The Study Of Aquaporin In Endolymphatic Sac Involved In The Pathogenesis Of Meniere Disease

OBJECTIVE The aim of this study was to explore the pathogenic mechanism of aquaporin(AQP)in the development of endolymph hydrops(EH)in Meniere’s Disease(MD)and the regulation of AQP2 expressed in endolymphatic sac(ES)via vasopressin(AVP)-vasopressin type 2 receptor(V2R)pathway and the differentially altered metabolites in ES,by analyzing the level of plasma arginine AVP in patients with MD,the expression and distribution of AQP-1,-2,-3,-5,-6 and V2 R in ES specimens from the patients with MD and patients with acoustic neuroma(AN)and the untargeted metabolomics analysis on ES luminal fluid(ELF)of MD and AN patients.METHOD ES samples were obtained from patients with AN during the removal of acoustic neuroma(AN)by way of the translabyrinthine approach,and immunohistochemistry was performed with AQP-1,-2,-3,-5,-6 and V2 R monoclonal antibodies in these ES tissue sections.(2)A total of 85 MD patients with unilateral or bilateral endolymph hydrops(EH)were selected as the experimental group and 90 normal subjects as the control group.The plasma AVP concentration in both MD group and control group was measured using Inverse C18 silica gel column radio-immunoassay.(3)After ES specimens were collected from patients with MD and AN,immunofluorescence was used to determine the distribution and subcellular localization of AQP-1,-2,-5 and V2 R in these ES specimens.Moreover,quantitative analysis of the fluorescence intensity of these individual AQPs was performed between the patients with MD and patients with AN.(4)Using a liquid chromatography-mass spectrometry(LC-MS),untargeted metabolomics analysis was performed on ELF of MD and AN patients collected during operation.RESULT(1)AQP1 was negatively expressed in the epithelial layer and weakly expressed in the subepithelial connective tissue,AQP2,AQP5 and V2 R were positively expressed in the ES epithelial layer,and negatively expressed in the subepithelial connective tissue,while AQP3 and AQP6 were negatively expressed both in the ES epithelial layer and subepithelial connective tissue.(2)The plasma AVP level between MD group(4.20±2.34pg/ m L)and control group(3.73±1.54pg/ml)was no statistical difference.The difference of plasma AVP level between unilateral MD group(4.25±2.42pg/m L)and bilateral MD group(3.74±1.41 pg/m L)was not statistically significant,and there was also no statistical difference between MD acute phase(4.32±2.43pg/m L)and remission phase(3.95±2.12pg/m L).Confocal microscopy analysis demonstrated that AQP1 was weakly expressed only in the subepithelial tissues,V2 R was expressed in the cytoplasm and membrane of the epithelial cells of the endolymph sac and there was no statistical difference in fluorescence intensity of AQP1 and V2 R between MD and AN groups.However,AQP2 was found to be mainly expressed in the basal lateral membrane of ES epithelial cells,while AQP5 was detected to be widely expressed in the apical membrane of ES epithelial cells,as well as in the cytoplasm and basal lateral membrane.The fluorescence intensity of AQP2 and AQP5 in the ES epithelial cells between MD and AN groups was statistically different.(4)The result of untargeted metabolomics showed ten differentially altered metabolites in the ELFs between MD group and AN group.The up-regulated metabolites in the ELF of MD group were as follows: 3-Amino-L-alanine,Hyaluronic acid,4-Hydroxynonenal,L-Capreomycidine,and D-Ribulose 1,5-bisphosphate,whereas the down-regulated metabolites in the ELF of MD group were as follows: D-Glucuronic acid,Inosine 5’-tetraphosphate,L-Arginine,N-Acetyl-D-glucosamine,D-Octopine.Immunohistochemisty showed that 4-Hydroxynonenal was positively expressed in the ES tissue sections of MD group,while negatively expressed in the ES tissue sections of AN group.Compared with that in the patients with AN,the expression of Hyaluronic Acid Synthase 2 in the ES epithelial cells of the patients with MD was upregulated,but there was no obvious change in the expression of hyaluronidase.Cluster of differentiation 44(CD44)of hyaluronic acid receptor was positively correlated with the expression of AQP2 in the ES epithelial cells of MD patients.CONCULSION In the present study,AQP2 and AQP5 were identified to be expressed in the epithelium of the human ES.Moreover,immunofluorescence analysis was used to further determine that the expression of AQP2 was mainly located in the basal lateral membrane of ES epithelial cells,which was presumed to be involved in the secretion of endolymph for maintaining the homeostasis of endolymph in the inner ear.Furthermore,the upregulated expression of AQP2 and AQP5 found in the ES of the patients with MD was possibly associated with the increased secretion of endolymph in ES that could has a causal link to the development of EH.However,there was no significant difference in plasma AVP level and the expression of V2 R in ES between MD group and control group,suggesting the upregulated expression of AQP2 found in the ES of the patients with MD was not associated with the regulation of the AVP-V2 R system.Finally,untargeted metabolomics analysis in the ELF between the MD group and control group showed that some differentially altered metabolites in the ES of MD may be potential biomarkers for regulating AQP2 expression independent of AVP.