Injury Of Podocyte Ultrastructure And Function In ASK3 Knockout Mice

Background and ObjectiveApoptosis signal-regulating kinase 3(ASK3,also called MAP3K15)is a new discovered protein kinase belonging to the mitogen activated protein kinase kinase kinase(MAP3K)family,and is conserved among various species.During clinical work,two brothers with pathology-confirmed idiopathic membranous nephropathy(IMN)were identified to have frame-shift mutation of ASK3.And ASK3 is proved to be down-regulated in kidney of propositus by immunohistochemistry.ASKS mainly expresses in renal tubules,and play roles in the regulation of cell volume by downstream pathway WNK1-SPAK/OSR1-NCC/NKCC.No research about the relationship of ASK3 and renal glomerular disease has been searched.Among the pathway,Odd-skipped related transcription factor 1(Osrl)is one of the earliest genes expressed in intermediate mesoderm.It plays an essential role in kidney organogenesis and is required for podocyte development.Therefore,we will explore the relationship between ASK3 and podocyte function by ASK3 knockout(ASK3-KO)mice,and further explore the susceptibility to adriamycin(ADR)-related podocyte injury in ASK3-KO mice.Methods1.Blood pressure,urine protein level,blood and urine electrolyte level,blood and urine biochemical indices,total renal weight/body weight ratio were tested on 9-10 week-old male ASK3-KO)mice and C57BL/6J mice(wild-type mice,WT mice).The HE staining,electron microscopic specimens of kidney were prepared.2.ADR injection(25mg/kg)was performed on 7-8 week-old female ASK3-KO and WT mice.Saline injection(12.5ml/kg)was performed on 7-8 week-old female ASK3-KO mice.Urine protein level was tested.3.RNA sequence and real-time PCR were used to identify the gene expression discrepancy between 9-10 week-old male ASK3-KO mice and WT mice.Results1.Systolic blood pressure is significantly elevated,24-hour urine calcium is significantly decreased in ASK3-KO mice,p<0.05.Serum sodium level,serum aldosterone level and 24-hour urine sodium have no significant difference.2.Total renal weight/body weight ratio is significantly decreased in ASK3-KO mice,p<0.05.Urine albumin creatinine ratio(ACR)has no significant difference.No structural change is found in HE staining of renal tissue.By electron microscopy,localized podocyte effacement was identified in ASK3-KO)mice.3.Day 1 ACR,Day 7 ACR,Day 9 ACR of ADR-injecton ASK3-KO mice are significantly higher than saline—injecton ASK3-KO mice,p<0.05.After ADR injection,Day 7 ACR of ASK3-KO)mice is significantly higher than WT mice,p<0.05.4.195 genes with expression discrepancy in kidney of ASK3-KO mice and WT mice are detected using RNA-sequence.Among them,PLAUR(plasminogen activator,urokinase receptor)is significantly down-regulated in ASK3-KO mice.Other podocyte related genes(OSR1,PAX2,WT1,NPHS1,NPHS2,SULT1B1,Synapo),genes in WNK1-SPAK/OSR1-NCC/NKCC pathway and renal ion channel genes(NCCr NKCC1,NKCC2,ENaC,NHE3,SGLT1)have no significant difference.5.No significant difference of renal OSR1,NCC,NKCC1,NKCC2,ENaC,NHE3,SGLT1 expression is found in real-time PCR between ASK3-KO mice and WT mice.PLAUR expressed significantly lower in ASK3-KO mice,p<0.05.ConclusionsThis is the first time that ASK3-KO mice are discovered to manifest as localized podocyte effacement.ASK3-KO mice are inclined to gain proteinuria after ADR injection than WT mice.Lower renal PLAUR expressing level is found in ASK3-KO mice.Our research provides new clues for podocyte injury in ASK3-KO mice.