Regulations Of Robo2 On Signal Transduction In Mouse Kidney Development

Objective:Kidney development defects may lead to severe urinary system structures abnormalities and kidney dysfunction. Study finds that Robo2 mutation can lead to reduced ureteric bud branching and number of glomeruli cells, podocyte lesions and other defects, but we are not clear about the regulations of Robo2 on signal transduction. The purpose of the study is to compare mRNA expression of Robo2 knockout and wild-type mice embryo kidney,find out the protein interacting with Robo2 during mouse development,and verify the function of the protein in kidney development and what’s the relationships between Robo2 and the protein.We will investigate the key molecular mechanism of Robo2 during the--metanephric mesenchyme and ureteric bud development.Methods:(1) Embryonic kidney RNA was extracted from timed-pregnant female mice at embryonic day 14.5(E14.5d).Comparing the RNA differences between Robo2 homozygous and wild-type mice kidney by RNA-Seq, qReal Time RT-PCR technique was used to measure the expression level of the important gene mRNA in the developing murine kidney.(2) Embryonic kidney protein was extracted from timed-pregnant female mice at E13.5d. Collect the protein by co-immunoprecipitation and SDS-PAGE gel electrophoresis, and then we use mass spectrometry to screen the proteins interacte with Robo2.(3) Verify interactions between retinal dehydrogenase Aldhla2 and Robo2: Embryonic kidney was extracted from timed-pregnant female mice at E12.5d-E18.5d and postnatal day 1(P1d),P3d,P5d,P7d,P14d,P21d and P35d, Immunofluorescence staining were used to examine the expression locations of Aldhla2 protein at different stages of kidney development in mice;Western blot was used to measure the expression level of Aldhla2 protein at different stages of kidney development in mice; If the pregnant mice have excessive consumption of retinoic acid, we will observe phenotypic changes in embryonic kidney and ureteral.Results:(1) There are 20872 different gene between Robo2 homozygous and wild-type embryo kidney through RNA-Seq.We analyse the distribution ratio of Robo2-/- gene expression in mouse kidney,and draw a molecular signal network about Robo2 in the kidney of mice, Compared to homozygous, the expression of K1c1,Clta,Pomgnt1-2,Fhl1,Robo2,Abil are more than 20 times in wild-type embryonic kidney by RT-PCRtest.(2) We find out 25 types of protein by mass spectrometry analysis,such as Aldhla2, Robol and so on,they may take part in regulating the metanephric mesenchyme and ureteric bud development with Robo2.(3) Aldhla2 Expression during kidney development: ①Western blot results: Compared to E12.5d group, Aldhla2 was highly expressed at E12.5d-E15.5d, but which were reduced from E17.5 and kept at very low level after birth.② Immunofluorescence staining revealed that Aldhla2 is mainly expressed in stromal cells of metanephric mesenchyme and cap mesenchyme, but not in ureteric bud. Aldhla2 protein expression could be observed in pretubule cell,comma body, s-shaped body and ultimately expressed in the visceral epithelial cell and mesangial cells of glomerular, which was also weakly expressed in part of the proximal tubular epithelial cells. Aldhla2 expression was disappeared at 5 weeks after birth.③ If the pregnant mice have excessive consumption of retinoic acid, we will observe embryonic kidney and ureteral abnormality,including "Horseshoe kidney" malformation, reduced in size of the kidney, renal pelvis dilation, bent dilatation deformity of the ureter, ureter and kidney connection exception, ureter and bladder connection exception.Conclusion:Klcl,Clta,Pomgntl,Fhll and Abil obviously reduce expression in Robo2 homozygous kidney,so Robo2 may play as a positive regulator to them,and they take part in the signal pathway of Robo2. We also find out 25 types of protein by mass spectrometry analysis,such as Aldhla2,Robol and so on,they may take interaction with Robo2. We show that Aldhla2 may be a key signal molecular from distribution and phenotype. If the pregnant mice have excessive consumption of retinoic acid, we will observe dilatation of embryonic kidney and ureter.