Expression And Function Of Protease Corin In Intestine

Chapter 1:Distribution of corin and ANP expression in mouse intestineObjective:Corin is a type Ⅱ transmembrane serine protease,originally found in the heart,which acts as an activated ANP by cleaving atrial natriuretic peptide(Pro-ANP),playing the function of dilating blood vessels and lowering blood pressure.So far,corin has been shown to be expressed in the kidney and skin sweat glands,which plays a role in promoting water and sodium excretion and participates in maintaining electrolyte homeostasis in the body.In addition to the kidney and skin,the intestine also plays an important role in maintaining electrolyte homeostasis.However,the distribution and function of corin in the mouse intestine,especially the function of colon in sodium absorption,are not yet known.In this chapter,we investigated the distribution of corin and ANP expression in the mouse intestine to provide a basis for subsequent studies on the function of corin in the colon.Methods:1.Messenger RNA(mRNA)from the heart,kidney,liver,duodenum,jejunum,ileum,cecum,proximal colon and distal colon were extracted from wild-type(WT)and corin knockout(KO)male mice aged 10-12 weeks.The expression of corin in tissues of WT and corin KO mice was detected by reverse transcription polymerase chain reaction(RT-PCR).2.The mRNA expressions of Nppa,Nprl,Scnnla and Cftr in WT and corin KO mice were detected by RT-PCR.3.Kidney and gut tissues of WT and corin KO mice were taken and fixed in 10%formalin.Hematoxylin and eosin(H&E)staining and immunohistochemistry(IHC)were performed to detect the expression and distribution of corin,ANP and NPRA proteins in mouse kidney and intestinal tissues.4.Immunofluorescence staining(IF)was used to detect the cellular localization of corin,ANP and NPR-A proteins in the mouse intestine.Results:1.Corin mRNA expression in mouse intestine:RT-PCR results showed that corin mRNA was expressed in all segments of WT mice intestines;No corin expression was detected in corin KO mice.2.Nppa,Nprl,Scnnla,and Cftr mRNA expression in mouse intestines:RT-PCR results showed that Nppa,Npr1,Scnn1a,and Cftr were positively expressed in all segments of the intestine both in WT and corin KO mice.3.Expression distribution of corin,ANP and NPR-A proteins in mouse kidney:IHC and IF results showed that in mouse kidney,corin was mainly expressed on the membrane surface of proximal tubules,ANP was mainly expressed in the cytoplasm of proximal tubules,and NPR-A was expressed in both cytoplasm and cell membrane.4.Expression distribution of corin,ANP and NPR-A proteins in intestinal tracts of mice:IHC results showed that corin and ANP proteins were mainly expressed in single cells,while NPR-A was mainly expressed in intestinal epithelial absorbing cells.IF results showed that corin and ANP proteins co-located in intestinal enteroendocrine cells,and NPR-A protein was mainly expressed on the surface of intestinal epithelial absorption membrane.Conclusions:In this chapter,expression and cell localization of corin,ANP and NPR-A proteins in the intestinal tracts of mice were revealed.These results suggest that corin and ANP may regulate corin-ANP signaling pathway through autosecretory/paracrine mechanism in intestinal endocrine cells,thus playing biological functions.Chapter 2:Effects of corin deficiency on mouse colon structure and intestinal sodium excretion functionObjective:In Chapter one,We found that corin,ANP and NPR-A were all expressed in mouse intestine,and the main function of the colon is to absorb electrolytes and water,form,store and excrete feces.so whether corin and ANP expressed in colon plays the function of regulating water and salt balance by regulating excretion of salt ions in feces?This chapter aims to explore the functions of corin and ANP in colon.Meanwhile,Chapter one suggested that corin and ANP may play regulatory functions in the intestine independent of cardiac and renal sources,so by constructing heart-conditional corin KO(hcKO)and kidney-conditional corin KO(kcKO)mice,to test whether corin in heart and kidney affects mouse colonic corin to perform physiological functions in the intestine.Methods:1.The colonic morphology of WT and corin KO SPF male mice aged 10-12 weeks was detected by H&E staining.Mice were given 0.3%or 4%salt diet for 3 weeks,and their feces were collected and dried.Then differences in fecal output were counted,and the concentration of Na+ and Cl-in feces solution was detected.2.Male SPF hcKO and kcKO mice aged 10-12 weeks were given 0.3%or 4%salt diet for 3 weeks.The feces were collected and dried to detect Na+and Clconcentrations in feces solution.3.It has been reported that aldosterone increases ENaC activity and promotes sodium absorption.We took 10-12 weeks old SPF grade WT,corin KO and kcKO male mice with subcutaneous buried aldosterone osmotic pump and collected feces to detect Na+ and Cl-excretion via feces in WT,corin KO and kcKO mice before and after aldosterone administration.Results:1.H&E staining showed no significant difference in colonic structure and morphology between WT and corin KO mice;there was also no significant difference in the amount of feces excreted between WT and corin KO mice(p=0.966).suggesting that corin deficiency does not affect the morphology of colonic tissue and defecation volume in mice.2.The concentrations of Na+ and Cl-in the feces of corin KO mice were significantly lower than those of WT mice under normal diet(p<0.001),suggesting that corin exerts a role in promoting intestinal sodium Na+and Cl-efflux in mice.3.In high-salt diet group,the concentrations of Na+and Cl-in mice feces were significantly higher compared with the normal salt diet,and the concentrations of Na+ and Cl-in the feces of corin KO mice were significantly lower than those of WT mice in the same group(p<0.001).It suggested that high salt diet promoted the efflux of salt ions via the intestine,and corin promoted the efflux of Na+ and Cl-more significantly under high salt conditions.4.Fecal Na+ and Cl-concentrations in hcKO mice under both normal and high salt diets were no significantly different compared to WT mice,suggesting that corin regulating Na+ and Cl-in feces are not dependent on the endocrine function of cardiac corin.5.Fecal Na+ and Cl-concentrations were significantly higher in kcKO mice than WT mice under normal salt and high salt diets,suggesting:after the absence of corin protein in the kidney,part of the salt excretion function that should be performed by the kidney is compensated by the colon,resulting in the excretion of more salt ions via the feces.6.The Na+ and Cl-concentrations in feces of mice given aldosterone under normal salt and high salt diets were compared.No significant differences were found in corin KO mice compared with those before the administration;the concentrations of Na+and Cl-in the feces of both WT and kcKO mice decreased compared with those before administration;the differences in the original salt ion concentrations in the feces of WT,corin KO and kcKO mice disappeared after the administration.It is suggested that in the intestine of mice,corin promotes the efflux of intestinal Na+ and Cl-by antagonizing aldosterone.Conclusions:Corin deficiency does not affect the structural morphology and defecation volume of the mouse colon,but the presence of corin promotes the efflux of transcatheter Na+and Cl-,and the Na+ and Cl-efflux effect is more pronounced under a high salt diet.There was no significant difference in intestinal efflux salt ion levels between WT and heart corin cKO mice,suggesting that it is not the heart-expressed corin that regulates the efflux of salt ions in the colon through endocrine action;renal corin cKO mice had significantly higher intestinal efflux salt ions than WT mice,suggesting that after corin deficiency in the kidney,colonic corin can regulate the intestinal efflux of more salt ions,thus achieve functional compensation.The difference in intestinal salt excretion between WT and corin KO mice disappeared after exogenous administration of aldosterone,suggesting that the corin-ANP signaling pathway acts through antagonism of aldosterone.Taken together,the results of chapter one showed corin,ANP and NPR-A were all expressed in the colon,suggesting that intestine-expressed corin regulates intestinal salt efflux through autocrine/paracrine action,thereby regulating the body’s water and salt homeostasis.Chapter 3:Effect of Corin deficiency on disease progression of colitis in miceObjective:Disturbances in intestinal homeostasis can induce a variety of intestinal diseases,such as inflammatory bowel disease(IBD),infectious diseases,diarrhea,and colorectalcancer.Some studies suggest that impaired absorption of intestinal water and ions(especially Na+and Cl-)is the main cause of IBD-associated diarrhea.In previous chapters,we found that corin,ANP and NPR-A are all expressed in the mouse colon and play a role in promoting intestinal salt ion efflux,thus regulating the body’s water and salt balance.So,does corin also play a regulatory role in the process of IBD?Methods:1.SPF WT mice and corin KO male mice aged 6-8 weeks(weighing 25 g±1g)were randomly assigned to two groups:Control group and DSS group.Mice in Control group were free to drink water,and mice in DSS group were free to drink 2.5%DSS aqueous solution.The body weight was recorded every day and the state of the mice was observed.The mice were killed 8 days later.2.The length of blind colon of mice in Control and DSS groups was recorded and counted and photographed;the terminal colon(0.5-1 cm,try to select the same part)was taken and part of it was made into paraffin sections for subsequent observation of histomorphology,and part of it was used to extract RNA for subsequent qPCR detection.3.To examine the morphology of the colon of mice using H&E staining and Alcian blue staining,and to score the pathology;to examine the tightly attached structures of mouse intestinal epithelial cells using immunofluorescence staining.4.In DSS group,body weight,colonic length,and colonic histomorphology of mice at 0,2,4,6 and 8 days after modeling were recorded to monitor dynamic lesion changes in mice.5.Changes of Tnf,Il-6,Il-10 and Il-1β inflammatory factors in mice colon were detected by qRT-PCR.Results:1.The body weight of WT and corin KO mice in the Control group remained stable and slowly increased state,and the body weight of DSS-KO mice decreased significantly less than that of DSS-WT mice from day 6.2.In the Control group,the colonic length of WT and corin KO mice was not statistically different;in the DSS group,the colonic length of mice was significantly shorter,but the colonic length of DSS-KO mice was larger than that of DSS-WT mice.3.H&E,Alcian blue and immunofluorescence staining showed that both WT and corin KO mice in the Control group were morphologically intact in the colon,and mice in the DSS group showed large ulcers and lesions in the colonic histomorphology,but the degree of colonic lesions in DSS-KO mice was lower than than that in DSS-WT mice.4.H&E staining demonstrated the dynamic process of colon tissue from intact to lesioned and ulcerated in DSS mice on days 0,2,4,6,and 8 after modeling,and the colon of DSS-WT mice showed obvious lesions from day 4 onward,and the dermal barrier was completely damaged and detached by day 8,and the degree of colon lesions in DSS-KO mice was relatively less.Conclusions:Corin gene deletion alleviates the severity of DSS-induced colitis in mice and attenuates the inflammatory response to colitis in mice.We hypothesize that corin deletion may reduce water-sodium loss through the intestine,regulate intestinal water-sodium balance and thereby alleviate colitis in mice and improve their health status.