| ObjectiveWith the improvement of human life and medical care level,the decline of birth rate and mortality rate in China has led to a significant trend of aging population,and the incidence rate of various elderly related diseases has also increased year by year.In the process of aging,the kidney is one of the organs with the most obvious changes in morphology and function.During the natural aging,the main structural changes of aged kidneys are renal parenchyma decreases,surface roughness increases,and cysts appear.The change of renal function is manifested by the decrease of glomerular filtration rate(GFR)at an average annual rate of 1%after 40 years old.Aging is a very complex process,regulated by many factors.At the same time,aging is also one of the important risk factors of kidney disease.Therefore,understanding the mechanism of renal aging and its degenerative progress is of great significance for delaying the development of renal aging and the progress of aging-related diseases,and improving the quality of life of the old population.The identity and function of each cell in the human body is determined by its gene expression program.Several steps in the gene expression process can be regulated,such as transcription,posttranscriptional processing,translation,and posttranslational modification.RNA splicing is a form of RNA processing in which a newly made precursor messenger RNA(mRNA)is transformed into a mature RNA by removing the non-coding sequences termed introns.Alternative splicing(AS)refers to the process of generating different splicing isoforms of mRNA through different splicing methods from the pre-mRNA.Nearly all human gene transcripts are alternatively spliced and can produce protein isoforms with divergent and even antagonistic properties that impact cell functions.AS is an important mechanism used to generate greater transcriptomic and proteomic diversity from a finite genome.The spliceosome,which is composed of five small nuclear RNAs(U1,U2,U4,U5,U6)and a variety of RNA-binding proteins,facilitates the splicing process.Bud31 protein(Bud31 homolog)was first discovered in Saccharomyces cerevisiae whole genome sequencing,and it is highly conservatively expressed in all eukaryotic cells.Proteomics studies based on mass spectrometry analysis show that Bud31,as an important splicing factor,participates in the formation of NTC(NineTeen Complex or Prp19,Pre-RNA processing factor 19)related complexes.In vitro biochemical analysis found that Bud31 played a role in promoting the splicing efficiency of precursor mRNA and catalyzing the assembly of spliceosome.Recent studies have shown that the abnormal splicing process is related to the occurrence and development of a variety of neurological aging-related diseases,such as Alzheimer’s disease(AD)and Parkinson’s disease(PD).However,the role and mechanism of alternative splicing in renal aging is still unclear.In this project,we will study the changes of alternative splicing in mouse aged kidney for the first time,and characterize the expression features of the splicing factor Bud31 in the process of renal aging.We will clarify the role of Bud31 in renal aging and renal tubular epithelial cell aging by using tubular epithelial cell-specific Bud31 knockout mice and targeted-inhibition of Bud31 expression in human renal tubular epithelial cell HK-2.This study will explore the new mechanism of renal aging at the level of posttranscriptional processing,which is of great significance for the prevention and treatment of renal aging and aging related-kidney diseases.Methods and Results1.Changes of alternative splicing events in kidney from aged mice compared with young mice.C57BL/6J male wild type(WT)mice aged 6 months and 18 months were collected.After perfusion,the renal cortex of mice was taken,paraffin embedded and sectioned,and the morphological damage of mice kidney was evaluated by H&E staining;senescence-associated β-galactosidase(SA-β-gal)staining was used to evaluate the cellular senescence in renal tissue.The results showed that compared with 6-month-old mice,18-month-old mice had more renal tubal dilatation,nuclear abscission,inflammatory cell infiltration and cellular senescence.Transcriptome sequencing(RNA-Seq)combined with differential gene and transcript analysis showed that the differential gene expression and alternative splicing events in the renal cortex of 18-month-old mice were significantly changed compared with those of 6-month-old mice.2.The expression level of splicing factor Bud31 in the kidney from mice of different ages.The total protein and RNA were extracted from the renal tissues of 3-,6-,12-and 18-month-old mice,and the paraffin sections were made.The expression of Bud31 in renal tissues of mice were detected by Western blot(WB),real-time quantitative PCR(real-time qPCR)and immunohistochemistry(IHC).The results showed that the protein level of renal Bud31 was decreased significantly in aged mice and IHC staining showed that the decreased Bud31 level was mainly expressed in the nucleus of renal tubular epithelial cells.3.The role of bud31 in renal aging3.1 Construction and verification of renal tubular epithelial cell specific Bud31 deficient mice.The renal tubular epithelial cell specific Bud31 deficient mice were constructed by Cre-loxP system,and DNA was extracted from mice tail.PCR was amplified and agarose gel electrophoresis was used to identify the genotype of mice,confirming that the conditional knockout mice were successfully constructed.Bud31fl/fl;Cdh16-Cre+/-mice was further confirmed by Western blot and immunofluorescence(IF);The expression of Bud31 in the kidney of Bud31fl/fl;Cdh16-Cre+/-mice was significantly decreased,and the deletion of Bud31 in renal tubular epithelial cells was successful.3.2 Renal tubular epithelial cell specific Bud31 deficiency accelerates renal aging in mice.The kidneys of 7.5-months-old Bud31fl/fl;Cdh16-Cre+/-mice and Bud31fl/fl;Cdh16-Cre-/-mice were perfused,and the rough surface of the kidney with cysts were observed in renal tubular epithelial cell specific Bud31 deficient mice.Compared with the control genotype mice,the renal tissue of the mice with specific Bud31 deficiency showed dilatation and vacuolation of renal tubular lumen,infiltration of inflammatory cells and shedding of renal tubular epithelial cells.Masson trichrome staining was used to observe the degree of renal fibrosis in mice.The results showed that the deletion of bud31 gene in renal tubular epithelial cells led to renal fibrosis in mice.The results of terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL)staining showed an accumulated cell death in kidney sections of Bud31fl/fl;Cdh16-Cre+/-mice.3.3 Renal tubular epithelial cell specific Bud31 deficiency promotes renal cellular senescence in mice.The frozen sections of kidney from 10-week-old mice were analyzed by SA-β-gal staining,more positive staining in renal tubules was observed in Bud31fl/fl;Cdh16-Cre+/-mice than Bud31fl/fl;Cdh16-Cre-/-mice.The mRNA expression levels of senescence-associated p16,p21 and p53,senescence associated secret phenotype(SASP)-associated pro-inflammatory molecules(IL-6,TNFα,TGF-β1 and MMP3)and pro-fibrosis molecules(CTGF and PAI-1)were detected by real-time qPCR.The results showed that the expression of senescence-associated genes and SASP-associated genes in the kidney of young mice was significantly increased by tubular epithelial cell specific Bud31 deficiency.WB detecting the expression of fibrosis-associated proteins(α-SMA and vimentin)and Masson’s trichrome staining showed that the degree of renal fibrosis was enhanced in the tubular Bud31 deficient mice.These results indicate that tubule specific bud31 deficiency promotes renal cellular senescence in young mice.3.4 Silencing the expression of bud31 in HK-2 cells promotes cellular senescence in vitro.After transfection of Bud31-specific siRNA(siRNA-Bud31)to silence the expression of Bud31 in HK-2 cells,the expressions of p16 and p53 were detected by WB and qPCR.The results showed that inhibition of bud31 expression led to the increase of senescence-associated proteins in HK-2 cells.SA-β-gal staining showed that β-gal positive staining of HK-2 cells with silenced Bud31 expression was more than that of control cells.Lamin A was detected by immunofluorescence assay.The results showed that the expression of lamin A in HK-2 cells was decreased after Bud31 knockdown,and the nucleus was flat and enlarged.These results suggest that knockdown of Bud31 in HK-2 cells promotes cellular senescence in vitro.3.5 Renal tubular epithelial cell specific Bud31 deficient mice showed premature aging phenotype.Compared with Bud31fl/fl;Cdh16-Cre-/-mice of the same age,7.5-month-old Bud31fl/fl;Cdh16-Cre+/-mice showed premature aging,such as hair loss,back rickets,gonadal atrophy and decreased stride.The systolic blood pressure(SBP),diastolic blood pressure(DBP),mean blood pressure(MBP)and heart rate(HR)were measured by tail cuff method.The blood pressure and heart rate of Bud31fl/fl;Cdh16+/-mice were significantly higher than those of Bud31fl/fl;Cdh16-Cre-/-mice.In addition,the urine of mice was collected by metabolic cage.The 24-hour urine volume of Bud31fl/fl;Cdh16-Cre+/-mice increased significantly.WB,qPCR and IHC were used to confirm that the expression level of Klotho in kidney of Renal tubular epithelial cell specific Bud31 deficient mice was significantly lower than that of control mice.It is suggested that the deletion of bud31 gene may lead to the decrease of Klotho secretion from kidney,thus promoting premature aging.ConclusionIn this study,the changes of alternative splicing events were found in mouse kidney of natural aging.The expression of the splicing factor Bud31 in the renal tubular epithelial cells was decreased,and the decrease or absence of Bud31 expression in the renal tubular epithelial cells accelerated the aging of the kidney and cause systemic premature aging phenotype.This study is to link renal aging with alternative splicing,an important posttranscriptional processing pattern of gene expression,and clarify the role of splicing factor Bud31 in renal aging.It is suggested that the abnormal changes of Bud31 and Bud31-mediated alternative splicing mechanism in renal tubular epithelial cells may be one of the key mechanisms of renal aging progress,which provides a new perspective for the study of renal aging,it also provides a new target for the prevention and treatment of aging-related kidney diseases. |
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