CN117589999A - Application of PFKP as chronic kidney disease treatment target and inhibitor thereof - Google Patents

Application of PFKP as chronic kidney disease treatment target and inhibitor thereof Download PDF

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CN117589999A
CN117589999A CN202311536915.XA CN202311536915A CN117589999A CN 117589999 A CN117589999 A CN 117589999A CN 202311536915 A CN202311536915 A CN 202311536915A CN 117589999 A CN117589999 A CN 117589999A
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pfkp
fibrosis
kidney
glycolysis
gene
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梁真
杨书
康林
杨广燕
李燕纯
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Shenzhen Peoples Hospital
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Shenzhen Peoples Hospital
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Abstract

The invention discloses an application of PFKP as a target point for treating chronic kidney diseases and an inhibitor thereof. In particular to an application of PFKP gene or PFKP protein serving as a target point in preparing a product for preventing or treating chronic kidney disease or kidney fibrosis, and an application of PFKP inhibitor in preparing a product for preventing or treating chronic kidney disease or kidney fibrosis. The invention discovers that PFKP is highly expressed in kidney tissues of patients with chronic kidney diseases for the first time, and induces the over-expression or the under-expression of PFKP of unilateral ureter occlusion mice through adeno-associated virus vectors, and the result shows that PFKP has the effect of regulating the degree of kidney fibrosis, the over-expression of PFKP in PTECs aggravates TGF-beta induced glycolysis and kidney fibrosis, and the down-regulation of PFKP weakens the glycolysis and the kidney fibrosis of the PTECs. PFKP inhibitors can inhibit glycolysis and renal fibrosis, and can be used for preparing medicines for treating chronic kidney disease or renal fibrosis.

Description

Application of PFKP as chronic kidney disease treatment target and inhibitor thereof
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to application of PFKP as a target point for treating chronic kidney diseases and an inhibitor thereof.
Background
The incidence and prevalence of Chronic Kidney Disease (CKD) is increasing annually worldwide. Diabetic nephropathy (DKD) is the most common cause of CKD, and is also the main cause of End Stage Renal Disease (ESRD), which severely threatens the life of the patient. Despite the many achievements achieved in CKD treatment, the treatment options for CKD remain limited and continue to be unsatisfactory using conventional methods. Renal fibrosis, including glomerulosclerosis and tubular interstitial fibrosis, is the major pathological change and common outcome for many CKDs. The tubular mesenchyme accounts for more than 90% of kidney parenchyma and has a plurality of important functions. Emerging evidence suggests that tubular injury occurs earlier than glomerular injury, which plays an important role in the progression of CKD. Furthermore, the development of tubular interstitial fibrosis is a key predictor of CKD progression to ESRD. CKD mediated tubular injury is a complex mechanism involving metabolic abnormalities, hemodynamic effects, and inflammatory responses.
Kidneys are one of the highest metabolic rate organs, driven mainly by Tubular Epithelial Cells (TECs). Kidneys need to maintain energy homeostasis, and abnormal energy metabolism can lead to cell dysfunction, cell death, and a variety of kidney diseases. Under physiological conditions, tubular Epithelial Cells (TECs) almost completely rely on mitochondrial Fatty Acid Oxidation (FAO) to produce energy, consuming large amounts of molecular oxygen. Following kidney injury, FAO capacity of Proximal Tubular Epithelial Cells (PTECs) is impaired, leading to reprogramming of cell metabolism to provide energy. Metabolic reprogramming, glycolysis from mitochondrial FAO to PETCs, is thought to play an important role in the progression of CKD. In diabetics, hypoxia, glycolysis and lipid accumulation of the tubules occurs due to alterations in metabolic substrates and oxygen transfer, leading to increased production of Reactive Oxygen Species (ROS), pro-inflammatory factors and pro-fibrotic factors, increased apoptosis of PTECs cells and increased renal fibrosis. Thus, improving energy metabolism is a new strategy for preventing and treating chronic kidney disease.
Disclosure of Invention
The technical problem to be solved by the invention is to provide the application of PFKP gene or PFKP protein as a target spot in preventing, improving or treating chronic kidney disease or kidney fibrosis, and/or to provide the application of PFKP inhibitor in preventing, improving or treating chronic kidney disease or kidney fibrosis. The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.
In order to solve the technical problems, the invention firstly provides any one of the following applications of PFKP genes or PFKP proteins:
a1 The use of the composition for controlling the degree of renal fibrosis;
a2 As a target in the preparation of a product for preventing, ameliorating or treating chronic kidney disease;
a3 As a target in the preparation of a product for preventing, ameliorating or treating renal fibrosis;
a4 As a target in the preparation of a product for inhibiting the occurrence and/or progression of renal fibrosis;
a5 As a target in the preparation of products for inhibiting glycolysis.
The modulation may be promotion or inhibition.
The PFKP gene encodes a PFKP protein, which may be human in origin, as well as the encoded protein thereof. The nucleotide sequence of the PFKP gene is GenBank Accession No. NM_002627.5 at position 3067548-3136802 (Update Date: feb 3, 2022), and the amino acid sequence of the PFKP protein is NCBI Reference Sequence: 3067596-3136579 (Update Date 18-Jul-2023) of NM_002627. The invention also provides any one of the following applications of the PFKP inhibitor:
B1 The use of a composition for the preparation of a product for the prevention, amelioration or treatment of chronic kidney disease;
b2 Use of a composition for the preparation of a product for the prevention, amelioration or treatment of renal fibrosis;
b3 Use of a composition for inhibiting the occurrence and/or progression of renal fibrosis;
b4 To a process for the preparation of a product for inhibiting glycolysis.
The PFKP inhibitor may have at least any one of the following effects:
d1 Inhibiting or reducing the expression or activity of PFKP gene;
d2 Inhibiting or reducing transcription of PFKP gene into mRNA;
d3 Inhibiting or reducing PFKP gene translation into a protein;
d4 Inhibit or reduce the activity or function of PFKP proteins.
In the above applications, the PFKP inhibitor may be a substance that inhibits PFKP gene expression, silences or knocks out PFKP gene, and/or a substance that reduces PFKP protein content and/or activity.
Further, the inhibition of PFKP gene expression, silencing or knocking out PFKP gene may be achieved by gene mutation, gene silencing, gene knockout, gene editing or gene knockdown techniques well known to those skilled in the art. Specific knockdown or shut down of expression of specific genes, for example, using RNA interference (RNAi) technology; the tool utilizing the gene editing technology may be, but is not limited to, CRISPR/Cas9 technology, zinc Finger Nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) technology, and the like.
Techniques for inactivating PFKP gene expression or silencing gene expression from the post-transcriptional or translational level using gene knock-down techniques are well known to those skilled in the art. Such gene knockdown techniques include, but are not limited to, RNA interference, morpholino interference, antisense nucleic acids, ribozymes, or dominant negative mutations.
Silencing of PFKP genes by inhibiting expression of PFKP genes using shRNA or siRNA expressed by viruses (e.g., lentiviruses, adeno-associated viruses) is well known to those skilled in the art.
In the above application, the substance may be one or more of a nucleic acid molecule, a carbohydrate, a lipid, a small molecule compound, an antibody, a polypeptide, a protein, a gene editing vector, a lentivirus, or an adeno-associated virus.
In the above applications, the nucleic acid molecules may comprise shRNA, microRNA, siRNA and/or antisense oligonucleotides.
Further, the shRNA (short hairpin RNA), microRNA (micro RNA), siRNA (small interfering RNA) and/or antisense oligonucleotide (e.g., antisense RNA) are used to inhibit expression of PFKP genes.
In the application, the target sequence of the shRNA can be shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3. The shRNA targets to interfere with the expression of PFKP genes.
In the above application, the small molecule compound may be isorhamnetin.
Further, the antibody may be an antibody against PFKP protein or a functional fragment thereof.
Further, the lentivirus or adeno-associated virus may be a recombinant lentivirus or recombinant adeno-associated virus expressing shRNA for knocking down PFKP gene.
Further, the lentivirus or adeno-associated virus may be a recombinant lentivirus or recombinant adeno-associated virus expressing the shRNA.
In one embodiment of the invention, the recombinant lentivirus is obtained by cloning and constructing a coding DNA molecule of interfering shRNA on a lentivirus vector AAV9-Ggt, and packaging the obtained lentivirus by using a lentivirus packaging system.
The shRNA, DNA molecule encoding the shRNA, or lentivirus or adeno-associated virus comprising the DNA molecule are also within the scope of the invention.
The present invention also provides pharmaceutical compositions, which may comprise any of the PFKP inhibitors described herein, which may have at least any of the following uses:
c1 Preventing, ameliorating or treating chronic kidney disease;
c2 Preventing, ameliorating or treating renal fibrosis;
C3 Inhibiting the occurrence and/or progression of renal fibrosis;
c4 Inhibition of glycolysis.
The active ingredient of the pharmaceutical composition may include a PFKP inhibitor as described herein.
Further, the pharmaceutical composition may also include one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier may be a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant, or lubricant.
The invention also provides the use of PFKP gene or PFKP protein in screening candidate drugs for the treatment of chronic kidney disease or kidney fibrosis, the method of screening may comprise: the PFKP is used as target to screen medicine or reagent to reduce PFKP gene expression level or PFKP protein content or activity as candidate medicine for treating chronic kidney disease or kidney fibrosis.
The invention also provides application of the PFKP gene or PFKP protein as a target for preventing or treating chronic kidney disease or kidney fibrosis.
The invention also provides the use of any one of the following PFKP inhibitors (such as interfering shRNA or isorhamnetin with target sequences of SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3):
E1 Inhibiting the expression of ECM proteins (COL 1A1, COL3A1 and CTGF) caused by renal fibrosis;
e2 Reduced expression water of Tgf- β1, col1α1, col1α2, col3α1, tip 1, mp9, fn1, serpin 1, α -SMA mRNA in kidney tissue caused by kidney fibrosis;
e3 Reducing the expression level of glycolytic related genes (e.g., phospho-LDHA, HIF-1α, HEK2, and phospho-PKM 2) caused by renal fibrosis;
e4 Reduced elevation of lactate concentration in Proximal Tubular Epithelial Cells (PTECs) caused by renal fibrosis.
Isorhamnetin (ISO) as described herein has a formula C 16 H 12 O 7 CAS number: 480-19-3, the structure of which is shown as a in figure 7.
The products described herein may be reagents, medicaments or pharmaceutical compositions.
The inhibition of glycolysis described herein may be inhibition of TGF-beta induced glycolysis.
The renal fibrosis described herein can be renal interstitial fibrosis.
Further, the renal interstitial fibrosis may be renal interstitial fibrosis resulting from unilateral ureteral obstruction.
The energy reprogramming of glycolysis is closely related to the development of chronic kidney disease. Although phosphofructokinase 1 (PFK) is reported to be a rate-limiting enzyme in glycolysis, the role of the platelet isomer of PFK (PFKP) in the development and progression of renal fibrosis is not yet known. The present invention investigated whether PFKP can mediate progression of renal interstitial fibrosis by modulating glycolysis of Proximal Tubular Epithelial Cells (PTECs). The over-expression or under-expression of the renal tubular PFKP of the unilateral ureteral occlusion mouse is induced by an adeno-associated virus vector (AAV), and the result shows that the over-expression of the proximal tubular-specific PFKP promotes tubular expansion, interstitial fibrosis area and renal glycolysis, and the down-regulation of the PFKP inhibits the tubular expansion, the interstitial fibrosis area and the renal glycolysis. Furthermore, down-regulation of PFKP inhibited PFKP expression, whereas over-expression of PFKP promoted TGF- β1-induced glycolysis of human kidney epithelial cell line HK 2. The Chip-qPCR results showed that TGF-. Beta.1 recruits the SMAD3/SP1 complex in the PFKP promoter, thereby enhancing PFKP expression. Treatment of mice with Isorhamnetin (ISO) (isorhamnetin) significantly improved increased ptec glycolysis and renal fibrosis. Thus, the results of the study demonstrate that PFKP leads to progression of renal interstitial fibrosis by modulating the glycolysis Jie Jie in PTECs.
The invention discovers that PFKP is highly expressed in kidney tissues of patients with Chronic Kidney Disease (CKD) for the first time, and can be used as a target for further researching the application of PFKP genes and encoding proteins thereof in preventing, improving or treating chronic kidney disease or kidney fibrosis. The invention adopts an in-situ injection method, utilizes adeno-associated virus vector (AAV) to induce PFKP to be over-expressed in a mouse body, and utilizes adenovirus-associated virus expressing shRNA to knock down PFKP in the mouse body, and results show that PFKP has the effect of regulating and controlling renal fibrosis degree, PFKP over-expression in PTECs aggravates TGF-beta induced glycolysis and renal fibrosis, and PFKP down-regulation weakens glycolysis and renal fibrosis of PTECs.
In conclusion, the PFKP (PFKP gene or PPFKP protein) of the present invention has the effect of regulating the degree of renal fibrosis. The PFKP inhibitor developed by the present invention can inhibit the expression of ECM proteins (COL 1A1, COL3A1 and CTGF) caused by renal fibrosis, reduce the elevation of lactic acid concentration in renal tissue caused by renal fibrosis, tgf-beta 1, col1 alpha 2, col3 alpha 1, tip 1, mmp9, fn1, serpin 1, alpha-SMA mRNA expression water, reduce the expression level of glycolysis-related genes (such as phospho-LDHA, HIF-1 alpha, HEK2 and phospho-PKM 2) caused by renal fibrosis, reduce the elevation of lactic acid concentration in Proximal Tubular Epithelial Cells (PTECs) caused by renal fibrosis, and further inhibit glycolysis and renal fibrosis, and can be used for preparing products for preventing, improving or treating chronic kidney diseases or renal fibrosis or products for inhibiting the occurrence and/or progress of renal fibrosis. The PFKP gene or PFKP protein as a target point can be used for screening candidate medicines for treating chronic kidney diseases or kidney fibrosis, developing new treatment methods and treatment medicines for the chronic kidney diseases or kidney fibrosis, and has wide clinical application value for preventing and treating the chronic kidney diseases or kidney fibrosis.
Drawings
Figure 1 shows that PFKP was significantly up-regulated in human and mouse fibrotic kidneys in example 1. A: re-analysis of microarray data of kidney biopsy specimens (GSE 66494) from CKD patients showed expression of PFKP and FN1, and correlation between PFKP and FN1 in kidney tissue. B: re-analysis of microarray data of kidney biopsy specimens (GSE 30122) from DKD patients showed PFKP and FN1 expression levels in the kidney tubules. C: correlation between PFKP and FN1, PFKP and evfr (GSE 30122). D: the Ju CKD tunets database covering multiple types of CKD shows the expression of PFKP and the correlation of PFKP with eGFR in the renal tubules. E: compared to sham-operated groups, UUO group mice kidney tissue PFKP and extracellular matrix (ECM) protein (FN 1 (Abcam, cat.#ab 209780), COL1A1 (Abcam, cat.#sc-59772), COL3A1 (Abcam, cat.#sc-271249)) levels. F: the quantitative result of panel E is displayed, n=3 (β -actin is used as load control). G: PFKP staining and Sirius red staining were performed immunohistochemistry in kidney tissues of the comparative UUO group and sham group. H: immunohistochemical staining (upper panel) and Sirius red staining (lower panel) of PFKP were quantified in 3 kidney sections at magnification x 100. Data are expressed as mean ± SD; n=6. * p <0.05, < p <0.01, < p <0.001.
FIG. 2 is a graph showing that PFKP overexpression aggravates UUO mouse kidney fibrosis in example 2. A: immunoblots showed protein levels of PFKP and ECM proteins (COL 1A1, COL3A1 and CTGF) in UUO and sham mice. Immunoblots showed protein levels of the epithelial marker E-cadherein (wuhan's eagle, cat.# 20874-1-AP) and the mesenchymal marker α -SMA (Abcam, cat.# ab 7817), n=6. B: the quantitative result of panel a is displayed, n=6 (β -actin is used as load control). C: UUO group mice kidney tissue Tgf- β1, col1 α1, col1 α2, col3 α1, tip 1, mp9, fn1, serpin 1 mRNA expression levels were compared to sham-operated groups, n=6. D: (left panel) Masson trichromatic staining and Sirius red staining were quantified in 3 kidney sections at magnification x 100, n=6. (right panel) immunofluorescent staining of E-cadherein and immunohistochemical staining of α -SMA were quantified in 3 kidney sections at magnification x 100, n=6. E: mRNA expression levels of TGF- β1, col1α1, col1α2, col3α 1,Tmip 1,Mmp9,Fn1 and serpin 1, n=6 in kidney tissues of UUO mice and sham operated groups. F: immunofluorescent staining of E-cadherein and immunohistochemical staining of alpha-SMA in the kidneys of mice, 3 fields of magnification of 200X per mouse. Data are expressed as mean±sd; * p <0.05, < p <0.01, < p <0.001.
FIG. 3 shows that PFKP reduction in example 3 reduces kidney fibrosis in UUO mice. A: immunoblots showed protein levels of PFKP and ECM proteins (COL 1A1, COL3A1 and CTGF) in UUO and sham mice. Immunoblots showed protein levels of the epithelial marker E-cadherin and the mesenchymal marker alpha-SMA. B: the quantitative result of panel a is displayed, n=6 (β -actin is used as load control). C: masson staining and Sirius red staining showed severity of kidney fibrosis in mice in UUO groups and sham groups. D: (left panel) Masson trichromatic staining and Sirius red staining were quantified in 3 kidney sections at magnification x 100, n=6. (right panel) immunofluorescent staining of E-cadherein and immunohistochemical staining of α -SMA were quantified in 3 kidney sections at magnification x 100, n=6. E: UUO group mice kidney tissue Tgf- β1, col1 α1, col1 α2, col3 α1, tip 1, mp9, fn1, serpin 1 mRNA expression levels were compared to sham-operated groups, n=6. F: immunofluorescent staining of E-cadherein and immunohistochemical staining of alpha-SMA in the kidneys of mice, 3 fields of magnification of 200X per mouse. Data are expressed as mean ± SD; * p <0.05, < p <0.01, < p <0.001.
FIG. 4 is a graph of PFKP regulation of renal glycolysis in example 4. A, C: immunoblots showed expression levels of phosphorylated LDHA, HIF-1α, HEK2 and phosphorylated PKM2 in UUO mice and sham mice. The right panel shows the quantitative results of immunoblotting, n=6 (β -actin was used as loading control). B: immunohistochemical staining showed expression levels of phosphorylated LDHA, HIF-1 a and HEK2 in UUO mice and sham surgery groups. D: immunohistochemical staining of phosphorylated LDHA, HIF-1 a and HEK2 was quantified in 3 kidney sections at magnification x 100, n=6. E: lactic acid concentration in tubular cells in UUO mice and sham groups, n=6. Data are expressed as mean ± SD; * p <0.05, < p <0.01, < p <0.001.
FIG. 5 shows that PFKP of example 5 plays an important role in TGF- β1-induced glycolysis of kidney TE cells. A: PFKP overexpression in the absence or presence of TGF-beta 1Lactic acid production by HK2 cells, n=5. Immunoblots showed PFKP protein levels. B-C: WT, PFKP over-expression (OE) and PFKP Knockdown (KD) cells were inoculated into hippocampal XF-24 cell culture microplates, respectively. Cells were allowed to stand overnight in 0.5% FBSDMEM and then treated with or without 10ng/ml TGF- β1 for 24h. All cells were incubated in glycolytic stress test medium without glucose and pyruvate, followed by sequential use of glucose (10 mM), oligomycin (5. Mu.g/ml), 2-deoxyglucose (2-DG; 50 mM). Real-time extracellular acidification rate (ECAR) was recorded as baseline (pre-glucose), glycolytic rate (post-glucose), glycolytic capacity (post-hypomycin) and glycolytic reserves; n=9. D: PTEC isolated from mice treated as shown in the figures. Immunoblots showed protein levels of PFKP, phosphorylated LDHA, LDHA, HIF-1 a, HEK2 and β -actin, right panels showed quantitative results of immunoblots, n=3 (β -actin was used as loading control). Data are expressed as mean ± SD; * P is p<0.05，**p<0.01，***p<0.001。
FIG. 6 is a graph showing that TGF-beta stimulation up-regulates PFKP expression in example 6. A: the Chip-seq database from Cistrome Data Browser was re-analyzed. B: immunoblots showed protein levels of PFKP in TGF-. Beta.1 (2 ng/mL) treated HK2 cells (upper panel). The bottom right panel shows the quantitative results of immunoblots, n=3 (β -actin was used as load control). The lower left panel shows the mRNA expression levels of PFKP in TGF- β1 (2 ng/mL) treated HK2 cells. C: SMAD3siRNA and SP1 siRNA alone or in combination with HK2 cells under TGF- β1 stimulation, PFKP mRNA levels were detected using qPCR, n=5. D: SMAD3siRNA, SP1 siRNA and TGF- β1 treated HK2 cells as shown. Immunoblot detection showed SMAD3 protein levels, SMAD3 phosphorylation levels, SP1 and PFKP protein levels in HK2 cells. E: a series of truncated PFKP promoters fused to luciferase reporter were co-transfected with Renilla plasmid into HEK293T cells, with or without TGF- β treatment, n=3. F: HK2 cells were incubated with TGF- β for 24h, DNA fragments containing the SP1 side region on PFKP promoter were immunoprecipitated with anti-SMAD3 or anti-SP1, and PCR amplification was performed, as shown in the following figures, n=5. G: nucleotide sequence of PFKP promoter-543 to-530 fragment. Predicted SP1 binding sites (SP 1), SBE and mutation sites (SBEm and Sp1 m) are all shown in the figure (left panel). Sp1 sites and SBEs in the PFKP promoter fused to a luciferase reporter gene were mutated either alone or in combination, co-transfected with the Renilla plasmid into HEK293T cells, followed by TGF- β stimulation (n=3).
FIG. 7 is a graph showing that isorhamnetin inhibits glycolysis of renal TECs by inhibiting TGF-beta-induced PFKP expression in example 7. A: chemical structure of Isorhamnetin (ISO). B: protein levels of PFKP, p-LDHA, LDHA, HIF-1α, HEK2 and β -actin after ISO treatment, with or without TGF- β1, and quantitative results of immunoblots are shown in the right panel, n=3 (β -actin is used as load control). C: lactic acid production of HK2 cells after ISO treatment in the absence or presence of TGF- β1, n=5. D. E: HK2 cells were inoculated into hippocampal XF-24 cell culture microplates with ISO or blank treatment, respectively. Cells were allowed to stand overnight in 0.5% FBSDMEM and then treated with or without 10ng/ml TGF- β1 for 24h. All cells were incubated in glycolytic stress test medium without glucose and pyruvate, followed by sequential use of glucose (10 mM), oligomycin (5. Mu.g/ml), 2-deoxyglucose (2-DG; 50 mM). Real-time extracellular acidification rate (ECAR) was recorded as baseline (pre-glucose), glycolytic rate (post-glucose), glycolytic capacity (post-hypomycin) and glycolytic reserves; n=9. Data are expressed as mean ± SD; * p <0.05, < p <0.01, < p <0.001.
FIG. 8 is a graph of ISO reducing UUO mouse kidney fibrosis and glycolysis in example 7. A: masson staining and Sirius red staining showed severity of kidney fibrosis in the UUO mice group and sham operated group with or without ISO treatment, 3 fields were observed under 200 x magnification per mouse, n=6. B: quantitative analysis under 100-fold magnification was performed on kidney sections of each mouse on Masson trichromatic staining and Sirius red staining, n=6. C: UUO mice and sham mice were treated with ISO or not, mRNA expression levels of Tgf- β1, col1 α1, col3 α1, fn1, α -SMA and serpin 1 in kidney tissue, n=6. D: immunoblots showed expression of glycolytic related genes (e.g., phosphorylating-LDHA, HIF-1 a, HEK2, and PDK 4) in kidney tissue in UUO mice and sham with or without ISO treatment. E: quantitative results of immunoblots of fig. D are shown, n=3 (β -actin was used as loading control). F: immunohistochemical staining for HIF-1 a and PFKP, ISO treatment or not, 3 fields were observed under 200 x magnification per mouse, n=6. G: quantitative analysis under 100-fold magnifying glass was performed for immunohistochemical staining detection of HIF-1 a and PFKP, n=6. H: lactic acid production in tubular epithelial cells of UUO group and sham group, ISO treated or not, n=6. Data are expressed as mean ± SD; * p <0.05, < p <0.01, < p <0.001.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The animal experiments in the following examples were approved by the animal ethics committee of Shenzhen people Hospital (Shenzhen, china) and met with the guidelines for nursing and use of laboratory animals published by the national institutes of health (NIH publication, eighth edition, 2011). Male C57BL/6 mice were purchased from university of Nanjing animal center (Nanjing, jiangsu, china) and placed in a temperature and humidity controlled environment to obtain food and drinking water freely. Mice were acclimatized 7 days prior to the experiment.
Materials and methods:
healthy male mice (C57 BL/6) at 8 weeks of age were randomly divided into 4 groups: sham group (Sham operation group), PFKP over-expression (PFKP OE/AVV-shPFKP group, UUO+AVV-Ctrl group, UUO+PFKP OE/AVV-shPFKP group. For surgery, mice were subjected to UUO surgery or sham surgery. The anesthetized mice were intraperitoneally injected with 2% sodium pentobarbital (4 ml/kg). In the UUO group, surgery is performed on one abdomen. The surgical procedure involves making two incisions, one through the skin and the other through the peritoneum, exposing the kidneys. The left ureter was then ligated twice with surgical silk and cut between the two ligations. The ligated kidneys were replaced and supplemented with sterile saline. The incision was sutured and mice were kept individually. False surgery is also performed in a similar manner, but without ureteral ligation. CO is adopted at different time points after operation 2 Mice were euthanized by inhalation, and kidney tissue was taken after PBS infusion for pathological examination, immunohistochemistry, and the like. The Unilateral Ureteral Obstruction (UUO) model constructed as described above (hereinafter also referred to as UUO mouse model) is used to induce renal fibrosis, and UUO is mainly characterized by tubular injury due to obstruction of urinary flow, resulting in oxidative stress, inflammation, and renal fibrosis.
Immunohistochemical staining:
the kidney specimens of mice were fixed with 4% paraformaldehyde and paraffin embedded. Sections with a thickness of 5 μm were prepared and antigen retrieval and peroxidase removal were performed. Subsequently, blocking was performed with 5% goat serum, and incubated overnight at-4 ℃ with PFKP, α -SMA (cat.#ab 7817), phospho-S6 (p-S6) (Abcam, cat.#ab 52903), HIF-1α (Abcam, cat.#ab 179483) and HK2 antibodies. Sections were then incubated with secondary antibodies and DAB stained (metasequoia gold bridge, cat.#pv-6000) followed by hematoxylin counterstain.
Cell culture:
human kidney proximal tubule epithelial cell line cells (HK 2 cells) were purchased from the American ATCC (ATCC number: CRL-2190). Cells were cultured in MEM medium containing 10% Fetal Bovine Serum (FBS), 100U/mL penicillin and 100. Mu.g/mL streptomycin at 37℃in an incubator containing 5% carbon dioxide and 95% air.
Histological staining:
mouse kidney tissue was embedded in paraffin, sections were prepared, and Masson staining and sirius red staining were performed with the kit. Stained areas were quantified using ImageJ software.
Western blotting:
the quick frozen tissue or cells are lysed with RIPA buffer containing phosphatase and protease inhibitors, and the protein is extracted from kidney tissue or HK-2 cells. After centrifugation, the supernatant is collected, electrophoresed on a sodium dodecyl sulfate-polyacrylamide electrophoresis gel of 8% -15% (depending on the target protein), and the protein is then transferred onto a polyvinylidene fluoride (PVDF) membrane by electrotransfer. PVDF membranes were blocked with 5% milk and incubated overnight with primary antibody. The next day, PVDF membrane was incubated with the corresponding secondary antibody and detected by chemiluminescence. The density of the immunoreactive bands was analyzed using ImageJ software.
The anti-bacterial agent comprises the following components: antibodies to PFKP (catalog No. ab 119796), CTGF (catalog No. ab 209780), HIF-1 a (catalog No. ab 179483), E-cadherein (catalog No. ab 231303), a-SMA (catalog No. ab 7817), hexokinease 2 (HEK 2) (catalog No. ab 209847), SMAD3 (catalog No. ab 8477), p-SMAD3 (catalog No. ab 52903), SP1 (catalog No. ab 227383), PDK4 (catalog No. ab 214938), β -actin were all purchased from Abcam (cambridge, uk). Antibodies to FN1 (catalog number 63779S), phospho-PKM 2 (p-PKM 2) (Tyr 105) (catalog number 3827S), PKM2 (catalog number 4053) and LDHA (catalog number 2012S) were all purchased from Cell Signaling Biotechnology (massachusetts, usa) as anti-mouse or rabbit IgG. Antibodies to COL1A1 (catalog No. sc-59772) and COL3A1 (catalog No. sc-271249) were from Santa Cruz Biotechnology (Shanghai) Inc. phosphorylating-LDHA (Tyr 10) (catalog number PA 5-105445) and FBS (fetal bovine serum) were both purchased from Invitrogen Life Technologies (Caliper, calif. USA). Etc. The secondary antibody is goat anti-rabbit or goat anti-mouse antibody.
Immunofluorescence:
the kidney specimens of mice were fixed with 4% paraformaldehyde and paraffin embedded. Sections 5 μm thick were deparaffinized from paraffin-embedded tissue. Antigen retrieval was performed by incubation in Target retrieval Solution buffer for 15 min at 95 ℃. The sections were then incubated with E-cadherein antibody overnight at 4 ℃. After washing, nuclei were counterstained with Alexa 488 fluorescence secondary antibody, DAPI. The stained sections were observed using a confocal microscope (Laica, weztlar, germany) and stained areas were quantified using ImageJ software.
RNA extraction and qPCR of cell samples:
total RNA was extracted from kidney tissue or cell samples using TRIzol according to standard procedures. Using PrimeScript TM The RT kit (Takala, cat. # RR047A) reverse transcribes RNA into cDNA. Use of TB on a LightCycler 480 InstrumentPremix Ex Taq TM II Mix was qPCR. The primer sequences are listed in Table 1 below. The data were analyzed using ΔΔct-method, normalized to angiogenin in kidney samples or GAPDH in cell samples as an internal reference.
The data in the examples below are all from at least three independent experiments. Each value is expressed as mean ± SD. All raw data initially obeyed normal distribution, single sample Kolmogorov-Smirnov non-parametric test analysis using SPSS22.0 software. For animal and cell experiments, two-tailed unpaired student t-test was used to compare the two groups. More than two groups were compared using a single-factor anova with Bonferroni post hoc test. Correlation coefficients were calculated using the Spearman correlation test. To avoid bias, all statistical analyses were performed using blind methods. * P <0.05, < P <0.01, < P <0.001.
Example 1, significant upregulation of PFKP in human and mouse fibrotic kidneys
Analysis of microarray data (derived from GEO, data set number: GSE 66494) of kidney biopsy specimens from CKD patients showed significantly elevated expression levels of PFKP (P < 0.001) and FN1 (a fibrotic gene) in kidney samples from CKD patients compared to healthy controls (a in fig. 1). There is a strong positive correlation between PFKP and FN1 (a in fig. 1). Furthermore, reanalysis of microarray data (derived from GEO, data set number: GSE 30122) obtained from kidney biopsy specimens of DKD patients showed the same result; the expression levels of PFKP and FN1 in the renal tubules were significantly elevated (B in fig. 1). PFKP exhibits a very significant positive correlation with FN 1; however, there is a negative correlation between PFKP and gfr (C in fig. 1). The Ju CKD tunent database (20) covers multiple types of CKD, and also shows that PFKP and evfr are inversely correlated in the tubular (D in fig. 1).
To investigate the potential relationship of PFKP in the pathogenesis of renal fibrosis, we first determined the protein expression levels of PFKP in Unilateral Ureteral Obstruction (UUO) -induced fibrotic mouse kidney tissue. As expected, immunoblots showed that the protein expression levels of PFKP and extracellular matrix (ECM) proteins (FN 1, COL1A1 and COL3 A1) were significantly up-regulated in the kidney tissue of UUO group compared to sham group (E, F in fig. 1). Immunohistochemistry and sirius red staining showed a significant increase in PFKP staining intensity of UUO group kidney tissue (G, H in fig. 1). Overall, the up-regulation of PFKP expression and its positive correlation with the fibrotic ECM protein gene observed in the kidney of fibrosed human or mouse suggests that PFKP may play a role in the pathogenesis of renal fibrosis.
Example 2 overexpression of PFKP aggravates UUO mouse model renal fibrosis
This example uses adeno-associated viral vectors (AAV) to induce kidney tubular PFKP overexpression in unilateral ureteral occlusion mice (UUO mice). To induce over-expression of PFKP in mice, PFKP was introduced into the kidneys using a type 9 serum adeno-associated virus (AAV 9) vector by in situ injection. The method comprises the following specific steps:
1. over-expression lentivirus construction
The Pfkp gene (GenBank Accession No. NM-002627.5, position 3067548-3136802 (Update Date: feb 3, 2022)) was cloned and constructed on the lentiviral expression vector AAV9-Ggt (product of Shanghai Ji Kai company) and packaged into lentiviruses (PFKP-overexpressing lentiviruses) using a lentivirus packaging system. In addition, empty vector virus expression vector plasmid is selected as a control, and is packaged into lentivirus by using a lentivirus packaging system for subsequent control experiments. The constructed lentivirus over-expressing PFKP was named AAV9-Ggt-PFKP, and the control lentivirus was named AAV9-Ggt-gfp. The constructed lentivirus AAV9-Ggt-PFKP is a recombinant virus which takes AAV9 as a vector and drives the PFKP gene to be overexpressed in tubular epithelial cells by a proximal tubular specific promoter Ggt.
2. In situ injection
The virus was diluted to 1X 10 with 200. Mu.L of physiological saline (0.15 mol/L NaCl) 12 vg/mL. Each group had 6C 57BL/6J mice. The back of the mouse is dehaired, the prone position is positioned on an operation table and the operation area is disinfected, the skin is cut at the position 1cm beside the left side of the spine of the back and 2cm below the ribs, yellow adipose tissue is visible, fascia is cut along the middle of the yellow adipose, fat is pulled out, the kidney is visible, the body position of the mouse is adjusted to be in the right lateral position, the kidney is extruded and the adipose tissue around the kidney is stripped, a faint yellow transparent/white small point is visible, the right side of the kidney is pressed downwards, a 30G injector is kept in the horizontal position to enter the renal pelvis, 100 μl of solution is injected in 3s, and the injector is pulled out after 10s of rest. Antibiotic is dripped, and the operation incision is sutured and disinfected.
The detection results are shown in FIG. 2. The AAV9-Ggt gene was used to introduce the Pfkp gene into the kidneys of WT mice and AAV9-Ggt-gfp was transfected as a control. After AAV transfection for 2 weeks, WT mice were subjected to UUO surgery. Immunoblots showed that Pfkp overexpression promoted expression of UUO mouse ECM proteins (COL 1A1, COL3A1 and CTGF) (A, B in fig. 2). Compared to sham-operated groups, the expression levels of Tgf- β1, col1α1, col1α2, col3α1, tmip1, mp9, fn1, serpin 1 mRNA were significantly increased in the kidney tissue of UUO group mice, and these mRNA expression levels were further increased after overexpression of Pfkp (E in fig. 2). Masson staining and Sirius red staining showed that kidney fibrosis was more pronounced in UUO mice groups and more severe after Pfkp overexpression (C, D in fig. 2) than in sham mice groups. Furthermore, immunoblots and immunohistochemistry showed that after Pfkp overexpression, the epithelial marker E-cadherein was inhibited and the mesenchymal marker α -SMA was upregulated, indicating that Pfkp induced PTECs epithelial-mesenchymal transition (EMT) (D, F in fig. 2). Taken together, these results indicate that Pfkp overexpression exacerbates kidney fibrosis in the UUO mouse model.
Example 3 knockdown of PFKP significantly reduces kidney fibrosis in UUO mouse models
In the embodiment, the application of taking PFKP as a target in reducing renal fibrosis is studied by knocking down PFKP by using adenovirus AAV9 expressing shRNA. To knock down PFKP in mice, shRNA (shPfkp) with PFKP specific for the specific tubule (AAV 9-Ggt) was introduced into wild-type (WT) mouse kidneys and AAV9-Ggt-gfp transfection was used as a control. The specific operation steps are as follows:
1. interfering lentiviral construction:
according to the 3067548-3136802 rd (Update Date: feb 3, 2022) sequence of mouse Pfkp gene (GenBank Accession No. NM-002627.5) and RNAi principle, cloning and constructing the encoding DNA molecule of the interfering shRNA and the encoding DNA molecule of the empty shRNA contrast to a lentivirus expression vector AAV9-Ggt, and packaging the lentivirus into lentiviruses by a lentivirus packaging system to respectively obtain a recombinant lentivirus for expressing the interfering shRNA and a recombinant lentivirus for expressing the empty shRNA.
Wherein, the target sequence of the interfering shRNA (shPfkp) is as follows:
5’-GTGGGTATGGTGGGCTCCATT-3’(SEQ ID No.1)，
5’-GATGTACAGAAGGCAATGGAT-3’(SEQ ID No.2)，
5’-TAGTATCAATGCCCTTCTGAT-3’；(SEQ ID No.3)。
the target sequence of the empty shRNA is: TTCTCCGAACGTGTCACGT.
2. In situ injection
The procedure is as in step 2 of example 2.
The detection results are shown in FIG. 3. shPfkp was introduced into WT mouse kidneys using AAV9-Ggt and AAV9-Ggt-gfp was transfected as a control. After AAV transfection for 2 weeks, mice were subjected to UUO surgery. Immunoblots showed that knockdown of Pfkp inhibited expression of UUO mouse ECM proteins (COL 1A1, COL3A1, and CTGF) (A, B in fig. 3). Furthermore, the expression levels of Tgf- β1, col1 α1, col1 α2, col3 α1, tip 1, mp9, fn1, serpin 1 mRNA were significantly increased in the kidney tissue of UUO group mice compared to sham group, but these mRNA expression levels were significantly decreased after knocking down Pfkp (fig. 3E). Masson staining and Sirius red staining showed that kidney fibrosis was more pronounced in UUO mice groups than in sham groups, and knocking down Pfkp significantly reduced the degree of kidney fibrosis (C, D in fig. 3). Furthermore, immunoblots and immunohistochemistry showed that the epithelial marker E-cadherein was upregulated and the interstitial marker α -SMA inhibited after knocking down Pfkp (D, F in fig. 3). Taken together, these results indicate that knocking down Pfkp reduces kidney fibrosis in the UUO mouse model.
Example 4 PFKP regulates renal glycolysis
1. Cell lactic acid assay and isolation of primary PT cells from mice
Lactate determination in cell or mouse PT cell samples was performed using an enzymatic lactate determination kit (Sigma-Aldrich, cat#MAK064). The method comprises the following steps: the sample was centrifuged at 10000g for 5 min at 4 ℃. The supernatant was collected and protein was removed with perchloric acid. The resulting supernatant and standard were added to 96-well microwell plates containing lactic acid assay mixtures. The lactic acid concentration was then calculated by incubating at 37℃for 30 minutes and then measuring the absorbance at 450nm using an instrument.
To isolate primary mouse PT cells, the protocols described in the literature (Jiang H, yamashita Y, nakamura A, croft K, ashida H. Quercetin and its metabolite isorhamnetin promote glucose uptake through different signalling pathways in myotubes. Scientific reports.2019;9 (1): 2690.) were followed. Kidneys were removed from mice and mechanically dissociated using GentleMACS cell dissociating agents. Cells were then isolated using anti-Promin-1 microbead binding antibodies and AutoMACS.
The experimental results are shown in FIG. 4. The energy reprogramming of glycolysis is closely related to the development of CKD. PFKP is the rate-limiting enzyme of glycolysis. Thus, the present invention investigated the role of PFKP in renal glycolysis regulation in vivo. Protein expression of key enzymes for glycolysis was analyzed in UUO groups and in sham surgery groups. Immunoblotting and immunohistochemical analysis showed that the expression of glycolysis-related genes, such as phospho-LDHA, HIF-1α, HEK2 and phospho-PKM2, was significantly increased in the kidney tissue of UUO group mice compared to sham group, and further increased after over-expression of PFKP (A, B, D in fig. 4). Conversely, PFKP knockdown showed the opposite result, with significantly reduced expression of glycolytic related genes. Furthermore, lactate concentration was significantly increased in PTE cells of UUO group, further increased after PFKP overexpression (C, D in fig. 4). However, after PFKP knockdown, the lactate concentration in UUO mouse PTE cells was significantly reduced (E in fig. 4).
Example 5 PFKP plays an important role in TGF-beta induced ptec glycolysis
1. To isolate primary proximal tubular cells in mice, we performed according to the methods described in the previous studies (Legouis D, ricksten S, faivre A, verissimo T, gariani K, verney C, galichon P, berchthold L, ferrille E, fernandez M, placer S, koppitch K, hertig A, martin P, naesens M, pugin J, mcMahon A, cipp. P, de Seigneux S.alternate proximal tubular cell glucose metabolism during acute kidney injury is associated with mole. Nature methodolism.2020; 2 (8): 732-743.). Kidneys were removed from mice and mechanically isolated using a GentleMACS cell dissociator (Miltenyl Biotec). Cells were then isolated using antibodies against Promin-1 bead binding and autoMACS (Miltenyl Biotec). After isolating cells and treating with 2ng/mL TGF-beta for 24 hours, cells were subjected to standard procedures using an enzymatic lactate assay kit (Sigma-Aldrich). Briefly, cell samples were centrifuged at 10,000g for 5 min at 4℃and the supernatant was collected and deproteinized using perchloric acid. The resulting supernatant and standard were added to 96-well microwell plates containing the lactic acid assay mixture. The plate was then incubated at 37℃for 30 minutes and absorbance was measured at 450nm using a microplate reader. The lactic acid concentration was calculated therefrom.
2. Extracellular acidification Rate (EACR)
To assess the activity of cellular glycolysis, extracellular acidification rate (ECAR) measurements were performed. ECAR is calculated from pH changes caused by proton release following lactate formation during glycolysis. ECAR values were obtained from real-time measurements by a Seahorse XF24 extracellular flux analyzer and then normalized to the protein content of each sample. Statistical analysis was performed using GraphPad Prism software and the results are expressed as mean ± Standard Deviation (SD). The significance of the differences between the groups was statistically significant using a two-tailed t-test, with p-values <0.05 as the differences.
TGF-beta signaling pathways play a critical role in fibrosis, particularly CKD renal fibrosis (Zhang Y, jin D, kang X, zhou R, sun Y, lian F, tong X.Signaling Pathways Involved in Diabetic Renal fibre, front Cell Dev biol.2021;9:696542.; meng XM, nikolic-Paterson DJ, lanHY.TGF-beta: the master regulator of fibre is. Nat Rev Nephrol.2016;12 (6): 325-338.). Srivastava et al demonstrated that TGF-. Beta.1 induces glycolysis in human PTECs. This example further analyzes whether PFKP is involved in the modulation of HK2 cell (human PTECs line) glycolytic activity by TGF- β. As a result, PFKP overexpression was found to significantly increase lactate production with or without TGF- β1 stimulation (a in fig. 5). ECAR measurements were further performed using a Seahorse X24 extracellular flux analyzer. Three cells (WT; PFKP over-expression, OE; and PFKP knockdown, KD) were incubated with TGF-. Beta.1 or without TGF-. Beta.1 in glycolytic stress test medium (without glucose and pyruvate). Next, the cells were continuously exposed to glucose, oligomycin and 2-deoxyglucose (2-DG; a glucose analog that inhibits glycolysis by competitive binding to HEK2, the first enzyme in the glycolysis pathway; FIG. 5B). Baseline ECAR, glycolytic rate, glycolytic capacity, and glycolytic reserves were determined. In the absence of TGF- β1 treatment, PFKP overexpression did not alter baseline ECAR, glycolytic rate, glycolytic capacity, or glycolytic reserves. PFKP knockdown significantly reduced glycolytic capacity and glycolytic reserves compared to WT cells (B, C in fig. 5). TGF- β1 significantly increased glycolysis, glycolytic capacity and glycolytic reserves in WT cells (B, C in fig. 5). The effect of TGF- β1 on ECAR was significantly enhanced by PFKP overexpression and attenuated by PFKP knockdown (B, C in fig. 5). Unlike the lactate assay, PFKP OE cells did not increase glycolysis in the absence of TGF- β1 stimulation (B, C in fig. 5). This is probably due to the fact that the basal medium used in the hippocampal experiments was free of glucose and pyruvate. Next, we wanted to verify the expression of glycolytic related genes in PTECs after kidney injury. Our results showed that expression of glycolytic related genes such as phosphorylated LDHA, HIF-1 a and HEK2 was significantly increased compared to isolated PTECs in Unilateral Ureteral Obstruction (UUO) mice, and further increased after Pfkp overexpression (fig. 5D). Taken together, PFKP is involved in TGF- β1 mediated upregulation of HK2 cell glycolytic capacity.
EXAMPLE 6 TGF- β1 upregulates the level of transcription of PFKP by recruiting the SMAD3-SP1 complex to the PFKP promoter
RNA extraction and real-time quantitative polymerase chain reaction (qPCR) of samples: total RNA was extracted from kidney tissue or cell samples using TRIzol (Invitrogen) following standard procedures. Using PrimeScript TM RT kit and gDNAEras (Takala, beijing, china; cat. # RR047A) reverse transcribe RNA into cDNA. Use of TB on the LightCycler 480 Instrument (Roche)Premix Ex Taq TM II Mix (Takala, cat. # RR820A) was subjected to qPCR. The primer sequences are listed in Table 1. The data were analyzed using the ΔΔct method with vinculin in kidney samples or GAPDH in cell samples as a standardized gene.
TABLE 1 list of primer sequences
Dual luciferase reporter assay:
cDNA is extracted from HEK293T cells of a human embryo kidney cell line, and the human PFKP gene is amplified by PCR. The promoter fragment of the target gene was cloned using PCR and inserted into pGL3 luciferase vector using primers in table 2. All constructs were verified using DNA sequencing analysis. And (5) performing double-luciferase reporter gene detection. HEK293T cells were transfected with the target gene promoter plasmid along with PFKP and Renilla luciferase. The firefly and mouse rabbit luciferase activities were quantitatively analyzed using a dual luciferase reporter system.
TABLE 2 primer sequence listing
Cistrome DB Toolkit factor binding, histone modification and chromatin accessibility can be looked up in any given genomic interval shorter than 2mb (Zheng R, wan C, mei S, qin Q, wu Q, sun H, chen CH, brown M, zhang X, meyer CA, liu XS. Cistrome Data Browser: expanded datasets and new tools for gene regulatory analysis.nucleic Acids Res.2019;47 (D1): D729-D735.). Thus, cistrome DB Toolkit was used to search for transcription factors that bind to the PFKP promoter, and SP1 was expected to bind directly to the PFKP promoter (a in fig. 6). It is well known that TGF-beta signaling regulates the expression of downstream genes by forming complexes between nuclear endosmad and dnas binding cofactors (e.g., SP 1) or transcriptional cofactors or cofactors (Shi Y, massagnus J. Mechanisms of TGF-beta signaling from cell membrane to the nucleic. Cell.2003;113 (6): 685-700.). In fact, TGF- β treatment increased PFKP mRNA and protein levels in HK2 cells (B in fig. 6). Furthermore, TGF- β induced up-regulation of PFKP was inhibited by siRNA SMAD3 and siRNA SP1 alone or in combination (C, D in fig. 6). A series of PFKP promoter deletion mutants were cloned into a luciferase reporter system (Luc 1-Luc4; FIG. 6E), and TGF- β stimulation was found to enhance PFKP activity of Luc1-3 structure, indicating that the smallest SMAD3-SP1 complex binding site within the PFKP promoter is between-3048 and-489 bp. Further ChIP analysis found that SMAD3 and SP1 were recruited to the-612 to-462 bp region of the PFKP promoter (F in fig. 6). Next, we define contributions of SMAD3 and SP1 binding elements to PFKP promoter-based transcription and TGF- β -induced transcription. Mutations were introduced to eliminate SMAD3 or SP1 binding (G in fig. 6). The results indicate that individual SBE mutations also inhibit TGF- β induced transcript levels, but have no significant effect on basal transcript levels. In addition, SP1 mutations reduced basal levels of transcription and TGF- β -induced transcription, whereas mutations at these two sites further reduced basal levels of transcription and abrogated TGF- β induction (G in FIG. 6). Overall, TGF- β1 increases the level of transcription of PFKP by recruiting SMAD3-SP1 complex to the PFKP promoter.
Example 7 use of PFKP inhibitors for reducing renal fibrosis and glycolysis
The PFKP inhibitor may be a substance that inhibits PFKP gene expression, silences or knocks out PFKP gene, or may be a substance that inhibits or reduces PFKP protein content and/or activity. In this example Isorhamnetin (ISO) was used as PFKP inhibitor to inhibit PFKP gene expression.
1. Isorhamnetin (ISO) inhibits glycolytic HK2 cells of kidney PTECs by inhibiting TGF-beta-induced PFKP expression, stood overnight in 0.5% FBSDMEM medium, then 10ng/ml TGF-beta was added for 1 hour, and protein and mRNA levels of PFKP were detected after ISO treatment.
The inventor has found through extensive and intensive studies that astragalus total flavonoids (total flavone of Astragalus membranaceus, TFA) can reduce atherosclerosis by dual inhibition of miR-33 and NF- κB pathways, and in part by inhibition of macrophage scavenger receptors. The main ingredients include calycosin, kaempferol, isoquercetin, isorhamnetin (ISO; FIG. 7A), formononetin, methylnisolone, ifolium and quercetin. Of these, only ISO inhibited TGF- β mediated up-regulation of PFKP (B in fig. 7). Mainly, ISO (C 16 H 12 O 7 CAS number: 480-19-3) is a flavone found in sea buckthorn and ginkgo fruits. It has a variety of biological activities including anti-inflammatory, anti-cancer, anti-oxidant and antibacterial activities. In addition, ISO has a protective effect on cardiovascular and neurodegenerative diseases. It also has pharmacodynamic effects against hyperuricemia, acute kidney injury and pulmonary fibrosis. The pharmacological actions of ISO are related to the regulation of NF-kappa B, PI K/AKT, MAPK and other signal pathways and downstream factors thereof. The HK2 cell lactic acid concentration was further examined. The TGF- β1 incubated HK2 cells had significantly elevated lactate concentration and ISO inhibited lactate concentration (C in fig. 7). In addition ECAR was used to evaluate the activity of cellular glycolysis. TGF-. Beta.1 treatment resulted in a significant increase in ECAR immediately, reflecting the enhancement of the acute glycolytic response of HK2 cells. However, EACR was significantly reduced after ISO treatment, reflecting reduced glycolytic response of HK2 cells (D, E in fig. 7). Overall, these results indicate that ISO inhibits glycolysis by inhibiting TGF- β induced PFKP expression, at least in part, in HK2 cells.
2. ISO reduces renal fibrosis and glycolysis in UUO mice
This example determines whether ISO intervention inhibited kidney fibrosis and elevated glycolysis in UUO mouse kidneys. The method comprises the following specific steps:
to investigate whether isorhamnetin (ISO; sigma-Aldrich, hamburg, germany) was able to alleviate kidney fibrosis in vivo, mice were randomly divided into five groups (n=6 per group), as follows: (i) control group, C57BL/6J mice received sham surgery; (ii) UUO group, C57BL/6J mice received UO surgery and only dimethyl sulfoxide treatment; (iii) UUO group, receiving 5mg/kg ISO treatment; (iv) UUO group, receiving 10mg/kg ISO treatment; (v) UFO group, receiving 30mg/kg ISO treatment. The basis for selecting an ISO dose of 10mg/kg was that the effect of ISO on GLUT4 levels in HFD-induced obese mouse models was studied in previous studies (Jiang H, yamashita Y, nakamura A, croft K, ashida H.Quercetin and its metabolite isorhamnetin promote glucose uptake through different signalling pathways in myotubes.scientific reports.2019;9 (1): 2690.). All treatments were taken orally once per day. After 7 days of treatment with CO 2 The chamber sacrifices the mice and kidneys were collected for ex vivo analysis.
The results are shown in FIG. 8. Masson staining and Sirius red staining showed that kidney fibrosis was more pronounced in UUO mice than in sham mice and that the degree of kidney fibrosis was significantly reduced after ISO treatment (A, B in fig. 8). In contrast, UFO mice Tgf-. Beta.1, col1a1, col3a1, fn1, α -SMA and Serpin 1 mRNA levels were elevated compared to sham mice, and this upregulation was inhibited following ISO intervention (FIG. 8C). Immunoblot analysis showed that the expression of glycolytic related genes like phospho-LDHA, HIF-1α, HEK2 and PDK4 was significantly increased in the kidney tissue of UUO mice compared to sham mice, further suppressed after ISO treatment (D, E in fig. 8). Notably, immunohistochemistry showed a significant increase in the renal HIF-1α and PFKP deposition in the UUO group compared to the control group, and improved after ISO treatment (F, G in fig. 8). Furthermore, in agreement with the in vitro study results, lactate concentration was also inhibited by ISO intervention (H in fig. 8). Taken together, the results indicate that ISO can inhibit PFKP expression, reduce glycolysis and renal fibrosis in UUO mice (fig. 7 and 8), i.e., that ISO can reduce renal fibrosis and glycolysis in vivo, suggesting that ISO may be a new strategy for treating renal fibrosis.
Taken together, the results of the present invention demonstrate that PFKP plays a key role in metabolic conversion of PTECs into glycolysis during the progression of fibrosis. PFKP lacks inhibitory effect on expansion of renal tubules, area of interstitial fibrosis and glycolysis in injured mice, whereas PFKP overexpression promotes expansion of renal tubules, area of interstitial fibrosis and glycolysis in injured mice. Mechanically, TGF-. Beta.1 increases the level of transcription of PFKP by recruiting the SMAD3/SP1 complex to the PFKP promoter of HK2 cells. Furthermore, ISO inhibition of glycolysis and PFKP expression may improve kidney fibrosis. These results provide a basis for further exploration of PFKP's therapeutic potential in CKD.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.

Claims (10)

  1. Use of the PFKP gene or PFKP protein for any of the following:
    a1 The use of the composition for controlling the degree of renal fibrosis;
    a2 As a target in the preparation of a product for preventing, ameliorating or treating chronic kidney disease;
    a3 As a target in the preparation of a product for preventing, ameliorating or treating renal fibrosis;
    a4 As a target in the preparation of a product for inhibiting the occurrence and/or progression of renal fibrosis;
    a5 As a target in the preparation of products for inhibiting glycolysis.
  2. Use of any one of the following pfkp inhibitors:
    b1 The use of a composition for the preparation of a product for the prevention, amelioration or treatment of chronic kidney disease;
    b2 Use of a composition for the preparation of a product for the prevention, amelioration or treatment of renal fibrosis;
    b3 Use of a composition for inhibiting the occurrence and/or progression of renal fibrosis;
    b4 To a process for the preparation of a product for inhibiting glycolysis.
  3. 3. The use according to claim 2, wherein the PFKP inhibitor is a substance that inhibits PFKP gene expression, silences or knocks out PFKP gene, and/or a substance that reduces PFKP protein content and/or activity.
  4. 4. The use according to claim 3, wherein the substance is one or more of a nucleic acid molecule, a carbohydrate, a lipid, a small molecule compound, an antibody, a polypeptide, a protein, a gene editing vector, a lentivirus or an adeno-associated virus.
  5. 5. The use according to claim 4, wherein the nucleic acid molecule comprises shRNA, microRNA, siRNA and/or antisense oligonucleotides.
  6. 6. The use according to claim 5, wherein the target sequence of the shRNA is shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3.
  7. 7. The use according to claim 4, wherein the small molecule compound is isorhamnetin.
  8. 8. The shRNA of claim 6, a DNA molecule encoding the shRNA of claim 6, or a lentivirus or adeno-associated virus comprising the DNA molecule.
  9. 9. A pharmaceutical composition comprising the PFKP inhibitor according to any one of claims 2-7, said pharmaceutical composition having at least any one of the following uses:
    c1 Preventing, ameliorating or treating chronic kidney disease;
    c2 Preventing, ameliorating or treating renal fibrosis;
    c3 Inhibiting the occurrence and/or progression of renal fibrosis;
    c4 Inhibition of glycolysis.
  10. Use of PFKP gene or PFKP protein in the selection of a candidate drug for the treatment of chronic kidney disease or kidney fibrosis, said selection method comprising: the PFKP is used as target to screen medicine or reagent to reduce PFKP gene expression level or PFKP protein content or activity as candidate medicine for treating chronic kidney disease or kidney fibrosis.
CN202311536915.XA 2023-11-17 2023-11-17 Application of PFKP as chronic kidney disease treatment target and inhibitor thereof Pending CN117589999A (en)

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