The Effect And Mechanism Of UTB On Sodium Ion Concentration In Cerebrospinal Fluid In Mice Fed On High Salt Diet

Background:Studies have found that[1]salt-sensitive hypertension is closely related to cerebrospinal fluid sodium ion concentration([Na⁺]_CSF),[Na⁺]_CSF elevation precedes elevated blood pressure which suggesting that [Na⁺]_CSF elevation plays an important role in the development of salt-sensitive hypertension.CSF is produced and secreted by epithelial cells in brain choroid plexus（CPECs）,ENa Cs is located in the apical and basal membranes of CPECs,and it mediates [Na⁺] transport from blood to CSF and participates in the balance of sodium ion.Our previous study found that [Na⁺]_CSF and blood pressure were significantly increased in UTB gene depletion mice for 4w high salt diet,and the expression of ENa C-α was significantly increased in choroid plexus,suggesting that UTB gene depletion is associated with increased expression of ENa Cs in choroid plexus.UTB is encoded by SLC14α1 gene and expressed in organs such as brain,kidney,red blood cells,testis and heart,it mainly involved in the regulation of urinary enrichment[2],sexual maturation[3]and cardiac hypertrophy[4].However,the role of the UTB gene in brain tissue is not yet clear.To investigate the role of UTB in brain tissue,and to elucidate the relationship between UTB and ENa Cs in the choroid plexus,In vitro experiments,mice CPECs were used as the research object,the expression of UTB protein was down-regulated by transfection of UTB-sh RNA plasmid,and then the effect of down-regulation of UTB protein on ENa C-α in mice CPECs were observed;In vivo experiments,UTB gene depletion mice were selected,the effect of high salt diet on ENa Cs of choroid plexus in UTB gene depletion mice were observed and the mechanisms were explained.The results from our study will provide experimental data and new therapeutic targets for the prevention and treatment of salt-sensitive hypertension.Objective:To investigate the effect of down-regulation of UTB expression on the expression of ENa C-α in epithelial cells of choroid plexus in mice,and to elucidate the possible mechanism by which UTB gene is involved in the regulation of sodium ion concentration in cerebrospinal fluid in mice fed on high salt diet.Methods and Results:（一）In vitro experiments: To investigate the effect of down-regulating the expression of UTB on ENa C-α in mice CPECs1.Identification of CPECsTTR is a protein specifically expressed in mice CPECs,the results of Immunofluorescence staining showed that TTR was expressed in the cytoplasm of CPECs,which could identify the mice CPECs.2.Down-regulation of UTB expression in mice CPECsThe mice CPECs were used as the cell model,in the experimental group,the sh RNA-UTB plasmid was transfected into the mice CPECs;In the negative control group,the sh RNA-scramble plasmid was transfected into the mice CPECs;In the positive control group,the UTB inhibitor（PU14）was added into CPECs media;Immunofluorescence results showed that the cell transfection efficiency was about60%,and the inhibition rate reached 65.7% in the group which sh RNA-UTB plasmid was transfected into CPECs for 48 h;The results of Immunofluorescence and Western-blot showed that the protein expression of UTB were significantly decreased after transfection of sh RNA-UTB plasmid for 48 h in mice CPECs（P<0.05）.3.The effect and mechanisms of down-regulation UTB on the expression of ENa C-α in mice CPECsThe results of Immunofluorescence staining、RT-PCR and Western-blot results showed that: the m RNA and protein expression of ENa C-α were significantly increased after transfection of sh RNA-UTB plasmid into CPECs for 48 h（P<0.05）.The expression of ENa C-α is regulated by s GK₁-Nedd4-2-Ena C-α signaling pathway.Our results showed that down-regulation of UTB induced increased expression of the s GK₁-Nedd4-2-ENa C-α pathway in mice CPECs,resulting in increased expression ofENa C-α.The protein expression of ENa C-α in CPECs added with EMD638683（s GK₁inhibitor）was not different compare to the control group.Compare to the control group,RT-PCR,cellular Immunofluorescence staining and Western-blot results showed that the phosphorylation levels of p-s GK₁（Ser422）/s GK₁,p-s GK₁（Thr256）/s GK₁ and p-Nedd4-2（Ser328）/Nedd4-2 were increased after transfection of sh RNA-UTB plasmid for 48 h in the mice CPECs;These results suggest that s GK₁-Nedd4-2-ENa C-α pathway is activated after down-regulating UTB expression in mice CPECs,phosphorylation of Ser422 and Thr256 are involved in the activation of s GK₁,which promote the phosphorylation of Ser328 at Nedd4-2,which leads to the enhancement of the expression of ENa C-α in mice CPECs.（二）In vivo experiment: To elucidate the mechanisms of UTB depletion on the sodium ion concentration of cerebrospinal fluid in mice fed on high salt diet.1.Identification of UTB gene depletion miceUsing heterozygous mice to obtain UTB gene depletion mice,genomic DNA was extracted for mice genotype identification,PCR results showed that wild-type mice showed a fragment at 400 bp;heterozygous mice showed two fragments at 250 bp and400 bp;while the UTB gene depletion mice showed a fragment at 250 bp.2.Effect of UTB gene depletion on sodium ion concentration of cerebrospinal fluid in mice fed on high salt dietThe sodium ion concentration of the cerebrospinal fluid was monitored by sodium ion kit and the results showed that the [Na⁺]_CSF of the UTB gene depletion mice was significantly increased（P<0.05）compared with the control group.3.Effect of UTB gene depletion on the expression of ENa C-α in choroid plexus of mice fed on high salt dietThe results of RT-PCR,Western-blot and Immunohistochemistry showed that the m RNA and protein expression of ENa C-α in choroid plexus were significantly increased compare to control group in UTB gene depletion mice fed on high salt diet for 4w（P<0.05）.4.The mechanisms of UTB gene depletion on the expression of ENa C-α in the choroid plexus in mice fed on high salt dietRT-PCR,Western-blot and Immunohistochemistry results showed that the phosphorylation levels of p-s GK₁（Ser422）/s GK₁,p-s GK₁（Thr256）/s GK₁ and p-Nedd4-2（Ser328）/Nedd4-2 were increased（P <0.05）in UTB gene depletion mice after administration of 4w high salt diet compare to the control group.These results confirmed that UTB gene depletion activates the s GK₁-Nedd4-2-ENa C-α pathway in the choroid plexus of mice with high salt diet,which leads to the phosphorylation of Ser422 and Thr256 involved in the activation of s GK₁,which promotes the phosphorylation of Ser328 in Nedd4-2,resulting in enhanced expression of ENa C-αin the choroid plexus of mice.This in turn causes an increase in [Na⁺]_CSF of mice.5.Effect of UTB gene depletion on water consumption and mean arterial blood pressure in mice fed on high salt dietThe changes of water consumption in mice of each group were monitored,the results showed that the water consumption of UTB gene depletion mice and wild-type mice increased significantly after 4w high salt diet（P<0.05）.The mean arterial blood pressure of the mice in each group was measured by non-invasive method,the results showed that the mean arterial blood pressure of the UTB gene depletion mice was significantly higher than that of the wild-type mice after 4w high salt diet（P<0.05）.Conclusion:（1）In vitro experiments: it confirmed that down-regulation of UTB expression in mice CPECs resulted in activation of s GK₁-Nedd4-2-ENa C-α pathway and increased expression of ENa C-α;（2）In vivo experiments: it confirmed that UTB gene depletion increased the expression of ENa C-α by activating the s GK₁-Nedd4-2-ENa C-α pathway in the mice choroid plexus,which in turn caused the increase of sodium ion concentration in the cerebrospinal fluid of mice with high salt diet,resulting in the occurrence of salt-sensitive hypertension in mice.