Podocytosis treatment target FLOT1 and application thereof

Abstract

The invention relates to a podocytosis treatment target FLOT1 and application thereof, and podocytes are epithelial cells covering glomerulus and have a kidney filtering function. Researches show that podocyte dysfunction can induce serious kidney diseases, namely podocyte diseases. Over activation of the mTORC1 signal has been proved to induce podocyte dysfunction, but the molecular mechanism of the upstream signal of mTORC1 activation in podocyte is not clear yet. According to the invention, the FLOT1 protein participates in inhibiting the activation process of mTORC1, so that the FLOT1 is a good therapeutic target for podocytosis.

Description

Technical Field

[0001] The present invention relates to the application of FLOT1 protein as a drug target in drugs for treating kidney diseases, and in particular to the application of FLOT1 protein as a drug target in treating mTOR1-dependent podocytosis. Background Art

[0002] Podocytes are terminally differentiated glomerular epithelial cells. The foot processes of adjacent podocytes interlace with each other to form a slit diaphragm, which maintains the normal filtration function of the glomerulus. The reduction in the number of podocytes or the loss of their function is an important cause of damage to the glomerular filtration function. In severe cases, it can lead to glomerular lesions and related kidney diseases, and this damage is irreversible. Kidney diseases caused by podocyte dysfunction are called podocytosis (REF).

[0003] Podocyte lesions occur for a variety of reasons. Dysregulated activity of mTORC1, a serine / threonine protein kinase that regulates cell growth and proliferation, is a hallmark of diabetic nephropathy. In the early stages of diabetes, mTORC1 is significantly activated. In response to mTOR activation, podocytes hypertrophy, foot processes efface, and eventually detach from the glomerular basement membrane (GBM), ultimately leading to a decrease in podocyte number and glomerulosclerosis. Therefore, the molecular mechanisms of mTORC1 activation in podocytes are crucial.

[0004] CDR can serve as a signaling platform for the AKT-mTORC1 pathway in glomerular podocytes. CDR is a circular dorsal fold induced by growth factors, which subsequently forms a "cup-shaped" structure, thereby activating PI3K to produce PIP3, further activating PLCγ and AKT. PLCγ produces DAG in the "cup-shaped" structure, thereby activating PKC and Ras, which simultaneously promote the closure of the "cup-shaped" structure to form macropinosomes. Extracellular nutrients are internalized into cells through the fusion of macropinosomes and lysosomes, inducing the activation of Rag in this process, and then mTORC1 is recruited to lysosomes. At the same time, activated AKT directly phosphorylates the TSC complex through a cytoplasmic pathway independent of macropinocytosis, leading to the activation of Rheb, which directly activates mTORC1 on the lysosomal membrane.

[0005] Macropinosomes (MPs) can be used as a good therapeutic target. EIPA, as an inhibitor of macropinocytosis, can effectively inhibit the production of macropinosomes, but the side effects are still unclear. Therefore, other targeted molecular inhibitors are needed.

[0006] The present application discovered FLOT1, a membrane protein enriched in cell membrane lipid rafts. The FLOT1 protein is involved in macropinocytosis. After the protein is knocked out, the number of macropinosomes (MPs) is reduced. Therefore, FLOT1 can be used as a drug target for the prevention and treatment of mTORC1-dependent podocyte disease. Summary of the invention

[0007] The purpose of the present invention is to provide a new podocytopathy therapeutic target FLOT1, and the FLOT1 protein can be used as a drug target for treating podocytopathy.

[0008] The present invention provides a method for using FLOT1 protein as a drug target in a drug for treating podocyte lesions, and provides cell biological and biochemical data, indicating that the loss of FLOT1 can inhibit the mTORC1 pathway without affecting cell viability.

[0009] Application of FLOT1 as a target in drugs for the prevention and / or treatment of podocytopathy.

[0010] FLOT1, a target that can inhibit mTORC1 activation.

[0011] Use of a substance for silencing, knocking out or mutating FLOT1 protein or a substance for inhibiting the expression of FLOT1 protein in any of the following a1)-a3):

[0012] a1) Preparation of products for the treatment or adjunctive treatment of podocytosis;

[0013] a2) preparing products for inhibiting podocytopathy;

[0014] a3) preparing products for inhibiting proliferation of podocytes.

[0015] Furthermore, FLOT1 knockout cells were constructed. During the knockout process, the primer sequences were designed to be any of the following:

[0016]

[0017] Furthermore, knockout of FLOT1 did not affect the number and size of CDRs, but the number of macropinosomes was significantly reduced.

[0018] Furthermore, loss of FLOT1 inhibited the mTORC1 pathway without affecting cell viability.

[0019] The substance that inhibits the activity of FLOT1 protein or the substance that reduces the content of FLOT1 protein is any one of the following a1)-a3)

[0020] Applications:

[0021] a1) Preparation of products for the treatment or adjunctive treatment of podocytosis;

[0022] a2) preparing products for inhibiting podocytopathy;

[0023] a3) preparing products for inhibiting proliferation of podocytes.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention uses CRISPR-Cas9 gene knockout technology to identify the role of FLOT1 in the mTORC1 activation mechanism. In podocytes, mTORC1 is regulated by the macropinocytosis process. The present invention confirms that FLOT1 is recruited into macropinocytosis, FLOT1 participates in the mechanism of macropinocytosis formation, and the loss of FLOT1 affects the efficiency of macropinocytosis closure to form macropinocytic bodies, while the loss of macropinocytic bodies weakens the activation of mTORC1, and thus can be used for the inhibition and treatment of podocyte pathology. At the same time, knocking out FLOT1 protein in podocytes does not affect the cell survival state. Therefore, the new target of the present invention can inhibit the pathology of podocytes without affecting the cell survival state. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Live cell imaging of the present invention shows that growth factor stimulation induces the formation of CDRs in podocytes. CDR produces 1-2 macropinosomes. (AB) EGF stimulation. (CD) PDGF stimulation.

[0027] Figure 2 Western blot analysis for the present invention showed that the macropinocytosis inhibitor EIPA blocked growth factor-stimulated pS6K, an output of mTORC1 activation.

[0028] Figure 3 Confocal microscopy showed that FLOT1 is localized in the growth factor-induced CDR of podocytes. Actin was used to label the CDR.

[0029] Figure 4 Western blot analysis confirmed the expression of GFP-FLOT1 in podocytes.

[0030] Figure 5 The expression of GFP-FLOT1 in podocytes was confirmed using FLOT1 antibody. (A) Confocal image of cells expressing GFP-FLOT1. (B) Confocal image of cells expressing GFP as a negative control.

[0031] Figure 6 GFP-FLOT1 is localized in the CDRs of podocytes.

[0032] Figure 7 Generation of FLOT1-KO podocytes. (A) Western blot analysis. (B) Confocal images.

[0033] Figure 8Knockdown of FLOT1 has no effect on the number and size of CDRs. (A) Phase images of growth factor-stimulated CDRs (arrows) in control (Scramble) and FLOT1KO cells. (B) Comparison of the number of CDRs in control and KO cells 60 minutes after stimulation. (C) Comparison of the size of CDRs in control and KO cells.

[0034] Fig. 9 Loss of FLOT1 affects the number of macropinosomes. (A) FDx / phase images of macropinosomes generated after growth factor stimulation (arrows indicate green positive vesicles). (B) Comparison of the number of macropinosomes in control and KO cells 60 minutes after stimulation.

[0035] Fig.10 Loss of FLOT1 affects growth factor-stimulated pS6K, an output of mTORC1.

[0036] (A) Representative western blot images of pS6K / S6K / FLOT1 / tubulin. (B) Quantification of pS6K / S6K ratio from three independent results.

[0037] Fig.11 .FLOT1 is not important for cell growth. Cell proliferation assays showed that cells without FLOT1 (KO) could still grow. DETAILED DESCRIPTION

[0038] See also Figures 1 to 11 , and as shown in Table 1:

[0039] Experiment 1: Live cell imaging confirms the generation of macropinosomes in podocytes ( Figure 1 )

[0040] (1) Cell culture, growth factor stimulation

[0041] The podocytes MPC5 were inoculated in a living cell dish, and after the cells adhered to the wall, growth factors EGF (16 nM) and PDGF (2 nM) were added for treatment, and then immediately observed under a microscope.

[0042] (2) Fully automatic living cell workstation microscope observation

[0043] ① Turn on the cell culture system controller switch to keep the carbon dioxide stable below 0.1Mpa.

[0044] ② Add an appropriate amount of high-pressure water to the cell culture system and place the cells in.

[0045] ③Open the imaging system control software and precool the CCD.

[0046] ④ Find the appropriate field of view under the microscope, set relevant parameters, and perform live cell imaging.

[0047] (3) Results:

[0048] Figure 1 Figure AB is a diagram of the process of growth factor EGF stimulation, and Figure CD is a diagram of the process of growth factor PDGF stimulation. Live cell imaging results show that after the addition of growth factors, a large annular wrinkled structure will first form on the surface of podocytes, which is called the circular dorsal wrinkle (CDR). Then this structure will continue to shrink toward the middle and finally close to form 1 to 2 bright vesicles, namely macropinosomes. The process from the generation of CDR to the formation of macropinosomes is called macropinocytosis.

[0049] Experiment 2: Western blot analysis confirmed that the macropinocytosis inhibitor EIPA blocked mTORC1 activation ( Figure 2 )

[0050] (1) Cell culture

[0051] The culture medium used for podocyte culture was RPMI-1640 medium, which contained 10wt% FBS and 1% vol penicillin-streptomycin. On days 10 to 14, podocytes were basically differentiated and mature. In this study, day 14 podocytes were used for protein extraction, immunofluorescence and other experiments.

[0052] According to the experimental requirements, 6-well plates were prepared and podocytes on day 12 were plated at a density of 60%-70%. The cells were divided into a control group and an EIPA-treated group, and each group was treated with growth factor EGF for 0, 30, and 60 minutes, respectively.

[0053] (2) Cell lysis and protein extraction

[0054] Prepare cell lysis buffer in advance (add 500 μL 2xCHAPS, 50 μL protease inhibitor, 450 μL ddHO for every 1 ml cell lysis buffer). 2 O), rinse the pretreated cells in the 6-well plate with PBS, add 120 μL lysis buffer when the cell density is about 80%, make it completely infiltrate the cells, let it stand on ice for 15 minutes, and collect the cells into the corresponding pre-cooled 1.5 mL EP tube with a scraper. Centrifuge at 4°C, 12000 rpm for 10 minutes, take the supernatant and add 5× SDS-PAGE loading buffer to dilute to 1×, mix well, and heat in a 100°C metal bath for 5 minutes to denature the protein.

[0055] (3) Western blotting

[0056] ① Electrophoresis: The electrophoresis gel used in the experiment is a 15-well plate precast gel. Before electrophoresis, place the protein sample in a 100℃ metal bath to heat and cook the sample for 5 minutes. Add the marker and the prepared sample to each well in turn, 10μL per well. Set the voltage to 80V first. When the marker migrates to a clear band, set the voltage to a constant voltage of 120V. When the sample is close to the bottom of the gel, turn off the power and prepare for membrane transfer.

[0057] ② Transfer: Place the transfer tank in a box containing an ice-water mixture, put in a transfer splint, and add ice bags to prevent the glue and film from being too hot.

[0058] ③ Blocking: Block in 5% skimmed milk powder solution at room temperature on a shaker for 1 hour.

[0059] ④ Primary antibody incubation: Add the primary antibody with 5% BSA into the incubation box, slowly put the cut PVDF membrane in, let the antibody completely cover the membrane, and incubate in a 4℃ refrigerator for 1 night. The next day, put the new PVDF membrane into a new incubation box and wash the membrane 6 times with 1×TBST, 8 minutes each time.

[0060] ⑤ Secondary antibody incubation: dilute the secondary antibody in a certain ratio (according to the existing technical instructions, such as 1:500) and add it to the small long box, incubate it at room temperature for 2 hours on a decolorizing shaker. Wash the membrane 6 times with 1×TBST solution, 8 minutes each time.

[0061] ⑥ Exposure: Take out the PVDF membrane with tweezers, place it on the imaging plate, press it slowly and gently until there are no bubbles, prepare ECL color developing solution (prepare it as needed, the specific formula is the existing technology), evenly add it onto the PVDF membrane, and use the gel imaging system for exposure.

[0062] ⑦After the experiment, seal the PVDF membrane with plastic wrap and store it at 4°C.

[0063] If the experiment needs to detect different proteins on the same membrane, wash the PVDF membrane with 1×stripping buffer for 20 minutes, wash the first incubated antibody, and then wash the membrane with 1×TBST solution. Block the PVDF membrane in 5% vol skim milk powder solution for 1 hour. Add another protein antibody dilution to be investigated and incubate in a refrigerator at 4℃ overnight. Take it out the next day and repeat the above steps ⑤~⑦.

[0064] (4) Results

[0065] WB results showed that after treatment with the macropinocytosis inhibitor EIPA, the phosphorylation level of S6K, a downstream signal of mTORC1, was significantly reduced, confirming that the macropinocytosis inhibitor EIPA could block the activation of mTORC1. At the same time, the pAKT level decreased slightly, while the pERK (phosphorylation-activated ERK) used as the control group was not significantly affected.

[0066] Experiment 3: Confocal microscopy was used to detect the expression of FLOT1 in podocyte CDRs. Cell immunofluorescence was used to test the production of IF and GFP-FLOT1 plasmid.

[0067] (1) Cell culture, GF stimulation (growth factor stimulation)

[0068] Prepare a 24-well plate, place a 14mm cell slide into the well, and rinse the slide twice with PBS. Inoculate wild-type podocytes into the 24-well plate at a density of 20%, culture overnight, and allow the cells to grow evenly on the slide. Treat with growth factors EGF and PDGF for 5 minutes, respectively, and use a control group without growth factor stimulation. Then perform immunofluorescence experiments.

[0069] (2) Fixation, staining, and confocal observation

[0070] ① Fix cells: Prepare a 4% vol paraformaldehyde fixative solution prepared in advance, add it to the pre-treated 24-well plate, and fix it at room temperature for 20 minutes. Wash the slides with sterile TBST solution to remove excess paraformaldehyde.

[0071] ② Penetration: Dilute TritonX-100 with sterile PBS solution to a concentration of 0.1% vol. Penetrate for 5 minutes at room temperature. After penetration, wash three times with sterile TBST solution, each time for 10 minutes.

[0072] ③ BSA blocking: Add freshly prepared 5% vol BSA solution filtered with a 0.22 μm filter membrane and incubate on a shaker at room temperature for 30 minutes.

[0073] ④ Primary antibody incubation: Dilute the antibody to be used in an appropriate ratio with 5% vol BSA, invert the cell surface of the cell slide on the antibody dilution solution, and incubate at 4°C for 18 hours. After overnight incubation, wash the cell slide three times with sterile TBST at room temperature, each time for 10 minutes.

[0074] ⑤ Fluorescent secondary antibody incubation: dilute the fluorescent secondary antibody to 1:500 using 5% vol BSA, incubate at room temperature in the dark for 2 hours, and wash the sample with sterile TBST.

[0075] ⑥Actin staining: After the fluorescent secondary antibody incubation, wash the sample three times with sterile TBST, 5 minutes each time, dilute Rhodamine with 5% volBSA at a ratio of 1:100, and incubate at room temperature in the dark for 1 hour. After incubation, wash the cell slides three times with sterile TBST at room temperature, 10 minutes each time.

[0076] ⑦Seal the slides: Use a sealing medium containing DAPI to seal the slides at room temperature in the dark.

[0077] ⑧Microscope observation: The samples were observed and detected using the confocal microscope Leica TCSSP5 on the large instrument platform of Nankai University.

[0078] (3) Production, transfection, fixation, staining, and confocal observation of GFP-FLOT1

[0079] ① Generation of GFP-FLOT1: The protein sequence of mouse FLOT1 was searched in the NCBI database, and primers were designed based on this sequence. The cDNA library obtained from podocytes was used as a template to amplify the FLOT1 gene fragment. Subsequently, two pairs of primers with partial repeat sequences were designed based on the sequences of FLOT1 and GFP plasmid vectors, and GFP-FLOT1 plasmid was obtained by homologous recombination.

[0080] ② Transfection: Wild-type podocytes were seeded in 24-well plates at a cell density of 30%. After 24 hours, 0.2 μg of GFP-FLOT1 plasmid and 0.8 μl of transfection reagent Genetwin were added to each well, incubated at 37°C for 6 hours, and then low-glucose culture medium was added for 18 hours.

[0081] ③ Fixation, staining, and confocal observation: Treat with growth factor EGF for 5 minutes, and set up a control group without growth factor stimulation. Add the 4% vol paraformaldehyde fixative prepared in advance to the pretreated 24-well plate and fix at room temperature for 20 minutes. Wash the slides with sterile TBST solution to remove excess paraformaldehyde. The staining and confocal observation steps are the same as in step (2) above.

[0082] (4) Results

[0083] Figure 3 This is an immunofluorescence image of the endogenous FLOT1 protein (obtained in step (2)). The results show that 5 minutes after the addition of growth factors EGF and PDGF, CDR can be induced on the surface of podocytes. The co-localization of FLOT1 and Actin suggests that FLOT1 plays a certain role in macropinocytosis. Figure 4 , Figure 5 This is the result of the effect verification of GFP-FLOT1. GFP-FLOT1 and endogenous FLOT1 showed a consistent distribution pattern. After exogenous expression of GFP-FLOT1 in podocytes, its role in CDR was verified again. Consistent with the results of endogenous FLOT1, GFP-FLOT1 can be recruited to the CDR structure after growth factor stimulation.

[0084] Experiment-4: Establishment of FLOT1KO cells to confirm that FLOT1 plays an important role in macropinosomes but has no effect on CDR ( Figure 7-9 )(Table 1).

[0085] Table 1 FLOT1KO primer list

[0086]

[0087] (1) Construction of FLOT1 knockout cells

[0088] ① Design primer sequences, as shown in Table 1

[0089] ② 9 μL of 100 μM forward and reverse primers and 2 μL of 10×T4 ligation buffer, place in a PCR instrument for annealing.

[0090] ③7μL annealing product, 1μL digested Lentiviral CRISPR, 1μL 10×T4 ligase, 1μT4 ligase, mix well and put into PCR instrument at 16℃ for 2 hours for ligation reaction.

[0091] ④Transformation and sequencing: The ligation product was transformed using DH5α, spread on LB solid culture medium, single clones were picked and shaken, and the plasmid was extracted from the bacterial solution with the correct sequence after bacterial solution sequencing.

[0092] ⑤ Plasmid transfection: After 24h of passage of HEK293T cells for plasmid transfection, plasmid transfection was performed when the cell density grew to 50%-60%. Prepare sgRNA plasmids for knockout of FLOT1 of different targets, use empty lentiCRISPRv2 plasmid as control vector, and packaging plasmids pMD2G and psPAX2. Take out the plasmid to be transfected from -20℃, melt at room temperature, and mix with a pipette. The transfection system is divided into Group A: add 500μL Opti-MEN reduced serum medium, then co-transfect 4μg sgRNA plasmid or empty lentiCRISPRv2 plasmid, 1μg pMD2G and 3μg psPAX2, and mix with a pipette. Group B: add 500μL Opti-MEN reduced serum medium, add Lipofectamine2000 transfection reagent according to the ratio of plasmids, and mix with a pipette. Then slowly add the liquid of group A to the corresponding group B and let it stand at room temperature for 20 minutes. During this time interval, replace the medium of HEK293T cells to be transfected, and replace 5mL of serum-free and dual-antibody-free DMEM medium for each culture dish. Slowly and evenly add the AB mixture to the cell culture dish and replace the medium after 6-8 hours.

[0093] ⑥ Packaging virus: Collect the DMEM medium used to culture HEK293T cells after plasmid transfection as the virus solution. 24 hours after the plasmid transfection medium change, collect the supernatant medium into a centrifuge tube and add new preheated DMEM medium. 48 hours after the plasmid transfection medium change, collect the supernatant medium again into the original centrifuge tube. Centrifuge the collected supernatant at 800g for 5 minutes at room temperature, and the supernatant is the virus solution. Use a 0.45μm filter membrane and a 10mL sterile syringe to filter the collected virus solution into a new centrifuge tube, aliquot and store in a -80℃ refrigerator.

[0094] ⑦ Plant the cells in a 6 cm cell culture dish, add 1.5 mL of virus stock solution, and add Polybrene at a ratio of 1:1000 to improve transfection efficiency. After 4-6 hours, replace with normal complete culture medium.

[0095] 5 μg / mL puromycin was added 36 hours after infection to select positive cells.

[0096] ⑧After positive cells were lysed and proteins were extracted, the expression level of FLOT1 protein was detected by Western Blot to verify the knockout efficiency.

[0097] (2)FDx

[0098] FLOT1 knockout cells and wild-type podocytes were plated into a 24-well plate with a cell slide. After the cells adhered, they were treated with low-glucose medium for 18 h, and then growth factor EGF and fluorescent marker FDx70 (0.5 mg / mL) were added. The cells were incubated at 37°C with a time gradient of 0, 5, 10, 15, 30, and 60 minutes.

[0099] (3) Fixation and observation

[0100] Add the pre-prepared 4% vol paraformaldehyde fixative to the pre-treated 24-well plate and fix at room temperature for 20 minutes. Wash the slides with sterile TBST solution to remove excess paraformaldehyde, seal the slides, and observe under a confocal microscope.

[0101] (4) Results

[0102] Figure 7 This is the result of the knockout efficiency verification of FLOT1. Combined with the results of WB and immunofluorescence, it shows that the FLOT1 knockout podocyte line was successfully constructed. Subsequently, in order to prove the role of FLOT1 in macropinocytosis, the number of CDRs in podocytes after FLOT1 knockout and wild-type podocytes was counted. Figure 8The results showed that after FLOT1 knockout, the number and size of CDRs did not change, but the number of CDRs internalized into macropinosomes changed. Therefore, FDx70 was used as a marker of macropinosomes to count the number of macropinosomes in podocytes after FLOT1 knockout and in wild-type podocytes. Fig. 9 The statistical results showed that 15 minutes after the addition of growth factors, FLOT1-knockout podocytes showed reduced macropinocytosis efficiency compared with wild-type podocytes, and this reduction effect was most obvious at 60 minutes.

[0103] Experiment 5: Western blot analysis confirmed that FLOT1 is important for mTORC1 activation ( Fig.10 )

[0104] (1) Growth factor treatment after cell culture

[0105] According to the experimental requirements, 6-well plates were prepared and podocytes were plated at a density of 60%-70% on day 12. According to the experimental design, the cells were divided into Scr group and FLOT1-KO group, and each group was treated with growth factor EGF for 0, 5, 10, 15, 30, and 60 minutes.

[0106] (2) Scrape and lyse the cells, extract the protein, and perform Western Blot.

[0107] (3) Results

[0108] The previous results ( Figure 2 ) have shown that inhibition of macropinocytosis affects the activity of mTORC1, resulting in decreased levels of pS6K. Fig. 9 The results showed that FLOT1 knockout inhibited macropinocytosis. In this example, WB was used to detect the downstream pS6K of mTORC1. The WB results ( Fig.10 ) showed that after FLOT1 knockout, pS6K was significantly reduced, that is, nTORC1 function was inhibited, thus determining that FLOT1 knockout would inhibit mTORC1.

[0109] Experiment 6: Cell growth experiments showed that FLOT1 is not important for cell growth. Fig.11 )

[0110] (1) Cell culture

[0111] 2×10 4 100 μL of cell suspension was inoculated in a 96-well plate with 3 replicate wells per group. Cell proliferation was detected at the same time point 1-4 days after the cells were inoculated in the 96-well plate.

[0112] (2) Cell growth assay

[0113] 10 μL of CCK8 solution was added to each well, and the cells were cultured in a 37°C cell culture incubator for 2 hours. The absorbance at 450 nm was measured using an enzyme-labeled instrument.

[0114] (3) Results

[0115] The results of CCK8 showed that although podocytes after FLOT1 knockout showed a slower growth rate compared with wild-type cells, FLOT1 knockout did not affect podocyte survival, suggesting the potential feasibility of FLOT1 as a possible therapeutic target for podocyte disease.

[0116] Any matters not described in the present invention are applicable to the prior art.

Claims

1. Application of FLOT1 as a target in drugs for the prevention and / or treatment of podocytopathy.

2. FLOT1, a target that can inhibit mTORC1 activation.

3. Use of a substance that silences, knocks out or mutates FLOT1 protein or a substance that inhibits the expression of FLOT1 protein in any of the following a1)-a3): a1) Preparation of products for the treatment or adjunctive treatment of podocytosis; a2) preparing products for inhibiting podocytopathy; a3) preparing products for inhibiting proliferation of podocytes.

4. The use according to claim 2, characterized in that: To construct FLOT1 knockout cells, during the knockout process, the primer sequence is designed to be any of the following:

5. The use according to any one of claims 1 to 4, characterized in that: FLOT1 knockout did not affect the number and size of CDRs, but the number of macropinosomes was significantly reduced.

6. The use according to claim 5, characterized in that: Loss of FLOT1 inhibits the mTORC1 pathway without affecting cell viability.

7. Use of a substance that inhibits FLOT1 protein activity or a substance that reduces FLOT1 protein content in any of the following a1)-a3): a1) Preparation of products for the treatment or adjunctive treatment of podocytosis; a2) preparing products for inhibiting podocytopathy; a3) preparing products for inhibiting proliferation of podocytes.

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