| The renal glomerulus is the important functional unit for renal filtration,which consists of multiple cellular barriers,including the fenestrated endothelium of glomerular capillaries,the glomerular basement membrane,and the slit diaphragm formed between the foot processes of adjacent podocytes.Defects in the podocyte development or podocyte injury impair the normal glomerular filtration barrier and lead to proteinuria,a common cause of kidney disease in humans.Studies have shown that signaling pathways such as WNT,NOTCH and TGF-β are involved in the regulation of podocyte development,which are crucial for podocyte development and glomerular homeostasis.Nonsense-mediated mRNA decay(NMD)is a widespread post-transcriptional regulatory mechanism of gene expression in eukaryotic cells.NMD can degrade the abnormal transcripts containing premature termination codon(PTC)and prevent the accumulation of truncated proteins.Meanwhile,NMD can regulate the abundance of certain mRNAs that can be translated into proteins with physiological functions,thus coordinating TGF-β,WNT,NOTCH and other signaling pathways in cells to regulate cell fate.There are two branches in the degradation process of NMD:One is the SMG5/7 mediated exonucleolytic branch,in which SMG5 and SMG7 form a heterodimer to recruit deadenylase complex and decapping complex for deadenylation and decapping of NMD targets;The second branch is the degradation branch mediated by SMG6,which can cleave the target mRNA in the vicinity of the PTC.The final step of NMD target clearance is executed by the XRN1 and exosome.NMD is an important regulatory mechanism for cell proliferation,differentiation and maturation.However,the functions and molecular mechanisms of NMD in the renal podocyte development remain unknown.Therefore,this research on the biological functions of SMG5 and SMG6 in the development of renal podocytes had been carried.First,a mouse model of podocyte-specific Smg5 knockout(Smg5F/F;NPHS2-Cre)was generated in this thesis.Podocyte-specific Smg5 knockout mice all died within 2-3 months after birth.The 3-week-old mutant mice began to develop proteinuria.Histological analysis of kidney identified glomerular capillary dilation,with reduced expression of podocyte markers.Protein casts,tubular dilation and cell proliferation were also observed in the kidneys of mutant mice.Ultrastructure analysis revealed the effacement of podocyte foot processes and the loss of slit diaphragm in the mutant mice.These results suggested that SMG5-mediated NMD was required for podocyte differentiation and maturation,and that SMG5 deficiency in podocytes led to early-onset renal failure and premature death in mice.Second,a mouse model of podocyte-specific Smg6 knockout(Smg6F/F;NPHS2-Cre)was established.Unlike podocytespecific Smg5 knockout mice,there were no significant differences in body weight,urinary protein content and glomerular morphology between the podocyte-specific Smg6 knockout mice and the controls.However,ultrastructure analysis showed that the glomerular basement membrane was thicker and the podocyte foot process was longer in the Smg6 mutant mice.These results indicated that NMD defects mediated by SMG6 did not affect the normal kidney function of mice,but had a slight effect on the podocyte development and the distribution and arrangement of foot processes.Furthermore,an ADR-induced nephropathy model based on the podocyte-specific Smg6 knockout mice was produced.The Smg6 mutant mice were found to develop more severe proteinuria compared to the controls,suggesting that the knockout of Smg6 exacerbated podocyte injury in mice.Taken together,this study preliminarily investigated the functions of SMG5 and SMG6 in podocyte development in mice,and revealed that SMG5 and SMG6 are essential for podocyte development.This study laid a solid foundation for further detailed characterizations on the molecular mechanisms of SMG5-NMD and SMG6-NMD in the podocyte development. |
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