Application of retinol saturase as target spot in treatment of diabetic kidney disease

Abstract

The invention relates to the technical field of biomedical engineering, provides application of retinol saturase as a target spot in treatment of diabetic kidney disease (DKD), and particularly relates to an effect of interfering RNA (Ribonucleic Acid) or antisense oligonucleotide or a recombinant expression vector of a targeted RetSat coding sequence in preparation of a product for treating DKD / or other complications. The invention also relates to a corresponding recombinant vector or pharmaceutical composition. The RetSat inhibitor can effectively reduce the levels of renal tubule injury markers and fibrosis related markers, has effective application value in relieving and treating the diabetic kidney disease, provides a new target spot for the treatment of the diabetic kidney disease, and has wide clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of retinol saturase (RetSat) as a target in the preparation of a drug for treating diabetic kidney disease (DKD). Background Art

[0002] Diabetic kidney disease (DKD) has become the leading cause of chronic kidney disease and end-stage renal disease worldwide. Studies have shown that approximately 40% of diabetic patients are at risk of developing DKD. It is estimated that by 2050, more than 1.31 billion people worldwide may be affected by diabetes, highlighting the growing challenges posed by DKD as an important clinical problem. Traditionally, it is believed that increased synthesis of mesangial collagen in the glomerulus leads to the accumulation of mesangial extracellular matrix (ECM), which plays an important role in the pathogenesis of DKD. However, more and more studies have found that the pathogenesis of DKD involves not only glomerular dysfunction, but also tubulointerstitial lesions. Recent studies have found that proximal tubular cells, rather than glomerular cells, play a key role in regulating glomerular filtration. Proximal tubular cells are the main contributors to kidney enlargement and hyperfiltration. In advanced DKD, the loss of tubular cells is closely associated with a decrease in eGFR. The pathological changes of DKD include nodular or diffuse glomerulosclerosis, tubular inflammation, atrophy and interstitial fibrosis. Among them, tubulointerstitial fibrosis (TIF) is an important pathological feature of chronic kidney disease and is considered to be a common final pathway leading to end-stage renal disease, which is characterized by complete loss of renal function. However, the molecular mechanism driving TIF in DKD is still not fully understood. Studies have shown that in the early stages of DKD, hypertrophy and proliferation of tubular epithelial cells occur simultaneously with thickening of the tubular basement membrane. As a result, due to the continuous increase in blood glucose levels, ischemia and hypoxia, tubular cell apoptosis, tubular atrophy and degeneration occur, which promotes the progression of TIF and ultimately leads to the progression of DKD to end-stage renal failure. Since tubular damage and subsequent TIF play a vital role in the occurrence and development of DKD, finding therapeutic targets for tubular damage in DKD has become a hope for the treatment of DKD.

[0003] Retinol saturase (RetSat) is an oxidoreductase with conserved protein sequences and genomes in human and rodent homologs and is highly expressed in adipose tissue, liver, and kidney. Intracellular RetSat protein is mainly localized in the endoplasmic reticulum and can catalyze the conversion of retinol to 13,14-dihydroretinol (13,14-dhretinol). The main transcriptional regulators of RetSat include nuclear peroxisome proliferator-activated receptor α (PPARα) in the liver, PPARγ in adipose tissue, and forkhead box O1 (FOXO1) in primary hepatocytes. However, recent studies have shown that the function of RetSat may be far beyond its traditional definition. Studies have found that RetSat expression levels are dynamically regulated in insulin resistance and type 2 diabetes-related diseases, and RetSat function is related to adipocyte differentiation, hepatic glucose and fatty acid metabolism, and macrophage phagocytosis. For example, RetSat can participate in the occurrence of fatty liver and regulate hepatic glucose and lipid metabolism by regulating carbohydrate response element binding protein (ChREBP), and the regulation of ChREBP by RetSat is independent of the formation of its enzymatic product dihydroretinol. In addition, RetSat expression in human liver is closely related to obesity, hepatic steatosis, and the expression of ChREBP target genes. Previous studies have also shown that high glucose-induced O-GlcNAc glycosylation-modified ChREBP is involved in mediating adipogenesis and fibrosis in mesangial cells.

[0004] Although RetSat plays an important role in the above-mentioned metabolic regulation, its specific role and regulatory mechanism in renal pathophysiology, especially in the pathogenesis of DKD, remain unclear. Therefore, there is an urgent need to develop new strategies for the relief and treatment of DKD. Summary of the invention

[0005] The present invention is aimed at the above problems and studies the application of retinol saturase as a target in the treatment of diabetic nephropathy: first, the adverse effects of abnormal expression of RetSat on DKD are confirmed, and then based on this target, the recovery of kidney cell damage after the abnormal expression of RetSat is eliminated or alleviated is observed, thereby determining the role of RetSat in the onset and progression of DKD.

[0006] The specific research process is as follows: First, compared with the control group, both DKD patients and DKD mouse models showed abnormal increases in tubular damage and fibrosis. At the same time, it was found that the expression of RetSat in the kidneys of DKD patients and mouse models was significantly upregulated compared with the control group. In order to further confirm the association between the expression level of RetSat and tubular damage in DKD, the expression of RetSat in tubular cells was detected again after stimulating the human tubular cell line HK2 with high glucose. The results showed that high glucose could induce the expression of RetSat in tubular cells HK2, accompanied by increased expression of tubular damage markers and fibrosis genes. Subsequently, RetSat was overexpressed in HK2 cells and siRNA was transfected to inhibit its expression. The results showed that RetSat overexpression could significantly increase the expression levels of tubular damage markers and fibrosis genes in tubular cells, and inhibiting its expression could effectively reduce tubular cell damage. Therefore, the study suggests that RetSat can participate in the occurrence and development of DKD by regulating renal tubular damage, and RetSat is expected to enter clinical application as a therapeutic target for renal tubular damage in DKD.

[0007] Based on the above research, the specific protection technical solution of the present invention is as follows:

[0008] The first aspect of the present invention provides use of a RetSat inhibitor in the preparation of a medicament for treating diabetic nephropathy or its complications.

[0009] Preferably, the RetSat inhibitor is selected from compounds that inhibit the activity or function of retinol saturase or interfering RNA or antisense oligonucleotides that target the RetSat coding sequence.

[0010] Further preferably, the sequence of the interfering RNA or antisense oligonucleotide targeting the RetSat coding sequence is any one of the following:

[0011] 5'-CCAACUACUAUGUUUACUATT-3' (SEQ ID NO. 1);

[0012] 5'-UAGUAAACATAGTTGTT-3' (SEQ ID NO. 2).

[0013] The results of cell experiments showed that RetSat overexpression could significantly increase the expression levels of tubular injury markers (RETSAT, FABP, KIM-1, NGAL) and fibrosis genes (TGFβ1, COL1A1, FN1) in renal tubular cells; after inhibiting RetSat expression, the expression levels of tubular injury markers and fibrosis genes in renal tubular cells were significantly downregulated.

[0014] In a second aspect, the present invention provides a recombinant vector targeting RetSat, comprising an expression vector and sh RNA or si RNA of the RetSat gene inserted into the expression vector, wherein the nucleic acid sequence of the sh RNA or si RNA is shown in SEQ ID NO. 1 or 2 above.

[0015] Wherein, the expression vector is a conventional vector such as a plasmid vector, a cosmid vector, a phage vector or a viral vector, and the specific type is selected from the prior art according to the actual situation. The "viral vector" includes adeno-associated virus and lentivirus. Suitable viral vectors are well known to those of ordinary skill in the art. The remaining "non-viral vectors" include liposomes or lipid complexes, cationic polymers, chitosan polymers and nanoparticle vectors. Suitable non-viral vectors are well known to those of ordinary skill in the art.

[0016] Correspondingly, the present invention also provides the use of a recombinant expression vector for inhibiting the RetSat gene in preparing a drug for treating diabetic nephropathy.

[0017] In a third aspect, the present invention provides a pharmaceutical composition for treating diabetic nephropathy, comprising an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises the RetSat inhibitor as described above, or a recombinant expression vector that inhibits the RetSat gene.

[0018] In terms of drug form, the drug or drug composition is suitable for administration by a method selected from the following group: oral administration, injection (such as direct naked DNA or protein injection, liposome-encapsulated DNA or mRNA injection), gold-coated gene gun bombardment, replication-defective adenovirus carrying target DNA or protein encoded by the target gene, enema administration, and transdermal administration.

[0019] Generally, liquid preparations can be kept stable for at least one year at 2°C-8°C, and freeze-dried preparations can be kept stable for at least six months at 30°C. The preparations can be suspensions, water injections, freeze-dried preparations and other preparations commonly used in the pharmaceutical field.

[0020] When the composition of the present invention is administered to animals including humans, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and administration route. The results of animal experiments and various circumstances can be referred to, and the total dosage cannot exceed a certain range.

[0021] Furthermore, the pharmaceutical composition of the present invention can be used in combination with other pharmaceutical compositions for treating diabetic nephropathy or other complications.

[0022] A fifth aspect of the present invention provides use of a reagent for detecting the expression level of RetSat in preparing a diabetic nephropathy detection kit or a drug screening kit.

[0023] The experimental results showed that the expression of RetSat was significantly upregulated in DKD patients, and the inhibition of RetSat expression alleviated the pathological manifestations in high glucose-induced renal tubular cells HK2, suggesting that RetSat is a promoter of DKD and has the effect of promoting the occurrence and development of DKD. Based on this, it can be inferred that RetSat can be used as a diagnostic marker or drug screening marker for DKD.

[0024] The method for drug screening using RetSat as a marker is as follows:

[0025] (A) treating cells, tissues or animals with DKD or mimicking DKD symptoms with a candidate substance;

[0026] (B) detecting the level of RetSat or a nucleic acid molecule encoding the protein in the cell, tissue or animal;

[0027] (C) After treatment with the candidate substance, if the level of RetSat or the nucleic acid molecule encoding the protein is lower than the expression level of RetSat in the DKD model tissue or cell, it indicates that the candidate substance has the effect of treating DKD by inhibiting RetSat.

[0028] In a sixth aspect of the present invention, a DKD diagnostic kit or drug screening kit is provided, which comprises a reagent for detecting the expression level of RetSat in a biological sample, and is composed of a reverse transcription system, a primer system, and an amplification system. Diagnosis or drug screening is achieved by detecting the nucleic acid expression level of RetSat.

[0029] Wherein, the biological sample is selected from kidney tissue or kidney cell HK2, and the primer sequences with specific detection of RetSat in kidney tissue are shown in SEQ ID NOs. 3 and 4 below; the primer sequences with specific detection of RetSat in kidney cell HK2 are shown in SEQ ID NOs. 19 and 20:

[0030] RetSat-F1: 5'-GTCTACGTGGGCCTTTACG-3' (SEQ ID NO. 3);

[0031] RetSat-R1: 5'-ACTTTCTTCCTAGCCTCCTGT-3' (SEQ ID NO.4);

[0032] RetSat-F2: 5'-CAGCATTGGCCGTTTTATCTTG-3' (SEQ ID NO. 19);

[0033] RetSat-R2: 5'-CCTTGAGGCCCTGAATGTAGG-3' (SEQ ID NO. 20).

[0034] Beneficial protection and effects of the present invention:

[0035] The present invention provides the use of a RetSat inhibitor in the preparation of a drug for treating DKD or its complications. Experiments have confirmed that both DKD patients and DKD mouse models show abnormal increase in renal tubular damage and fibrosis. It is also found that the expression of renal RetSat in DKD patients and mouse models is significantly upregulated compared with the control group. Inhibiting its expression can effectively reduce renal tubular cell damage, indicating that RetSat is a pathogenic factor of DKD and has the effect of aggravating the pathological damage of DKD. Therefore, the present invention provides a new target and basis for the treatment of DKD by targeting the inhibition of RetSat, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The results showed that the expression of RetSat in the kidney tissue of DKD mice was significantly increased: A. HE, PAS, PASM, Sirius red staining of kidney tissue sections of DKD and NC group mice, as well as semi-quantitative analysis of Sirius red staining and mesangial expansion; B. Heat map analysis of differential protein proteomics in the kidneys of DKD and NC group mice.

[0037] Figure 2 The expression levels of RetSat in kidney tissues of mice in the DKD and NC groups are shown: A. RetSat mRNA expression level; B. RetSat protein level and semi-quantitative analysis; C. mRNA expression level of renal tubular injury markers; D. mRNA expression level of fibrosis-related markers.

[0038] Figure 3 The expression levels of RetSat, renal tubular injury markers and fibrosis-related markers in HK2 cells treated with high glucose (HG) are shown: A. RetSat mRNA expression level in HK2 cells; B. RetSat protein level and semi-quantitative analysis in HK2 cells; C. mRNA expression level of renal tubular injury markers in HK2 cells. D. mRNA expression level of fibrosis-related markers in HK2 cells.

[0039] Figure 4 The expression of RetSat in the kidney of DKD patients is shown: A. Heat map analysis of differentially expressed genes in the kidney of DKD patients and control groups in the GEO database; B. HE, PAS, PASM, Sirius red staining and Sirius red staining and semi-quantitative analysis of mesangial expansion in renal biopsy tissue sections of MCD and DKD patients; C. Immunofluorescence staining and semi-quantitative analysis of RetSat in renal biopsy tissue of MCD and DKD patients.

[0040] Figure 5The results show that overexpression of RetSat or inhibition of RetSat expression in HK2 cells promotes changes in the expression levels of RetSat, tubular injury markers and fibrosis-related markers: A. RetSat protein level and mRNA expression level in HK2 cells after overexpression of RetSat; B. mRNA expression level of tubular injury markers in HK2 cells after overexpression of RetSat; C. mRNA expression level of fibrosis-related markers in HK2 cells after overexpression of RetSat; D. RetSat protein level and mRNA expression level in HK2 cells after inhibition of RetSat expression; E. mRNA expression level of tubular injury markers in HK2 cells after inhibition of RetSat expression; F. mRNA expression level of fibrosis-related markers in HK2 cells after inhibition of RetSat expression. DETAILED DESCRIPTION

[0041] The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include a detailed description of conventional methods, such as PCR methods, methods for constructing vectors and plasmids, methods for inserting protein-encoding genes into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and are described in many publications, including Sambrook, J., Fritsch, E. F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold spring Harbor Laboratory Press.

[0042] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention, and the preferred implementation methods and materials described in the specific implementation methods are for demonstration purposes only.

[0043] Example 1 RetSat is significantly overexpressed in kidney tissues of DKD mice and HK2 cells treated with high glucose (HG) 1. Construction of DKD mouse model

[0044] Eight-week-old male C57BL / 6J experimental mice were housed in the SPF animal room of the Experimental Animal Center of Tongji Hospital Affiliated to Tongji University. After one week of adaptive feeding, they were divided into a normal control group (NC) and a DKD group. The mice in the NC group (n=10) were fed with ordinary feed, and the mice in the DKD group (n=10) were fed with HFD. After 4 weeks, the mice fed with HFD were given a one-time low-dose intraperitoneal injection of STZ (90 mg / kg), while the NC group was injected with an equal volume of citric acid buffer. After one week, the mice were fasted for 8 hours. If the fasting blood glucose of the mice was ≥11.1 mmol / L twice, the T2DM model was considered to be successfully established. The above diet was continued for 6 weeks to establish the DKD model.

[0045] 2. High glucose treatment of HK2 cells

[0046] HK2 cells were measured at 2.0*10 5 / well of a 6-well plate, and after 24 hours, DMEM culture medium was added according to a concentration gradient of 5mM and 30mM. After 72 hours of culture, the cells were collected for subsequent PCR detection.

[0047] 3. RetSat Detection

[0048] 3.1 Renal pathology

[0049] (1) Preparation of paraffin sections: ① Immerse the kidney tissue block in paraformaldehyde for 24 hours; ② Dehydrate in a dehydrator; ③ Place in xylene to make it transparent. ④ Place the transparent kidney tissue block in the melted paraffin and embed it. ⑤ Cool at -20℃, place in a paraffin slicer and cut into 4μm thick white slices, bake in a 60℃ oven, dry and place at room temperature for later use.

[0050] (2) HE staining: ① Paraffin sections of kidney tissue were placed in xylene, anhydrous ethanol and 75% alcohol in turn for dewaxing and washing. ② Stain with hematoxylin solution for 3 minutes to color the cell nuclei and rinse with double distilled water. ③ Remove the excess dye with differentiation solution and rinse with double distilled water. ④ Stain with eosin solution for 5 minutes. ⑤ Soak in anhydrous ethanol 3 times, 5 minutes each time, soak in xylene 2 times, 5 minutes each time to make it transparent, and finally seal with neutral gum. ⑥ Take pictures and analyze with an upright optical microscope.

[0051] (3) PAS staining: ① Treat the slices with 3% hydrogen peroxide for 10-15 minutes to remove endogenous peroxidase in the tissue. ② Immerse the slices in periodic acid solution and stain for 5-10 minutes. ③ Rinse with double distilled water. ④ Treat the slices with sodium chloride solution for 5 minutes to enhance the staining effect. ⑤ Immerse the slices in Schiff reagent and stain for 15-30 minutes. ⑥ Rinse with double distilled water to remove excess Schiff reagent. ⑦ Dehydrate with gradient alcohol (70%, 80%, 90%, 95%, 100%) in sequence, and then transparentize with xylene. ⑧ Seal the slices with neutral gum, and after drying, take pictures and analyze them under an upright optical microscope.

[0052] (4) PASM staining: ① Immerse the sections in 0.5% periodic acid solution for 5-10 minutes. ② Rinse the sections with double distilled water to remove periodic acid residues. ③ Immerse the sections in Schiff reagent and stain for 15-30 minutes. ④ Rinse the sections with double distilled water to remove excess Schiff reagent. ⑤ Immerse the sections in a staining solution containing ammine silver solution for 15-30 minutes. ⑥ Treat the silver-stained sections with sodium chloride, and the color development effect is adjusted according to the color development intensity. ⑦ Rinse the sections with double distilled water to remove excess silver salt. ⑧ Use hematoxylin to contrast stain the sections for 2-5 minutes. ⑨ Rinse the sections with double distilled water. ⑩ Dehydrate with gradient alcohol (70%, 80%, 90%, 95%, 100%) in sequence, and then transparentize with xylene. Seal the slides with neutral gum, wait for them to dry, and then take photos under an upright optical microscope for analysis.

[0053] (5) Sirius red staining: ① Immerse the slices in Sirius red staining solution and stain for 10-15 minutes. ② Rinse the slices with double distilled water to remove excess dye. ③ Dehydrate with graded alcohol (70%, 80%, 90%, 95%, 100%) in sequence, and then transparentize with xylene. ④ Seal the slices with neutral gum and wait for them to dry naturally. After they are dry, take photos under an upright optical microscope for analysis.

[0054] (6) Immunofluorescence staining: ① Dewax the paraffin sections in xylene twice, 10-15 minutes each time. Then dehydrate with gradient alcohol (100%, 95%, 90%, 80%, 70%) in sequence, and then rinse with double distilled water. ② Place the sections in sodium citrate buffer (pH 6.0), heat in high pressure steam for 10-20 minutes, and then cool naturally. ③ Block with 10% goat serum and block the sections at room temperature for 1 hour. ④ Incubate with RetSat antibody (1:200) at 4°C overnight. ⑤ Incubate with fluorescently labeled secondary antibodies matching the primary antibody at room temperature for 1 hour. ⑥ Wash the sections 3 times with TBS, 5-10 minutes each time. ⑦ Stain with DAPI to mark the cell nucleus for 5-10 minutes, and then wash with TBS. ⑧ Seal the stained tissue sections with anti-fluorescence attenuation sealing fluid and observe under a fluorescence microscope.

[0055] 3.2 Protein expression detection

[0056] Take 50-100 mg of kidney tissue in an EP tube, prepare RIPA lysis buffer and PMSF at a dose of 100:1, take 300 μL of lysis buffer for each kidney tissue sample, and then put it into a grinder for full homogenization. After centrifugation at 1000 rpm for 5 minutes, wash twice with PBS, discard PBS, add 1 mL of cell lysis buffer containing protease inhibitors, and ice bath for 30 minutes. During this period, use a pipette to blow repeatedly to ensure that the cells are completely lysed, centrifuge at 12000 rpm for 10 minutes at 4°C, and extract the supernatant as the protein solution.

[0057] Take 0.8mL of protein standard solution and add it to 20mg BSA. After fully dissolving, prepare a 25mg / mL protein standard solution. Take 20μL of 25mg / mL protein standard solution and add 980μL PBS to dilute it to 0.5mg / mL protein standard. Add 0, 1, 2, 4, 8, 12, 16 and 20μL of the standard to a 96-well plate, and then add PBS to make up to a total volume of 20μL, which is equivalent to a standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5mg / mL, respectively. Add 4μL of sample to a 96-well plate and make up to a total volume of 20μL with PBS. Add 200μL of BCA working solution to each well, place at 37℃ for 30 minutes, and detect the OD value at 560nm with an enzyme reader. Calculate the protein concentration of each sample based on the standard curve. The protein sample was mixed with protein loading buffer (5×) in a ratio of 4:1, heated at 100°C for 10 min to fully denature the protein, and then stored at -20°C.

[0058] Add 4 μL of protein marker or a sample containing 20 μg of total protein to the SDS-PAGE gel, and adjust the voltage to 100 V after 80 V electrophoresis for 30 minutes and continue electrophoresis for 1 hour. Cover the gel with a methanol-activated PVDF membrane and electrophoresed at 400A for 40 minutes. After the transfer, wash the PVDF membrane with TBST 3 times, 5 minutes each time. Then put it in WB fast blocking solution for 15 minutes, and wash it with TBST 3 times, 5 minutes each time. Then put it in 1:1000 diluted anti-RetSat antibody, anti-Smurf2, anti-ChREBP or 1:5000 diluted anti-β-actin antibody and incubate it at 4°C overnight. The next day, take out the PVDF membrane, wash it with TBST 3 times, 5 minutes each time, and then put it in the secondary antibody and incubate it at room temperature for 1 hour. After incubation, wash it with TBST 3 times, 5 minutes each time. After washing, develop it with supersensitive ECL chemiluminescence working solution.

[0059] 3.3qPCR detection

[0060] 3.3.1 Extraction of total RNA from HK2 cells

[0061] 1. Add 500 μL TRIzol reagent to each well of the 6-well plate cells and pipette. After standing at room temperature for 5 minutes, centrifuge at 12000g for 10 minutes at 4°C, and transfer the supernatant to an RNase-free EP tube;

[0062] 2. Add 100 μL of chloroform, shake vigorously for 15 seconds to mix, let stand at room temperature for 3 minutes, and centrifuge at 12000g for 15 minutes at 4°C;

[0063] 3. Carefully transfer the upper aqueous phase to another RNase-free EP tube, add an equal volume of isopropanol, gently invert to mix, let stand at room temperature for 10 minutes, and centrifuge at 4°C, 12,000 g for 10 minutes;

[0064] 4. Discard the supernatant, add 1 mL of pre-cooled 75% ethanol to the precipitate, mix thoroughly by inversion, centrifuge at 7500 g for 5 min at 4°C, repeat twice;

[0065] 5. After discarding the liquid, place the EP tube upside down on dust-free paper and air dry for 30 minutes;

[0066] 6. Add 20 μL RNase Free H2O and dissolve at 4°C for half an hour. Determine the concentration and purity of total RNA, requiring the A260 / A280 ratio to be between 1.9-2.0; A260 / A230 to be greater than 2 and to be a single peak.

[0067] 3.3.2 Extraction of total RNA from mouse kidney tissue

[0068] 1. Take 30 mg of kidney tissue, add 1 mL of Trizol reagent, grind and homogenize at 4°C, centrifuge at 12000g for 10 min at 4°C, and transfer the supernatant to an RNase-free EP tube;

[0069] 2. Add 200 μL of chloroform, shake vigorously for 15 seconds to mix, let stand at room temperature for 3 minutes, and centrifuge at 12000g for 15 minutes at 4°C;

[0070] 3. Carefully transfer the upper aqueous phase to another RNase-free EP tube, add an equal volume of isopropanol, gently invert to mix, let stand at room temperature for 10 minutes, and centrifuge at 12000g at 4°C for 10 minutes.

[0071] 4. Discard the supernatant, add 1 mL of pre-cooled 75% ethanol to the precipitate, mix thoroughly by inversion, centrifuge at 7500 g for 5 min at 4°C, repeat twice;

[0072] 5. After discarding the liquid, place the EP tube upside down on dust-free paper and air dry for 30 minutes;

[0073] 6. Add 20 μL RNase Free H2O and dissolve at 4°C for half an hour. Determine the concentration and purity of total RNA, requiring the A260 / A280 ratio to be between 1.9-2.0; A260 / A230 to be greater than 2 and to be a single peak.

[0074] 3.3.3 RNA reverse transcription

[0075] HK2 cell RNA was reverse transcribed using PrimeScript RT Master Mix kit (Takara RR036A, Japan). The reaction system (10 μL) is shown in Table 1 below:

[0076] Table 1 Reverse transcription reaction system

[0077]

[0078] After gentle mixing, reverse transcription was performed with the following procedure: 37°C for 15 min, 85°C for 5 sec, 4°C forever.

[0079] 3.3.4 qPCR reaction

[0080] use Premix Ex Taq TM Ⅱ kit (Takara RR820A) was used for quantitative analysis by ABI real-time PCR instrument (Applied Biosystems). The reaction system (10 μL) is shown in Table 2 below:

[0081] Table 2 qPCR reaction system

[0082]

[0083] The reaction procedure is shown in Table 3 below:

[0084] Table 3 qPCR reaction procedure

[0085]

[0086] The qPCR primer sequences are shown in Tables 4 and 5 below:

[0087] Table 4 Primer sequences for mouse kidney tissue

[0088]

[0089] Table 5 Primer sequences for HK2

[0090]

[0091]

[0092] IV. Results Analysis

[0093] The results of the mouse DKD model showed that compared with the control group, the kidneys of DKD mice showed significant mesangial expansion and collagen deposition, confirming that the DKD mouse model was successfully established ( Figure 1 A). Further proteomic analysis revealed differentially expressed proteins between the kidney tissues of NC and DKD mice, showing that RetSat was significantly upregulated in the DKD group ( Figure 1 B). qPCR and WB verification analysis showed that compared with the NC group, the gene and protein expressions of RetSat in the kidney tissue of the DKD group mice were significantly increased ( Figure 2 AB); it was also found that compared with the NC group, the expression of renal tubular injury markers and renal fibrosis genes in the kidneys of DKD mice was significantly increased ( Figure 2 CD).

[0094] To further confirm the association between the expression level of RetSat and tubular damage in DKD, we stimulated the human tubular cell line HK2 with high glucose and then detected the expression of RetSat in tubular cells again. The results showed that high glucose could induce the expression of RetSat in HK2 tubular cells ( Figure 3 AB), and the expression levels of renal tubular injury markers and renal tubular fibrosis markers were significantly increased ( Figure 3 CD).

[0095] Example 2: RetSat expression in the kidneys of DKD patients is significantly upregulated

[0096] Using bioinformatics tools, differentially expressed genes between DKD patients and controls were analyzed in the GEO database. Figure 4 As shown in Figure 2, compared with the control group, the expression of RetSat in the kidney of the DKD patient group was significantly upregulated ( Figure 4 A). Then, the results of the GEO database were verified by tissue testing using renal biopsy tissues of patients with minimal change kidney disease (MCD) and DKD. It was found that the mesangial expansion and collagen deposition in the kidney tissues of DKD patients were higher than those in the MCD group ( Figure 4 B), and then immunofluorescence staining confirmed that RetSat was mainly expressed in the renal tubules, and the expression of RetSat protein in the kidneys of DKD patients was significantly higher than that in the MCD group ( Figure 4 C).

[0097] Example 3 Interference in the expression of RetSat can inhibit DKD pathological damage

[0098] RetSat small interfering RNA (siRNA) was constructed. HK2 cells were cultured at 2.0*10 5 / well of a 6-well plate, and transfect when the cell density of HK2 cells in the 6-well plate increases to 60-70%. Take a sterile centrifuge tube, add 125μL Opti-MEMTM, 200pmol siRNA (sequence 5′-CCAACUACUAUGUUUACUATT-3′ (SEQ ID NO.1), 5′-UAGUAAACATAGTAGTTGGTT-3′ (SEQ ID NO.2)) and 4μL Lipo 8000 reagent, and mix gently. Take 250μL and add it to a 6-well plate. Then put it in a cell culture incubator and transfect for 48 hours. Then collect the transfected cells for subsequent PCR and western detection.

[0099] RetSat overexpression plasmid (Flag-RetSat) was constructed. HK2 cells were cultured at 2.0*10 5 / well of a 6-well plate. When the cell density of HK2 cells in the 6-well plate increases to 60-70%, transfect. Take a sterile centrifuge tube, add 125μL Opti-MEMTM, 2.5μg Flag-RetSat and 4μL Lipo 8000 reagent, and mix gently. Take 250μL and add it to the 6-well plate. Then put it in a cell culture incubator and transfect for 48 hours. Then collect the transfected cells for subsequent PCR and western detection.

[0100] The results are as follows Figure 5 As shown in the figure, RetSat overexpression can significantly increase the expression levels of tubular injury markers (FABP, KIM-1, NGAL) and fibrosis genes (TGFβ1, COL1A1, FN1) in renal tubular cells ( Figure 5 AC); after inhibiting RetSat expression, the expression levels of tubular injury markers and fibrosis genes in renal tubular cells were significantly downregulated ( Figure 5 DF).

[0101] In summary, multi-level experiments were conducted on the above-mentioned DKD patients, DKD mouse models and in vitro tubular cell models, and the results all suggested that RetSat may be a new potential drug target for the treatment of DKD.

[0102] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Use of RetSat inhibitors in the preparation of drugs for treating diabetic nephropathy or its complications.

2. The use according to claim 1, characterized in that: The RetSat inhibitor is selected from compounds that inhibit the activity or function of retinol saturase or interfering RNA or antisense oligonucleotides that target the RetSat coding sequence.

3. The use according to claim 2, characterized in that: The sequence of the interfering RNA or antisense oligonucleotide targeting the RetSat coding sequence is shown in any one of SEQ ID NO.1 to SEQ ID NO.

2.

4. A recombinant vector targeting RetSat, characterized in that: It comprises an expression vector and an interfering RNA or antisense oligonucleotide inserted into the expression vector and targeting a RetSat coding sequence, wherein the sequence is shown in any one of SEQ ID NO.1 to SEQ ID NO.

2.

5. The recombinant vector targeting RetSat according to claim 4, characterized in that: in, The expression vector is a plasmid vector, a cosmid vector, a phage vector or a virus vector.

6. Use of the recombinant vector targeting RetSat according to claim 4 or 5 in the preparation of a drug for treating diabetic nephropathy.

7. A pharmaceutical composition for treating diabetic nephropathy, characterized in that: The pharmaceutical composition comprises an active ingredient and a medically acceptable excipient, carrier or diluent. Wherein, the active component is the RetSat inhibitor according to any one of claims 1 to 3 or the recombinant vector targeting RetSat according to any one of claims 4 to 5.

8. Use of a reagent for detecting the expression level of RetSat in the preparation of a diabetic nephropathy detection kit or a drug screening kit, characterized in that: The kit comprises primers with detection specificity to kidney tissue or cells.

9. The use according to claim 8, characterized in that: The primer sequences with specific detection of RetSat in kidney tissue are shown in SEQ ID NOs. 3 and 4; the primer sequences with specific detection of RetSat in kidney cells HK2 are shown in SEQ ID NOs. 19 and 20.

10. A diabetic nephropathy diagnostic kit or drug screening kit, the kit comprising a reagent for detecting the expression level of RetSat in a biological sample, the reagent comprising primers with detection specificity for RetSat in kidney tissue, the sequences of which are shown in SEQ ID NOs. 3 and 4; and primers with detection specificity for RetSat in kidney cells HK2, the sequences of which are shown in SEQ ID NOs. 19 and 20.

Images