Adaptive Responsive Mechanisms Of The Spotted Scat(Scatophagus Argus) Kidney To Salinity Change Based On Proteomics

Scatophagus argus is an estuarine fish that can tolerate salinity fluctuation throughout its life,which make it a ideal novel to study osmoregulatory mechanisms.Kidney plays a vital role in osmoregulation,which has specific osmotic reception cells that can monitor the change of environmental osmotic factor concentration,regulate its endocrine function cells to secrete corresponding hormones,and make corresponding responses.In order to study the regulatory function of kidney on salinity stress,the proteomic analysis of renal cells and tissues under different salinity stress was carried out.Primary renal cell was treated with hypoosmotic(150 mOsm/L)and hyperosmotic(600 mOsm/L)medium for 24 h,and then the proteome library of renal cells under different osmotic pressure was constructed by label-free.A total of 3,787 proteins were screened.14 and 31 significant difference proteins were found in hypo-osmotic and hyper-osmotic group compared to the control group,respectively.Gene Ontology(GO)functional annotation,GO enrichment analysis,and protein clustering analysis were performed on these differential proteins.It was found that hypotonic stress mainly affected the expression of cytoskeleton related proteins,inclouding tubulin alpha-4A chain?tubulin beta chain?myosin-11 and type I cytoskeletal 18.However,most of the differentially expressed proteins in hyperosmotic group were extracellular interstitial proteins,like fibronectin?collagen and so on.In order to further explore the expression changes of these proteins,we selected 10 proteins related to cytoskeleton,energy biosynthesis and metabolism from the differential proteins for RT-qPCR verification.Our results were basically consistent with the proteome data.Among the selected hypertonic groups,the mRNA level of 3 proteins was opposite to the protein level,like procollagen-lysine,2-oxoglutarate 5-dioxygenase 2(PLOD2)?fibronectin(FN)and ADP-ribosylation factor-like protein 1(ARF1).Therefore,it is assumed that after hypotonic stimulation,the intracellular osmotic pressure is higher than the extracellular osmotic pressure,and the cells expand immediately,and then express cytoskeleton related proteins in large quantities,thus affecting other ion channels.Under hyperosmotic stimulation,the cells were in a state of water loss and morphologic contraction,which affected the adhesion between cells.However,the increase of fn and plod2 showed that the cells were repairing the damaged protein.This provides a new perspective for the study of the mechanism of osmotic pressure regulation at the cellular level.S.argus acclimated under different salinity were used as experimental subjects in vivo experiment.And proteomic analysis was performed using iTRAQ quantification.A total of 18,541 peptides and 6,305 proteins were identified.25‰salinity was set as the control group,while 0,10,40,50‰salinity were experimental groups.Compared to the control group,there were 232 up-regulated proteins and 334 down-regulated proteins.Differential proteins were analyzed by GO and KEGG pathway mapping,and 59 significant differential proteins related to dopamine(DA)metabolism were selected for heat map analysis.16 KEGGs related to dopamine metabolism were selected to obtain a Protein-protein interaction(PPI)network analyzed by STRING database,which contained 103 differentially expressed proteins.According to the results,we selected five important enzymes in DA anabolism:tyrosine hydroxylase(TH),Parkinson disease protein 7(DJ-1),monoamine oxidase A(MAOA),monoamine oxidase B(MAOB),and catechol O-methyltransferaes(COMT)and plotted the expression pattern of these proteins.The concentrations of dopamine,dopamine precursors(L-dopa)and metabolites(Homovanillic acid,HVA)in serum and kidney were detected by LC-MS/MS.In high salinity group,the concentration of DA in serum increased significantly and HVA decreased significantly.However,HVA was significantly down-regulated in hypo-salinity group.In kidney,the concentration of DA increased remarkably at 0‰salinity;L-DOPA decreased significantly under hyper-salinity stress;HVA concentrations all increased significantly at 10‰,40‰and 50‰salinity.Protein expression of renal Na~+/K~+-ATPase?(NKA?)and dopamine receptor 1(SaDRD1)was examined by Western blot.The results revealed the same expression pattern of NKA?compared to proteomic analysis,but protein expression of SaDRD1 was down-regulated when salinity increased.Based on proteomic analysis,we screened the proteins associated with DA metabolism and analyzed the causes of the changes for these proteins,providing new data for studying the role of dopamine systems in osmoregulation.