2 cells infected with LRP5 adenovirus for 24 hours and then treated with PA for 24 hours; (c, d) Western blot and grayscale statistical analysis of the levels of PGC-1α, PGC-1β and PPARα in HK2 cells infected with LRP5 adenovirus for 24 hours and then treated with PA for 24 hours; (e, f) Western blot and grayscale statistical analysis of the expression levels of p-ERK1/2 and ERK1/2 in the kidneys of each group.

DETAILED DESCRIPTION

In view of the existing research on diabetes and nephropathy, the present invention has discovered a new targeting molecule LRP5, the amino acid sequence of the targeting molecule is shown in SEQ ID NO: 1, and the DNA sequence is shown in SEQ ID NO: 2. The targeting molecule of the present invention can treat both diabetic nephropathy and hyperlipidemia.

Example

The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

The examples involve the addition amounts, contents and concentrations of various substances, wherein the percentages described therein, unless otherwise specified, are all by mass percentages.

Experimental methods:

1. Experimental Animals

The experimental animals used in this invention were bred in the SPF barrier environment of Hemu Building, North Campus of Sun Yat-sen University. All animal experiments were approved by the Experimental Animal Management Committee of Sun Yat-sen University. All experimental animals were raised in the barrier facility of the Experimental Animal Center of the North Campus of Sun Yat-sen University in a clean environment with a temperature of (21±2)°C, humidity of (35±2)%, 12h-12h continuous lighting, free access to food and water, and the drinking water was sterile water prepared by the Experimental Animal Center.

1) Lrp5 knockout mice were constructed. Twenty WT and KO mice were randomly divided into two groups (10 mice/group). They were fed with normal chow (NCD) and high-fat diet (HFD), respectively. Random blood glucose levels and body weight were monitored every two weeks.

2) When a difference in fasting or postprandial blood glucose levels was detected, the mice were subjected to glucose tolerance test (GTT), in vitro islet perfusion and insulin tolerance test (ITT). The GTT test method was to starve the mice for 12-16 hours, intraperitoneally inject 1g/kg body weight of glucose, and measure blood glucose levels at 0, 15, 30, 60, and 120 minutes. The ITT test method was to starve the mice for 4-6 hours, intraperitoneally inject 1U/kg body weight of insulin, and measure blood glucose levels at 0, 15, 30, 60, and 90 minutes.

2. RNA Sequencing

Total RNA was extracted from mouse kidney tissue, and the constructed library was subjected to RNA sequencing using the Illumina Novasek 6000 sequencing platform (Jinneng, China).

3. Total RNA Extraction

Total RNA was isolated from kidney tissues and cultured cells using an RNA extraction kit (Takara, USA).

4. Real-time PCR

RNA was reverse transcribed using HiScript IIQ RT SuperMixfor qPCR (+gDNA wiper) (Vazyme, China). qPCR experiments were performed using AceQ qPCR SYBR Green Master Mix (Vazyme, China). The specific primer sequences were normalized by β-actin as shown in Table 1:

Table 1. Real-time PCR primer sequences

5. Fluorescence staining

Fresh kidney samples were fixed in 4% PFA, then embedded in paraffin and sliced. After a series of dewaxing and rehydration steps, the paraffin sections were placed in sodium citrate solution and boiled for 15 minutes for antigen repair. Washed twice with PBS, 0.3% Triton solution was added to permeabilize the membrane for 30 minutes, 5% goat serum was added to block at room temperature for 1 hour, primary antibody was added to incubate at 4 degrees overnight, and secondary antibody was added to incubate at room temperature for 2 hours. Antifade sealing agent was added for sealing. Photographed under a laser confocal microscope or an inverted microscope.

6. Cell line sources and cell culture

Mouse renal proximal tubule epithelial cell line (HK2) was kindly provided by Professor Nan Ning of the Third Affiliated Hospital of Sun Yat-sen University. HK2 cells were cultured in DMEM/F12 complete medium (containing 10% FBS, 1% penicillin-streptomycin) at 37°C and 5% CO2 in a cell culture incubator.

7. Adenovirus infection of HK2 cells

The LRP5 overexpression adenovirus (Ad-LRP5) was synthesized by Shanghai Jikai Biotechnology Co., Ltd., and the LRP5 knockdown adenovirus (Ad-shLRP5) was synthesized by Shanghai Jima Co., Ltd. After HK2 was plated overnight, adenovirus (MOI: 50) was mixed with polybrene (5ug/ml) and added to the cells for 24h, and then 300μM palmitic acid (PA) was added for 24h treatment to terminate the experimental detection indicators.

8. Western blot experiment

Total protein of cells or tissues was extracted with Western blot lysis buffer containing PMSF, and SDS-PAGE was performed, then transferred to PVDF membrane, and the membrane was blocked with 5% skim milk powder. The membrane was incubated with specific primary antibody at 4°C overnight, and the secondary antibody (Bio-rad, USA) was incubated at room temperature for 2h. Finally, the rinsed PVDF membrane was placed in an exposure instrument and chemiluminescent developer (Bio-rad, USA) was added for development. The specific antibodies used are shown in Table 2:

Table 2. Antibody list

9. Statistical processing

Image J and Graphpad software were used to analyze the data, and all experiments were repeated more than 3 times. Data were expressed as mean ± standard deviation, and t-test was used for pairwise comparison between groups, and one-way analysis of variance was used for comparison between multiple groups. P < 0.05 was considered statistically significant.

Example 1: LRP5 regulates blood lipid and blood glucose levels in DKD mice

As shown in Figures 1 to 3, Lrp5 ^-/- -HFD (high-fat diet-induced type II diabetes) mice showed hyperlipidemia but lower blood glucose levels. The results further confirmed that Lrp5 knockout inhibited the expression of renal proximal tubule glucose transporter (SGLT2) but upregulated the level of glucose reabsorption transporter (GLUT2). These data indicate that LRP5 is an important molecule that can lower blood lipids and inhibit renal glucose reabsorption, and is a target with great clinical application prospects.

Example 2: LRP5 is a new target for the treatment of diabetic nephropathy

As shown in Figures 4 to 10, the above results of the present invention prove that Lrp5 knockout increases renal tubular damage and fibrosis, and the specific mechanism is to reduce the activity of the PPAR pathway and inhibit the expression of fatty acid oxidase in the proximal tubules of the kidney, thereby causing proximal tubule apoptosis and tubulointerstitial fibrosis.

Claims (10)

1. A targeting molecule LRP5, characterized in that the amino acid sequence of the targeting molecule is shown in SEQ ID NO: 1. 2. Use of the targeting molecule according to claim 1 in the preparation of drugs for treating diabetic nephropathy and hyperlipidemia. 3. The use according to claim 2, characterized in that the diabetes is one or more of type 1 diabetes, type 2 diabetes, special types of diabetes and gestational diabetes. 4. The use according to claim 2, characterized in that the targeting molecule treats diabetic nephropathy by regulating the expression level and transcriptional activity of the PPAR pathway. 5. The use as claimed in claim 2, characterized in that the targeting molecule can reduce the body's blood lipid level, enhance the fatty acid oxidation capacity of the renal proximal tubules, improve the lipid metabolism function of the proximal tubules, inhibit proximal tubule lesions and renal fibrosis, and achieve the goal of improving diabetic nephropathy. 6. A drug that can simultaneously reduce the body's blood lipid level and inhibit the kidney's reabsorption of glucose, thereby treating diabetic nephropathy, characterized in that the drug contains a targeting molecule LRP5 and a pharmaceutically acceptable carrier. 7. The drug according to claim 6, characterized in that the drug is administered by one or more of subcutaneous injection, intravenous injection, intramuscular injection or nasal administration. 8. The drug according to claim 6, wherein the pharmaceutically acceptable carrier is a delivery carrier. 9. The drug according to claim 8, characterized in that the delivery vector comprises at least one of a plasmid vector, a liposome, a viral vector, a nanovesicle, a membrane-penetrating peptide modification or electroporation. 10. The drug according to claim 6, characterized in that the drug may also contain other drugs for treating diabetes and/or kidney disease.