The Effect Of Colonic Dysbiosis From UT-B Gene Depletion On Salt Sensitive Hypertension In Mice

Background:Studies have found that gastrointestinal microbiome imbalance is closely related to salt-sensitive hypertension.Nicola Wilck[1]et al.found that systolic and diastolic blood pressure increased significantly after 14 days of high salt intake in salt-sensitive hypertensive mice,and that The species content of 8 kind of gastrointestinal microbiome represented by Lactobacillus was significantly reduced.After administration of salt-sensitive hypertensive mice with Lactobacillus muesli,the elevated systolic and diastolic blood pressures were significantly reduced,with diastolic blood pressure reaching normal levels.It is suggested that the maintenance of gastrointestinal microbiome balance has an important role in stabilizing blood pressure.The nitrogen required by the gastrointestinal microbiome originates from urea in the colon.A decreasing of the urea in the colon will cause an imbalance in the gastrointestinal microbiome Transportation of urea in the colon is carried by UT-B,which is expressed on the cell membrane of colonic epithelial cellsOur team's previous results found that the mice with UT-B gene deletion significantly increased their mean arterial pressure after feeding high salt diet for 4 weeks.UT-B is a urea transporter encoded by the SLC14al gene.It is widely present in kidney,red blood cells,brain,testis,intestines[2],bladder[3]and other tissues,and is mainly responsible for urine concentration and other processes[4].Studies have shown that UT-B is expressed on the cell membrane of colonic epithelial cells,but there are fewer studies on its physiological functions.Given that UT-B is a membrane protein that mediates the transmembrane transport of urea,UT-B expressed on the cell membrane of colonic epithelial cells mediates the entry of urea into the colon,thereby providing the nitrogen source needed for survival of the colonic microbiome and maintaining balance of the colonic microbiome.We speculate that if the UT-B gene is deleted,it will cause change of urea concentration in the colon,leading an imbalance in the colonic microbiome,therefore the occurrence of salt-sensitive hypertension in miceIn order to verify the relationship between the UT-B gene expressed in the colon and the gastrointestinal microbiome imbalance,the role and related mechanisms of gastrointestinal microbiome imbalance based on UT-B gene deletion in mice with salt-sensitive hypertension were elucidated.In this study,UT-B gene-deficient mice were selected as the research object,and wild-type mice were used as control.They were given ordinary diet and high-salt diet 4 weeks,respectively,and observed changes in water consumption and blood pressure of the mice.And we measured the urea and short-chain fatty acid content,microbiome and GPR41,Olfr78 expression in the colon.Based on the analysis of the role of colonic microbiome and its metabolites in mice with salt-sensitive hypertension,SCFAs were given to mice for 4 weeks,and the effect of SCFAs on salt-sensitive hypertension in mice was observed.,which might provide new therapeutic targets on the prevention and treatment of hypertensionResearch purposes:To determine the expression and role of UT-B gene in mice colon.To discuss the effect of UT-B gene deletion on mice colonic microbiome balance.To elucidate the effect of gastrointestinal dysbiosis basing on UT-B gene deletion on salt sensitive hypertension in mice and analyze relating mechanisms,finally providing new ideas and targets for the prevention and treatment of salt-sensitive hypertensionMethods and results:(1)Part 1:Using UT-B gene-deficient mice as the research object,to analyze the effect of UT-B gene deletion on the balance of intestinal microbiome in mice,and to explore the effect of gastrointestinal microbiome imbalance basing on UT-B gene deletion on salt-sensitive hypertension in mice.1.Obtaining UT-B gene-deficient miceHeterozygous mice were used for mating to obtain homozygous UT-B gene-deficient mice and wild-type mice with the same genetic background.Subsequently,mice of different genotypes were genetically identified.The PCR results of UT-B gene deletion-type homozygous,wild-type and heterozygous mice were only one band at 250bp,only one band at 400bp,and one band at 250bp and 400bp,respectively2.The expression and localization of UT-B gene in mice colonThe same segmental colon of UT-B gene-deficient mice and wild mice were obtained,and the expression and localization of UT-B gene in the colons of wild-type mice were detected by RT-PCR,Western-blot,and immunohistochemical methods.UT-B gene was expressed in the apical membrane and glandular foci of colon epithelial cells in wild-type mice,but no expression was found in UT-B gene-deficient mice3.Determination of urea content in the colon of mice in each groupThe same segment of colon of UT-B gene-deficient mice and wild mice were taken,and the urea content was measured by a urea kit.As a result,it was found that the colonic urea content was significantly lower in the UT-B gene-deficient mice than the wild-type mice.(P<0.001)4.To observe the effects of high-salt diet on water consumption and mean arterial pressure of UT-B gene-deficient mice,using UT-B gene-deficient mice as research objects and wild-type mice as controls.Male mice of the same age were divided into 4 groups:UT-B+/+(high-salt diet group)UT-B+/+(general diet group)UT-B-/-high-salt diet group)UT-B-/-(General diet group).At the same time,the blood pressure status of the mice was measured by the instrument,the daily water consumption of the mice was observed,and the average arterial pressure of the mice in each group was measured by the "non-invasive method".The results showed that:The water intake of the high-salt group was significantly higher than that of the ordinary diet group,UT-B-/-(high-salt diet group)blood pressure value was significantly higher than other groups5.Effects of UT-B gene deficiency on mice colonic microbiome balanceThe wild-type mice were used as controls.the colonic microbiome of the mice was measured after the UT-B gene was deleted.The experimental group was set as mentioned before.After 4 weeks,the colonic contents of the same segment of mice in each group were taken for 16S ribosomal DNA identification(16SrDNA),the sequencing was used to compare the richness,uniformity,and content of each intestinal microbiome.The results showed that compared with the wild-type mice fed the normal diet and the high-salt diet group,the intestinal microbiome abundance,uniformity,and diversity of the UT-B gene-deficient mice fed a high-salt diet for 4 weeks were significantly reduced.At the same time,it was found that the ratio of Firmicutes to Bacteroides(F/B)was significantly higher than that of Other groupings,which suggested the existence of hypertension at the level of the intestinal flora.6.Effects of a high-salt diet on colonic acetate content in UT-B gene-deleted miceSCFAs are metabolites of the intestinal microbiome and are closely related to the production and development of salt-sensitive hypertension.Based on the above results,we found that mice with UT-B gene deficiency fed a high-salt diet could cause imbalance in the colonic microbiome.Gas chromatography was used to detect changes in acetate which is one of the intestinal microbiome 's metabolites.The results showed that compared with wild-type mice,UT-B gene-deficient mice had significantly lower acetic acid content in the colon after being fed a high-salt diet(P<0.01).(?)Part ?:Effect of magnesium acetate supplementation on the salt-sensitive hypertension in UT-B gene-deficient mice fed with high-salt diet.1.Effect of magnesium acetate supplementation on colonic acetate content in mice of each groupsTake male mice of the same month age and group them as follows:UT-B+/+(high-salt diet);UT-B+/+(general diet);UT-B-/-(high-salt diet);UT-B-/-(Common diet);UT-B-/-(high-salt diet 4 weeks+acetate 4 weeks);UT-B-/-(common diet 4 weeks+acetate 4 weeks).Gas chromatographic test results showed that the content of magnesium acetate in the colon of mice in the UT-B-/-(high-salt diet)group was significantly lower than that in other groups of mice;after 4 weeks of supplementation with magnesium acetate solution,UT-B-/-(high The salt diet 4 weeks+acetate 4 weeks)mice significantly increased acetic acid content(P<0.05).2.Effect of magnesium acetate supplementation on the expression of SCFAs receptors in the colon of mice of each groupSCFAs regulate blood pressure by binding to their receptors.Therefore,we examined the expression of SCFAs receptors in each group of mice.RT-PCR results showed that after supplementation with magnesium acetate for 4 weeks,the expression of Olfr78 mRNA in the UT-B-/-H group was the strongest,while in the UT-B-/-H+acetate group was the weakest;GPR41 mRNA in the UT-B-/-H+acetate expressed the most strongly,while the expression in the UT-B-/-H group was the weakest.Western blot results showed that the Olfr78 protein was the strongest in expression of the UT-B-/-H group,and the UT-B-/-H+acetate group was the weakest;the expression of GPR41 protein was strongest in the UT-B-/-H+acetate group,while the weakest expression of which in the UT-B-/-H group.Immunohistochemical results showed that GPR41 protein expression was localized in colonic endocrine cells L cells,the strongest expression was in the UT-B-/-H+ acetate group,and the weakest expression in the UT-B-/-H group;Olfr78 protein expression localization Epithelial cells lining the colonic crypts had the strongest expression in the UT-B-/-H group and the weakest expression in the UT-B-/-H+acetate group.It was confirmed that after administration of magnesium acetate diet,the expression of Olfr78,a hypertensive receptor,can be reduced,and the expression of GPR41 receptor,which lowers blood pressure,can be enhanced(P<0.05)3.Effect of magnesium acetate supplementation on mean arterial pressure of mice of each groupTake blood pressure of the mice in the non-invasive manner,the results showed that:fed with magnesium acetate solution for 2 weeks,blood pressure of the mice in the UT-B-/-H+magnesium acetate group began to drop;after 4 weeks,the UT-B-/-H+magnesium acetate group mice Blood pressure dropped to normal level(from 157±2.3mmHg to 119±1.7mmHg),and continued feeding with magnesium acetate,blood pressure did not drop below normal levels,and blood pressure of mice in the control group remained unchanged.It was confirmed that acetate supplementation could relieve the salt-sensitive hypertension in mice caused by UT-B gene deletionConclusions:(1)The imbalance of the gastrointestinal microbiome caused by the deletion of the UT-B gene leads to a decrease in the content of short-chain fatty acids(SCFAs)which are the metabolites of the gastrointestinal microbiome(2)The decrease in the content of short chain fatty acids(SCFAs)in the intestinal flora of mice induces the generation of salt-sensitive hypertension in mice caused by the deletion of the UT-B gene(3)Acetate supplementation can alleviate the salt-sensitive hypertension in mice caused by UT-B gene deletion.