| Glomerular disease is a kind of kidney disease with proteinuria,edema,hematuria,and hypertension as the main clinical manifestations.It is the main cause of chronic kidney disease(CKD)in China.Proteinuria is the most basic clinical manifestation of glomerular disease and an independent risk factor for the progression of chronic kidney disease.In mammalian kidney,the outermost filtration membrane composed of podocytes and the slit diaphragm between podocytes is mainly involved in regulating the filtration of proteins during the blood circulation in the glomerulus.The main function of the proximal renal tubules is to reabsorb albumin filtered from the glomerular filtration barrier.In Drosophila,pericardial nephrocyte and mammalian podocytes have significant similarities in structure,molecule and function.They both have the function of filtering and absorbing proteins,which can be used as a good model to research podocyte-related diseases.The structure of the mammalian podocyte slit diaphragm is the main component of the glomerular filtration barrier,and its structural damage is closely related to the occurrence of proteinuria and the disease progression of nephrotic syndrome.Podocyte slit diaphragm is not only a physical barrier for glomerular filtration of blood components,but also an important signal transduction platform for podocytes,which can dynamically change its signal transduction and endocytosis through the phosphorylation of slit diaphragm protein nephrin.In cultured podocytes,dephosphorylation of Y1193,a conserved residue of nephrin,can trigger βarrestin2-mediated clathrin dependent endocytosis of nephrin.In the study of mouse model,it was found that the damage of podocyte specific dynamin,synaptojanin and endophilin would lead to the defect of endocytosis,foot process fusion,and heave proteinuria in mice,which eventually developed into renal failure.Recent studies have confirmed that several gene mutations related to endocytosis were found in the gene research of patients with nephrotic syndrome,suggesting that endocytosis may play an important role in maintaining normal glomerular function.However,due to the limitation of rodent model,it is very difficult to explore the structure and function of slit diaphragm in mammalian kidney with high resolution image.Therefore,it is not clear whether endocytosis is involved in maintaining the structure and function of slit diaphragm in vivo,and what the specific mechanism is.Part one:Role of clathrin mediated endocytosis in the endocytosis of slit diaphragm proteins in Drosophila nephrocyte and its molecular mechanismObjectiveIn Drosophila nephrocyte model system,we specifically silenced the coding genes of CME key factors,observed the effect of CME on the expression and localization of slit diaphragm protein,detected whether the function and ultrastructure of nephrocyte changed,and comprehensively analyzed the role of CME pathway in maintaining slit diaphragm integrity.Methods1.Bioinformatics methods were used to search papers,gene ontology,Harward DRSC,Gene 2 Function,MARRVEL,OMIM,FlyBase and other database resources to screen the genes possibly related to clathrin mediated endocytosis(CME)and clathrin independent endocytosis(CIE)and their homologous genes in Drosophila.2.Dot-GAL4 system was used to hybridize with UAS-geneRNAi system.Gene mutation was induced by transgenic Drosophila to silence the expression of key genes of CIE pathway.The filtration and absorption function of nephrocyte was detected by endogenous fluorescent fusion protein tracing and exogenous 10 KD Texas-Dextran uptake.The expression and localization of slit diaphragm were detected by immunofluorescence staining.3.Dot-GAL4/UAS-geneRNAi system was used to silence the expression of clathrin and its regulatory factors in fly nephrocyte.The function of nephrocyte was detected by tracing endogenous and exogenous fluorescent proteins.The expression and distribution of slit diaphragm protein was detected by immunofluorescence.The ultrastructural changes of nephrocyte were observed by transmission electron microscope.4.The activation time of RNAi in GAL4 system was controlled by GAL80ts,a temperature sensitive driver,to selectively silence the expression of key factors of CME pathway in Drosophila adult nephrocyte.The localization and expression of slit diaphragm protein were observed by immunofluorescence.Results1.After specifically silencing Arf79F,one of the key factors of CLIC/GEEC pathway,the results of immunofluorescence showed that Pyd protein was regularly distributed on the surface of cell membrane,showing a fingerprint like pattern,and the expression and localization of Pyd protein had no significant change.2.After silencing the expression of clathrin light chain(Clc),clathrin heavy chain(Chc)and shibire,immunofluorescence results showed that Pyd and Ssn were disorderly distributed on the cell surface,and multiple aggregation regions of Pyd and Sns were observed in the cytoplasm.The results of fluorescent protein tracing showed that the nephrocyte absorbed endogenous haemolymph protein and exogenous dextran particles decreased significantly,and the nephrocyte showed obvious dysfunction.3.Specifically silencing the expression of shibire in nephrocyte,and then Immunofluorescence co-staining of Pyd and Clc in nephrocyte.The results showed that the relationship between slit diaphragm Pyd and clathrin structure changed,and Pyd was encapsulated by the ring formed by Clc,which directly confirmed the endocytosis of Pyd through CME pathway.4.The expression of AP-2,AP-1,lap,aux and hsc70-4 was specifically silenced.The results of immunofluorescence and fluorescent protein tracing showed that after interfering with the expression of AP-2,aux and hsc70-4,the localization of Pyd and Sns was abnormal,and the uptake and absorption function of nephrocyte was seriously impaired.After interference on the expression of AP-1,the expression and localization of slit diaphragm protein and the function of nephrocyte were not significantly improved.However,the distribution of Pyd and Sns on the surface of nephrocyte was not affected.5.Using the GAL80ts system to control the phase of gene interference targeting clathrin in nephrogenocytes,the results of immunofluorescence showed that the expression of clathrin light chain silenced by gene silencing led to the abnormal distribution of Pyd on the cell surface,and some proteins were mislocated in the cytoplasm.6.The results of transmission electron microscopy showed that the ultrastructural changes of nephrocyte were obvious after RNA interference on shi and Clc in fly nephrocyte,which were the key factors of CME pathway.At the same time,multiple vesicles could be seen in the nephrocyte cytoplasm,suggesting that CIE pathway might be involved in the endocytosis of slit diaphragm proteins.Conclusions1.The Drosophila nephrocyte model is a suitable model system for studying the endocytosis mechanism of podocyte slit diaphragm proteins.2.Specifically silencing the key genes of clathrin mediated endocytosis can lead to the irregular distribution of slit diaphragm proteins Pyd and Sns on the cell surface,and mislocalization in the cytoplasm,resulting in dysfunction of Drosophila nephrocyte.3.Specific silencing of key genes involved in clathrin mediated endocytosis can lead to significant ultrastructural changes in Drosophila nephrocyte.4.Clathrin mediated endocytosis is a necessary condition for mature nephrocyte to maintain the structural and functional integrity of slit diaphragm.5.It can not be ruled out that clathrin-independent endocytosis is involved in maintaining the dynamic balance of slit diaphragm.Part two:Role of ESCRT complex in the intracellular transport and sorting of slit diaphragm protein and its molecular mechanismObjectiveThe expression of ESCRT-0,Ⅰ,Ⅱ,Ⅲ complexes and their regulatory factors were specifically silenced by Drosophila nephrocyte model.The structure of slit diaphragm and the expression distribution of slit diaphragm protein were detected,and the function of nephrocyte was evaluated.The main regulatory factors of recognition and sorting after slit diaphragm protein endocytosis were analyzed.Methods1.Through literature and database search,we screened the genes that might be involved in the recognition and sorting of slit diaphragm protein after endocytosis and the homologous genes in Drosophila.2.Dot-GAL4 was used as the driver to specifically silence the coding genes of ESCRT-0 complex in Drosophila nephrocytes.Immunofluorescence colocalization staining was used to detect the distribution of ubiquitin and slit diaphragm protein and their relationship.The endogenous fusion protein MHC-ANF-RFP and exogenous 10 KD dextran were used to trace the filtration and absorption function of Drosophila nephrocytes.3.Dot-GAL4 was used as the driver,RNAi was used to silence the coding genes of ESCRT-Ⅰ,ESCRT-Ⅱ and ESCRT-Ⅲ complexes.The distribution of ubiquitin and slit diaphragm protein was detected by immunofluorescence,and the function of nephrocyte was evaluated by endogenous and exogenous fluorescent protein tracing.Results1.After silencing the expression of Hrs or Stam,immunofluorescence showed that a large number of ubiquitin molecules were abnormally aggregated on thenephrocyte,and the distribution of slit diaphragm protein was disordered,suggesting that ubiquitin recognition was impaired and could not be further sorted and transported.The results of endogenous and exogenous tracer tracing experiments showed that the filtration function of nephrocyte was seriously damaged and the cell volume was significantly increased.2.After silencing the expression of the regulatory factors of ESCRT-0,Mop and Ubpy,the filtration function of nephrocyte decreased significantly,the cell volume increased,the distribution of slit diaphragm protein was abnormal,and the structure of slit diaphragm was destroyed.3,After the specific silencing of the genes encoding Vps37a,the subunit of ESCRT-Ⅰ,Vps36 and Vps25,the subunits of ESCRT-Ⅱ,and shrb,the subunit of ESCRT-Ⅲ,the abnormal aggregation of ubiquitin molecules on the cells,the abnormal distribution and mislocalization of slit diaphragm protein,the dysfunction of filtration and uptake,and the significant increase of cell volume were observed.4.After silencing the expression of genes encoding other subunits of ESCRTs,there was no significant change in the function and the structure of slit diaphragm.Conclusions1.ESCRT-0 and its regulatory factors Mop and Ubpy are involved in the recognition and sorting of slit diaphragm proteins after endocytosis.2.The subunits Vps37a,Vps36,Vps25 and shrb of ESCRTs complexes are necessary to maintain the structural and functional integrity of the slit diaphragm.3.Shrb may be an important factor involved in the degradation pathway of metabolites in Drosophila nephrocyte.4.Drosophila nephrocyte model is an efficient model for large-scale screening of pathogenic genes of kidney disease.Part three:the role and regulation of endosomes and exocysts complex in the transport and circulation of slit diaphragm proteinsObjectiveUsing Drosophila model,the coding genes of Rab GTPases and exocysts complex were specifically silenced in nephrocyte,and the effects of Rab GTPases and exocysts on the structure and function of slit diaphragm were studied.Methods1.Bioinformatics methods were used to screen the genes that may be related to the transport and recycling pathway of slit diaphragm proteins and their homologous genes in fly.2.Dot-GAL4 was used as a specific driver.RNAi was used to interfere with the expression of key factors of early endosome,late endosome,retromer and recycling endosome in nephrocyte.Fluorescent protein uptake tracing and immunofluorescence were used to detect the function of nephrocyte,the basic structure and protein distribution of slit diaphragm,and the main transport and sorting pathways of slit diaphragm after protein endocytosis were analyzed.3.Using Dot-GAL4/UAS-geneRNAi system,the coding genes of exocysts complex were specifically silenced in nephrocytes.The function of nephrocytes was evaluated by tracing the endogenous and exogenous fluorescent proteins.The basic structure and membrane protein distribution of the slit diaphragm were detected by immunofluorescence.Results1.After specific silencing of Rab5 gene expression in early endosome,immunofluorescence showed that Pyd was disorderly distributed on the cell surface and mislocated in the cytoplasm.Fluorescent protein uptake tracing showed that the function of early endosome was seriously damaged.2.After the specific interference the expression of Rab7,Vps29,Vps26 and Vps35,the key factors of late endosome and retromer,the fluorescent protein uptake tracing experiment showed that the filtration and reabsorption function of nephrocyte was normal.The immunofluorescence results showed that the distribution of Pyd on the surface of nephrocyte was uniform and regular,and the structure of slit membrane was complete,suggesting that late endosome to the Lysosome pathway and retromer to Golgi pathway are not the main transport direction of slit diaphragm proteins in nephrocyte.3.The results showed that after silencing Sec5,Sec6,Sec10,Sec15 and Exo84 genes expression,immunofluorescence showed that the cell surface distribution of slit diaphragm protein Pyd was abnormal,and part of Pyd protein mislocated in the cytoplasm.Exogenous dye particles and endogenous protein uptake tracing experiments showed that the filtration and absorption function of nephrocyte was decreased significantly.Conclusions1.The slit diaphragm of Drosophila nephrocyte needs a balanced endocytosis and recycling pathway to maintain its structural integrity.2.After physiological endocytosis from the plasma membrane,slit diaphragm proteins enter the early endosome under the regulation of Rab5,and then are mainly sorted and transported to the recycling endosome controlled by Rab11.3.Lysosomal degradation and Golgi transport are not the main transfer pathways after slit diaphragm protein endocytosis.4.Rab11 interacts with exocysts to make the recycling endosomes tethered to the nephrocyte membrane.After membrane fusion,the slit diaphragm proteins return to the slit diaphragm structure. |
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