Use of lctl as a therapeutic target for kidney injury in chronic kidney disease

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to the use of lactase-like protein (LCTL) as a target for treating kidney injury of chronic kidney disease. The present application also provides the use of a medicament for increasing the expression of LCTL gene or the amount of LCTL protein in the preparation of a medicament for treating kidney injury of chronic kidney disease (CKD). The present application first shows that the knockout of LCTL gene can aggravate the kidney injury degree of a CKD mouse model. The present application first uses overexpression lentivirus to overexpress the LCTL gene in vivo, which can reduce the kidney injury degree of a CKD mouse model. The present application first synthesizes a secretory LCTL, and in vitro, it is verified that the secretory LCTL can reduce the injury degree of a kidney tubular cell under a disease state. In summary, the present application provides more treatment strategies for treating kidney injury of chronic kidney disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of LCTL as a therapeutic target for kidney damage in chronic kidney disease. Background Technology

[0002] Chronic kidney disease (CKD) is a common chronic disease characterized by damage to the kidney structure or decline in kidney function.

[0003] LCTL (Lactase-like protein), also known as γ-Klotho (KLγ) or Klotho / lactoderm-phlorizin hydrolase-associated protein (KLPH), belongs to the Klotho family. This family includes α-Klotho (KLα), β-Klotho (KLβ), and LCTL proteins. Among the three members of the Klotho protein family, KLα is the most extensively and thoroughly studied renal protective protein, and is therefore also directly referred to as Klotho protein. KLα was initially defined as an anti-aging gene, primarily expressed in the kidneys, parathyroid glands, brain, and heart. Most studies have shown that membranous and secretory KLα play a protective role in kidney disease (e.g., see Lin Y, Kuro-o M, Sun Z. Genetic deficiency of anti-aging gene klotho exacerbates early nephropathy in STZ-induced diabetes in malemice. Endocrinology 2013; 154(10):3855-63). LCTL is highly homologous to KLα and is primarily expressed in the kidneys, skin, and lens. LCTL consists of 14 exons and translates into a 567-amino acid protein, exhibiting high homology between humans and mice. Similar to KLα, LCTL comprises a large extracellular domain including an extracellular segment of 519 amino acid residues, followed by a single transmembrane domain and an intracellular segment of 5 amino acid residues. LCTL is a new member of the Klotho protein family discovered in recent years based on homology analysis of KLα (e.g., see Ito S, Kinoshita S, Shiraishi N, Nakagawa S, Sekine S, Fujimori T, Nabeshima YI. Molecular cloning and expression analyses of mouse betaklotho, which encodes a novel Klotho family protein. Mech Dev 2000; 98(1-2):115-9). As it is a newly discovered protein, there is very little research on LCTL, its function is not yet clear, and no studies have been found on its application in kidney diseases. Summary of the Invention

[0004] To fill the gap in the prior art, this invention is the first to discover the use of LCTL as a therapeutic target for kidney damage in chronic kidney disease.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides the use of lactase-like protein (LCTL) as a therapeutic target for kidney injury in chronic kidney disease.

[0007] In a second aspect, the present invention provides the use of LCTL as a target in the development, screening, or preparation of medicaments for the treatment of kidney damage in chronic kidney disease.

[0008] In a third aspect, the present invention provides the use of an agent that increases LCTL gene expression or LCTL protein levels in the preparation of a medicament for treating chronic kidney disease injury.

[0009] Alternatively, the agents for increasing LCTL gene expression or LCTL protein quantity as described above may include one or more of the following: nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, peptides, proteins, gene editing vectors, lentiviruses, or adeno-associated viruses that increase LCTL expression.

[0010] Alternatively, in the above-described uses, the agent that increases LCTL gene expression or LCTL protein quantity is an adeno-associated virus or recombinant LCTL protein that increases LCTL expression.

[0011] Alternatively, in the above-described uses, the amino acid sequence of the recombinant LCTL protein is as shown in SEQ ID No. 1.

[0012] Alternatively, in the above-described uses, the drug may further comprise a pharmaceutically acceptable carrier or excipient.

[0013] Alternatively, in the above-described uses, the renal injury in chronic kidney disease is selected from one or more of the following: chronic kidney injury caused by ischemia-reperfusion, chronic kidney injury caused by drug toxicity, chronic kidney injury caused by bacterial infection, or chronic kidney injury caused by immune stress.

[0014] Alternatively, in the above-described uses, the renal injury in chronic kidney disease is selected from one or more of the following: diabetic nephropathy, hypertensive nephropathy, or end-stage renal disease.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] 1. This invention is the first to demonstrate that LCTL gene knockout can aggravate kidney damage in a CKD mouse model. Animal studies have found that in a chronic kidney disease model (diabetic nephropathy) using LCTL gene knockout mice, the degree of kidney function damage, proteinuria level, and fibrosis are aggravated, and LCTL gene deletion can aggravate kidney damage in the mouse model.

[0017] 2. This invention is the first to utilize lentivirus overexpression to reduce the degree of kidney damage in a CKD mouse model by overexpressing the LCTL gene in vivo. Animal studies have found that inducing LCTL gene overexpression through lentivirus injection can reduce kidney function damage in CKD mouse models (folate nephropathy, UUO mouse models), which may serve as a therapeutic strategy for kidney damage.

[0018] 3. This invention is the first to synthesize secretory LCTL and demonstrates in vitro that it reduces the degree of damage to renal tubular cells in disease states. The in vitro synthesized recombinant LCTL protein can reduce the degree of damage to renal tubular cells, and the recombinant LCTL protein may serve as a therapeutic strategy for kidney injury. Attached Figure Description

[0019] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0020] Figure 1 Construction of LCTL gene knockout mouse strains. (A) Schematic diagram of LCTL gene knockout; (B) PCR-based genomic analysis comparing LCTL knockout (KO), heterozygous knockout (HZ), and endogenous (EN) mice; (C) RT-qPCR analysis of LCTL mRNA levels in the kidneys of LCTL KO mice and littermate wild-type (EN) mice; (D) Western blot analysis of LCTL protein levels in the kidneys of LCTL KO mice and littermate EN mice; and (E) Quantitative analysis (n=6).

[0021] Figure 2LCTL gene knockout significantly aggravated renal function impairment in diabetic nephropathy mice. A diabetic nephropathy (STZ-DN) model was induced in wild-type (EN, LCTL+ / +) and LCTL knockout (KO, LCTL- / -) mice via intraperitoneal injection of streptozotocin, while normal control (Buffer) mice were injected intraperitoneally with buffer. The following data were collected for each group of mice: (A) urinary albumin / creatinine ratio; (B) serum creatinine level; (C) serum blood urea nitrogen (BUN) level; (D) serum cysteine ​​C level (n=6); (E) Periodic Acid-Schiff (PAS) staining of the kidneys of STZ-DN mice with LCTL EN or LCTL KO genes or normal control mice. Scale bar, 25 μm; (F) Quantification of the proportion of glomerular mesangial matrix deposition in PAS staining (n=6); (G) Immunostaining and (H) quantitative analysis of the podocyte marker synaptic protein (Syn) (n=6), scale bar, 25 μm; (I) Transmission electron microscopy (TEM) image of podocyte foot processes, scale bar, 1 μm; (J) Average number of foot processes per 1 μm of basement membrane (GBM) length (n=6).

[0022] Figure 3 Figure: Synthesized plasmid GPAAV-CMV-Mouse_Lctl-WPRE.

[0023] Figure 4 LCTL overexpression alleviates kidney damage in folate-induced nephropathy (FAN) mice. Folate-induced nephropathy (FAN) models were induced in wild-type LCTL (KLγ) (CTL) and LCTL-overexpressing (OE) mice via intraperitoneal injection of folic acid, while normal control (Buffer) mice were injected intraperitoneally with buffer. The following data are presented for each group of mice: (A) Cystatin C 2 days after folic acid injection; (B) Cystatin levels 28 days after folic acid injection; (C) Serum blood urea nitrogen (BUN) levels; and (D) Serum creatinine levels (n=6); (E) Immunoblotting results showing the expression levels of LCTL (KLγ), α-SMA, col-I, and Fn in each group of mice; and (F) corresponding statistical results; (G) Masson staining of the kidneys of folate-induced nephropathy mice or normal control mice in each group. Scale bar: 200 μm.

[0024] Figure 5 LCTL overexpression alleviates kidney damage in UUO mice. (A) Expression levels of LCTL(KLγ), α-SMA, and Vimentin in UUO and sham-operated mice in wild-type (CTL) and LCTL (OE) mice; (B) and corresponding statistical results; (C) Masson staining of kidneys in UUO or normal control mice in each group, scale bar, 200 μm.

[0025] Figure 6 LCTL is the full-length amino acid sequence of an amino acid, which includes a signal peptide, an extracellular segment, a transmembrane segment, and an intracellular segment.

[0026] Figure 7 The amino acid sequence of the LCTL recombinant protein of the present invention is the extracellular segment of the LCTL with a his tag.

[0027] Figure 8 : Recombinant LCTL protein synthesized in vitro. Wherein, NPE: supernatant; DPE: inclusion body protein; MW: molecular weight; Φ: uninduced strain (negative control). Expression assay conditions: 1 mM IPTG was used to induce expression at 16℃ / 16h and 37℃ / 4h, respectively. No.1: BL21(DE3) strain, No.2: T7E strain.

[0028] Figure 9 LCTL recombinant protein reduces the degree of fibrosis in tubular cells. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the materials and reagents used are commercially available. Example 1: LCTL gene knockout aggravates kidney damage in diabetic nephropathy mice 1. Experimental methods

[0030] LCTL gene knockout mice were constructed, and a diabetic nephropathy mouse model was induced by intraperitoneal injection of streptozotocin (STZ). The renal function, degree of kidney damage, and degree of renal podocyte damage in the mice were then examined.

[0031] 1.1 Constructing LCTL gene knockout mice

[0032] A pair of male and female LCTL+ / -(HZ) mice with a C57BL / 6 background were used to design sgRNAs using CRISPR / Cas9 technology. The fertilized eggs were fertilized via high-throughput electroporation, and the resulting LCTL+ / - mice were identified by PCR to obtain the first LCTL+ / - mice. These mice were then bred at the Experimental Animal Center of Sun Yat-sen University using LCTL-HZ male and female mice. The offspring were identified by PCR to obtain LCTL- / -(KO) and LCTL+ / +(WT) mice suitable for experimental use.

[0033] 1.2 Establishment and grouping of streptozotocin-induced diabetic nephropathy mouse model (DN)

[0034] Streptozotocin (STZ) was used to induce diabetic nephropathy (DN) in LCTL- / - (KO) and LCTL+ / + (WT) mice with a C57BL / 6 background. After a 4-hour fast, 55 mg / kg STZ was dissolved in 0.1 mmol / L sodium citrate solution (pH 4.5) and administered intraperitoneally for 5 consecutive days. Two weeks after STZ induction, fasting blood glucose was measured. Mice with a fasting blood glucose level greater than 16.7 mmol / L were considered to have successfully induced a diabetic diabetic (DM) model, and those with insufficient fasting blood glucose were sacrificed. Two weeks after DM modeling, glycated hemoglobin (HbA1c) was measured. Mice with HbA1c >7% were considered to have successfully induced a DM model, and those with insufficient HbA1c were sacrificed. The remaining mice (DN group) were considered to have successfully induced a DM model and were sacrificed 12 weeks after DM modeling. Control mice were given the same dose of sodium citrate solution as a solvent control group (NC group).

[0035] After DM modeling, random 4-hour urine samples were collected in a metabolic cage at week 12 for urinary albumin detection.

[0036] KO and WT mice were randomly divided into 4 groups using a random number table, with 8-15 mice in each group, as follows:

[0037] (1) LCTL wild type, solvent group (WT-NC group);

[0038] (2) LCTL wild-type, diabetic nephropathy group (WT-DN group);

[0039] (3) LCTL gene knockout, solvent group (KO-NC group);

[0040] (4) LCTL gene knockout, diabetic nephropathy (KO-DN group).

[0041] 1.3 General condition and renal function test of mice

[0042] In animal experiments, blood samples were collected and serum cystatin C (CysC), blood urea nitrogen, and serum creatinine levels were measured using ELISA and a fully automated biochemical analyzer to assess renal function in mice.

[0043] 1.4 Assessment of glomerular damage

[0044] (1) By using PAS staining, the relevant renal tissue pathological damage scores (such as average mesangial area width) were recorded to determine the degree of glomerular damage;

[0045] (2) The expression of synaptopodin, a marker of glomerular podocytes, was detected by immunofluorescence (IF) staining to determine the degree of glomerular sclerosis.

[0046] 1.5 Detection of podocyte damage in animal experiments

[0047] The morphology of podocytes was observed using a transmission electron microscope (TEM), and the pathological damage scores of podocytes were recorded, including the average number of podocytes in the glomerulus, the average number of foot processes, and the average width of the foot processes.

[0048] 2. Experimental Results

[0049] 2.1 Successful construction of LCTL gene knockout mice

[0050] like Figure 1 As shown, the knockout of the LCTL gene and protein in LCTL KO mice was confirmed at the DNA, mRNA, and protein levels by knocking out the Exon3-Exon7 fragment of LCTL exons.

[0051] 2.2 LCTL gene knockout significantly aggravated renal function damage in diabetic nephropathy mice.

[0052] like Figure 2 As shown, using a diabetic nephropathy mouse model, LCTL gene knockout significantly aggravated proteinuria and renal function impairment in diabetic mice, manifested by elevated levels of serum creatinine, blood urea nitrogen, and cystatin C. Kidney pathology revealed significantly increased mesangial matrix deposition in LCTL knockout mice, and fluorescence staining showed a significant decrease in the expression of the podocyte marker Synaptopodin. Kidney electron microscopy showed that LCTL gene knockout significantly aggravated podocyte fusion and exacerbated podocyte damage. These results indicate that LCTL gene knockout can significantly aggravate renal function impairment in diabetic nephropathy mice.

[0053] Example 2: LCTL overexpression reduces kidney damage in mice with chronic kidney disease. 1. Experimental methods

[0054] An overexpression virus of LCTL was constructed and a CKD mouse model (folate nephropathy and UUO mouse model) was established by tail vein injection.

[0055] 1.1 Construction and grouping of folate nephropathy mice

[0056] The method for inducing folate-induced nephropathy (FAN) in mice using folic acid (FA) employs a single, high-dose injection to induce a toxic tubular necrotizing folate nephropathy model. FA was dissolved in 0.3 mol / L sodium bicarbonate solution at a concentration of 0.25 mg / g and administered intraperitoneally once. Two days after folic acid induction, the mice were weighed. A small blood sample was collected via the medial canthal vein under anesthesia, and the serum was obtained by centrifugation for measuring serum CysC levels. A serum CysC level greater than 1000 μg / L two days after folic acid induction was considered a successful model; mice that failed to establish the model were sacrificed.

[0057] LCTL KO and wild-type (WT) mice were randomly divided into 4 groups using a random number table, with 6 mice in each group, as follows:

[0058] (1) LCTL wild type, solvent group (WT-NC group);

[0059] (2) LCTL wild-type, folate nephropathy group (WT-FAN group);

[0060] (3) LCTL gene knockout, solvent group (KO-NC group);

[0061] (4) LCTL gene knockout, folate nephropathy group (KO-FAN group).

[0062] 1.2 Establishment and grouping of a unilateral ureteral ligation mouse model (UUO)

[0063] Modeling Methods for Unilateral Ureteral Ligation (UUO) Mouse Model: Mice in the surgical group (UUO) were anesthetized with pentobarbital (45 mg / kg). After routine surgical disinfection and preparation, an incision was made on the right thigh near the midline of the abdomen. The skin and muscle tissue were dissected sequentially to expose the abdominal cavity. The obliquely running ureter was located along the posterior abdominal wall, and after dissecting the ureter, it was ligated at both the proximal and distal ends before being cut. The abdominal organs were repositioned, and the muscle and skin layers were sutured sequentially. In the sham surgery group (Sham), the ureter was dissected but not ligated; the remaining procedures were the same as in the UUO group. Seven days post-surgery, the mice were euthanized.

[0064] LCTL KO and wild-type (WT) mice were randomly divided into 4 groups using a random number table, with 6 mice in each group, as follows:

[0065] (1) LCTL wild type, Sham group (WT-Sham group);

[0066] (2) LCTL wild type, UUO group (WT-UUO group);

[0067] (3) LCTL gene knockout, Sham group (KO-Sham group);

[0068] (4) LCTL gene knockout, UUO group (KO-UUO group).

[0069] 1.3 Preparation of LCTL overexpression virus

[0070] The packaging of mouse Lctl gene overexpression AAV virus mainly includes: the construction of Lctl overexpression vector and the packaging of Lctl overexpression adeno-associated virus. The specific steps are as follows:

[0071] 1.3.1 Construction of Lctl overexpression vector:

[0072] (1) The target gene was constructed into the GPAAV-CMV-MCS-WPRE vector. Primers were designed and synthesized to amplify the target fragment, which was then ligated into the enzyme-digested vector.

[0073] (2) The ligation product is transferred into the prepared bacterial competent cells, and the resulting single-clone colonies are sent to a sequencing company for sequencing. The correct clone is the vector that has been successfully constructed.

[0074] The synthesized plasmid GPAAV-CMV-Mouse_Lctl-WPRE has the following structure: Figure 3 As shown.

[0075] The nucleotide sequence of the plasmid constructed above is shown in SEQ ID No. 2.

[0076] 1.3.2 Packaging of Lctl-overexpressing adeno-associated virus:

[0077] The target plasmid obtained above was transfected, amplified, and the viral fluid was collected, concentrated, and purified. Finally, the viral titer was determined.

[0078] 1.4LCTL overexpressing virus via tail vein injection

[0079] Two days after folic acid injection, the folate nephropathy mouse model was injected via tail vein with LCTL overexpressing virus (3e11vg), while control mice were given the same amount of empty vector control virus.

[0080] UUO model mice were injected with LCTL overexpressing virus (3e11vg) via tail vein 10 days before surgical modeling, while control mice were given the same amount of empty control virus.

[0081] 2. Experimental Results

[0082] 2.1 LCTL gene overexpression reduces the degree of renal function impairment in folate nephropathy mice

[0083] like Figure 4 As shown, LCTL overexpression significantly reduced the degree of renal function damage in folate-induced nephropathy (FORD) mice. Two days after modeling, LCTL overexpression did not affect renal function in FORD mice, but at 28 days post-modeling, LCTL overexpression significantly reduced the degree of renal function damage, as evidenced by significantly lower levels of serum cystatin C, serum BUN, and serum creatinine in LCTL-overexpressing FORD mice. Immunoblotting results showed that LCTL overexpression significantly reduced the expression of fibrosis markers in FORD mice. Masson staining results showed that LCTL overexpression significantly reduced the degree of renal fibrosis in FORD mice.

[0084] 2.2 LCTL gene overexpression reduces renal fibrosis in UUO mice

[0085] like Figure 5 As shown, LCTL overexpression significantly reduced the degree of renal fibrosis in UUO mice. Western blotting results indicated that LCTL overexpression significantly reduced the expression of fibrosis markers in UUO mice. Masson staining results also showed that LCTL overexpression significantly reduced the degree of renal fibrosis in UUO mice.

[0086] Example 3: Preparation of recombinant LCTL protein and its effect on the degree of damage to renal tubular cells in disease states.

[0087] 1. Experimental Methods

[0088] Recombinant LCTL protein was synthesized and stimulated in renal tubular epithelial cell lines.

[0089] The full-length amino acid sequence of LCTL includes a signal peptide, an extracellular segment, a transmembrane segment, and an intracellular segment, as shown in SEQ ID NO.3. Additionally, as... Figure 6 As shown. The amino acid sequence of the recombinant LCTL protein of the present invention is a his-tagged extracellular segment of LCTL, as shown in SEQ ID No. 1. Additionally, as... Figure 7 As shown.

[0090] Human renal tubular epithelial cell line (HK2) was purchased from ATCC (CRL-2190, https: / / www.atcc.org / products / crl-2190). HK2 cells were stimulated with 10 ng / mL of fibrotic TGF-β1. With or without TGF-β1 stimulation, 500 ng / mL of synthetic LCTL recombinant protein was added. After co-stimulation for 48 h, cell protein lysates were collected for analysis.

[0091] 2. Experimental Results

[0092] like Figure 8 As shown, the LCTL recombinant protein (rLCTL) was successfully synthesized and purified, and the concentration and purity of the synthesized LCTL recombinant protein were verified by electrophoresis.

[0093] like Figure 9 As shown, in in vitro experiments, using a renal tubular epithelial cell line, LCTL recombinant protein was found to significantly reduce tubular cell damage under TGF-β stimulation.

[0094] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several modifications or equivalent substitutions can be made to the technical solution without departing from the principle of the present invention, and these modifications or equivalent substitutions should also be considered within the scope of protection of the present invention.

Claims

1. The use of adeno-associated virus with increased LCTL expression in the preparation of medicaments for treating chronic kidney disease injury, characterized in that: The chronic kidney disease damage mentioned is folic acid nephropathy.

2. The use according to claim 1, characterized in that: The drug also contains a pharmaceutically acceptable carrier.

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