| The mammalian kidney is one of the most complex organs in the body,mainly composed of a fibrous renal capsule,peripheral cortex and inner medulla.The kidney can filter the fluid and metabolites in the blood to form urine.It is the main organ necessary for adjusting pH and electrolytes and maintaining the overall fluid balance.In addition to these excretion functions,the kidney also produces a variety of hormones and various secretory factors,such as renin,erythropoietin,calcitriol(1,25-dihydroxycholecalciferol)and prostaglandin.Generally,during the embryonic kidney development of higher vertebrates,the pronephrosis,midnephrosis,and metanephrosis will be formed sequentially from beginning to end.The pronephros and mesonephros are temporary organs that absorb and degenerate in the embryonic stage,and the metanephros will eventually develop into mature and permanent urinary organs.Metanephros development is the key to kidney development.Kidney development originates from the mesoderm.Pronephrosis occurs on the 8th day of pregnancy in mice.During embryonic development,except for fish and amphibians,pronephrosis does not have any excretion function in vertebral animals and gradually degenerates.The mesorenal in mice occurred on the 9.5th day of pregnancy,and the mesorenal had no obvious excretion function.Similar to the pronephrosis,as the embryonic development progresses,the mesonephros will be absorbed and degenerated,but the mesonephros and mesonephal tubules will still be left after degeneration,which will be used in the development of the gonads and reproductive tracts of both sexes.Make an important contribution.The metanephros in mice occurs on the 10.5th day of pregnancy,and the metanephros has begun to gradually form during the development of the mesorenal.Metanephrosis will eventually form a mature kidney after growth,development and differentiation.The metanephros contains only ureteral buds,postnatal renal mesenchymal cells and stromal cells.On the 11th day of pregnancy,the ureteral bud invaded the metanephric mesenchyme,and the metanephric mesenchymal cells around the top of the ureteral bud formed cap-like mesenchymal aggregates around the ureteral bud through signal induction,and the induction continued Differentiate into epithelial renal capsules,proliferate and differentiate into comma-shaped bodies,and then elongate into S-shaped bodies.S-shaped body stromal cells near the ureteral bud differentiate into distal renal tubules,and the contralateral epithelial cells differentiate into foot Cells and renal capsules form glomeruli,while the stromal cells at both ends of the S-shaped bodies gradually differentiate into proximal tubules,distal tubules and medullary loops.Similarly,metanephric stromal cells can also induce the branching circulation of ureteral buds through cytokines,thereby developing a complete collecting duct system.The remaining stromal cells in the kidney eventually develop into the kidney capsule,intrarenal mesenchyme and fibrous connective tissue.The metanephrosis of the mouse does not develop into a fully functional mature kidney until the 28th day after birth.The transcription of genes related to development in the kidney is almost stagnant after birth,while the transcription of genes related to kidney differentiation gradually increases,such as the kidney.Functional detoxification related genes and various macromolecular transport genes.When the kidney develops to the adult kidney on the 28th day after birth,the transcription of genes related to kidney differentiation gradually ceases,and some genes in the intercellular signaling pathway begin to increase their own transcription.The normal growth and development of the kidney is very important to the body,and abnormal kidney development is the main cause of kidney disease.In the process of kidney development,there are about hundreds of genes and proteins expressed,mainly various types of transcription factors,growth factors,and adhesion molecules.Studies have shown that abnormal splicing is closely related to the occurrence and development of kidney development-related diseases.For example,splicing defects caused by exon mutations in PKD1 are a new mechanism for the pathogenesis of autosomal dominant polycystic kidney disease.Knockout of the epithelial-specific splicing factor Esrp1 resulted in ureteral bud branching defects and a decrease in the number of nephrons.Splicing mutations due to intron deletion can cause polycystic kidney disease.BUD31 protein(BUD31 homolog)was first discovered in Saccharomyces cerevisiae whole genome sequencing.It is located in the nucleus and is highly conserved in all eukaryotic cells.Proteomics studies based on mass spectrometry have shown that it is an important splicing factor that participates in the formation of NTC(NineTeen Complex or Prp19,Pre-RNA processing factor 19)related complexes.The highly conserved NTC protein complex can stabilize the interaction between snRNA and protein at an early stage through the spliceosome assembly pathway.The splicing factor BUD31 plays an important role in the occurrence and development of a variety of tumors.At present,there are relatively few studies on the role of splicing factor in the development of the kidney and its mechanism.This study provides new clues for the understanding of the molecular level of splicing regulation of kidney development,and also provides new ideas for the early prevention and more precise targeted therapy of chronic kidney disease caused by various kidney development abnormalities.Research content1.The expression of BUD31 in the early kidney development of mice after birth showed a trend of first increasing and then decreasingKidneys of wild-type male mice of different ages(19.5 days of embryonic stage,3 days,2 weeks,and 6 weeks after birth)were collected and sent to the company for sequencing.The overall expression profile of the obtained sequencing data was analyzed by heat map and found that there were 134 The change of BUD31 in the master splicing factor showed a trend of first increasing and then decreasing,and the expression was highest at 2 weeks after birth.The kidney tissues of wild-type mice of different ages were taken,and the mRNA and protein expression of BUD31 were determined by qPCR and Western blotting.The results showed that the expression of BUD31 in the mouse kidney showed a trend of first increasing and then decreasing with the time of mouse development.,And the highest expression level was at 2 weeks.Immunohistochemical staining and immunofluorescence staining,it is also obvious that the expression of BUD31 showed a trend of increasing first and then decreasing.2.Construction of renal tubule-specific knockout mice with Bud31 geneThe Bud31fl/fl female mice constructed in the early stage of the research group were mated with Cdh16-Cre male mice to obtain F1 generation Bud31fl/+;Cdh16-Cre heterozygous male mice were mated with Bud31fl/fl female mice to obtain F2 generation Bud31fl/fl;Cdh16-Cre homozygous knockout mice.Bud31fl/fl mice of the same sex in the same cage were used as controls.After identification of the mouse tail,the kidneys of the homozygous knockout mice and control mice were obtained,and RNA and total protein were extracted.After qPCR and Western blotting experiments,it was found that the expression of BUD31 in the kidneys of homozygous knockout mice was significantly reduced.After immunofluorescence staining and immunohistochemical staining of the kidney tissue,it was seen that the expression of BUD31 in the kidney was significantly reduced.3.Growth retardation of Bud31 renal tubular specific knockout miceFrom the 5th day after birth,the weight of homozygous knockout mice and control mice in the same cage was counted.It was found that the body weight of knockout mice was continuously lower than that of control mice in the same cage.The homozygous knockout mice of different ages also found that their body weight was lower than that of the control mice,and the average kidney weight was also significantly reduced.4.The kidney of Bud31 renal tubule-specific knockout mice has morphological damageThe kidney tissues of 4-week-old homozygous knockout mice and control mice were collected,and the morphological damage of the mouse kidneys was assessed by HE staining.The kidney sections of the kidney tubule-specific knockout Bud31 mice showed increased renal tubule lumen.It is large,and the nucleus of the epithelial cells of the renal tubules has fallen off,showing a phenomenon of vacuolation.PAS staining of kidney tissue sections revealed that the renal pelvic cavity was significantly dilated.By injecting bromophenol blue dye into the renal pelvic cavity,the dye normally flows from the renal pelvis through the ureter and then into the bladder.This proves that the expansion of the renal pelvic cavity is not due to ureteral blockage,but may be due to the kidney.Excessive urine produced by filtration cannot be discharged from the body in time,which causes dilation of the renal pelvis.Inulin was injected through the tail vein,and the glomerular filtration rate of homozygous knockout mice was also decreased according to the glomerular clearance rate of inulin per unit time.At the same time,the urine creatinine also has a decreasing trend,and the blood urea nitrogen increases.5.The number of collecting ducts in the renal parenchyma of Bud31 renal tubule-specific knockout mice decreasesThe kidneys of 6-week-old knockout mice were collected,and the collecting duct marker AQP3,the proximal convoluted tubule marker AQP1 and the distal convoluted tubule marker Calbindin were used to observe the immunohistochemical staining of kidney tissue sections.It was found that the degree of immunohistochemical staining of the collecting duct marker AQP3 in homozygous knockout mice was significantly reduced,while the renal tubule-related markers did not change significantly.6.Bud31 renal tubule-specific knockout mice have obvious polyuria and decreased AQP2 expressionCollect homozygous knockout mice of different ages and control mice in the same cage.Through a 24h metabolic cage experiment,it was found that knockout mice had significantly more urine than control mice.The kidney tissues of 6-week-old homozygous knockout mice and control mice in the same cage were collected,RNA and total protein were extracted,and the expression of AQP2 was reduced by qPCR and Western blotting experiments.Further AQP2-specific immunohistochemical staining and immunofluorescence staining of kidney tissue sections also showed that the expression of AQP2 in the kidney of homozygous knockout mice was significantly reduced,and the reduction of AQP2 was accompanied by the reduction of co-localized AQP3.7.BUD31 affects the proliferation of mouse kidney collecting duct cell line M-1The overexpression and underexpression of BUD31 based on the Tet-on system were constructed and transferred to the M-1 mouse collecting duct cell line,and the in vitro function experiments were carried out.Plate cloning experiments proved that overexpression of BUD31 can significantly promote the ability of collecting duct cells to form colonies and promote the proliferation of collecting duct cells;knockdown of BUD31 can inhibit the ability of collecting duct cells to form colonies and inhibit the proliferation of collecting duct cells.The EdU incorporation experiment again verified that overexpression of BUD31 increases the proportion of EdU-positive cells,while knocking down BUD31 can reduce the proportion of EdU-positive cells.8.Changes in the expression of solute vectors related to urine concentration in the kidney of Bud31 renal tubule-specific knockout miceThe pH measurement of the instant urine of homozygous knockout mice and the same cage control mice found that the urine pH of knockout mice generally decreased.Take the mouse kidney tissue on the 0th day of birth and send it to RNA-seq sequencing.Make a heat map of the relative expression of all the genes whose expression has changed,and select some genes related to urine concentration,and pass the changed genes.The qPCR verification found that NCC and Pendrin had a significant and stable decrease.The three subunits αβγ of the Na+transporter ENaC were also reduced by qPCR verification,and ENaC-specific immunofluorescence staining also showed less ENaC-specific fluorescence.9.Bud31 conditional knockout mice reduce the expression of genes related to kidney developmentThe sequencing results were further analyzed for genes whose expression changed after Bud31 was knocked out,and genes related to kidney development were screened for further verification.The RNA and total protein were extracted from the kidney tissue of homozygous knockout mice.The expression of SHH gene related to mouse kidney development was found to be significantly reduced by qPCR and Western blotting.The kidney tissue was embedded in paraffin,tissue sectioned,and passed Immunohistochemical staining and immunofluorescence staining also found that the expression of SHH gene in conditioned knockout mice was significantly reduced.Knockdown the BUD31 stably transfected cell line to collect the total protein of the constructed mouse kidney collecting duct,and the SHH was also reduced by Western blotting experiment.Research conclusion1.The expression of BUD31 in the early kidney development of mice after birth showed a trend of first increasing and then decreasing;2.Renal tubule-specific Bud31 gene knockout mice have slow growth and development,and the kidneys have morphological damage;3.Tubular-specific Bud31 knockout mice have obvious symptoms of polyuria,their renal parenchymal collecting ducts are abnormally developed,and the expression of AQP2 is reduced;4.Knockdown of BUD31 can inhibit the proliferation of mouse kidney collecting duct cell line M-1;... |
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