Role Of Thioredoxin1in The Impaired Renal Sodium Excretion In D₅F173L Transgenic Mice

With the development of people’s living level, cardiovascular diseases become theleading cause of human death worldwide and the prevalence of cardiovascular disease wasrising[1][2]. Essential hypertension is one of the most common healthy risk factors in bothdeveloped and developing countries, affecting about33.5%of the middle-aged adultpopulation[3]. The past studies have shown that the long-term regulation of blood pressurerests on renal mechanisms[4]. Renal dopamine receptors have been shown as importantregulators of blood pressure, sodium balance in hypertension[5]. Dopamine have counterregulatory effects on cellular signal transduction, production of reactive oxygen species,renal sodium excretion, and blood pressure via renal dopamine receptors[5][6]. Compare tothe other dopamine receptors, Dopamine D5receptor (D5R) has a relatively high affnity fordopamine and can be activated in the absence or presence of low concentrations ofendogenous agonist[7]. Therefore, The D5receptor has aroused our signifcant interest.Previous studies have found that D5R can upregulate the production of Cyclic adenosinemonophosphate (cAMP)[8]and the elevating blood pressure of D5-/-mice was related tohigh AT1receptor levels[9][10].We know that the function of D5dopamine receptor isweakened but not abolished completely in hypertension patients. So we stud y D5mutant mice but not D5-/-mice. D5R gene locus (chromos ome4p1511-1611) polymorphism weresignificantly correlated with essential hypertension. The mutant sites of D5R gene includeL88F, F173L, A269V, P330Q, C335X, N351D, S390G, and S453G[1]. F173L mutation sitewas related to hypertension particularly[1]. To investigate the role of dopamine D5R in thedevelopment of hypertension, we established mutant D5F173L transgenic mice expressedhuman D5receptor F173L mutant gene, which exhibited deficient o f D5receptor function,may result in elevating blood pressure[2]. However, the mechanism of elevated bloodpressure in D5F173L transgenic mice remains not clear. Since the espression amount ofrenal D5receptor is only20%in D1-receptor[3], D5receptors may regulate blood pressurethrough other mechanisms.The previous studies reported that D5receptors closely related with oxidative stressand the D5receptor inhibits the production of ROS by inhibition of PLD and NADPHoxidase expression and activity[4]. Then, we selected some genes related to oxidative stressand found the mRNA and protein expression of Trx1was significantly decreased in thekidney of D5F173L transgenic mice. Thioredoxin system, an important intrinsicanti-oxidant system in mammalian cells. There are two Trxs including cytosolic (Trx1) andmitochondrial (Trx2) in humans. Previous studies show that Trx1plays an essential role inthe antioxidant process[5].Trx1system plays an important role in the development of avariety of cardiovascular diseases. The expression of Trx1is decreased in hypertensivepatients[6]. Although previous studies suggest thioredoxin system can reduce blood pressureby increasing NO levels, downregulating the growth rate of VSMCs, antioxidants, anti-inflammatory and regulating NADPH activity[7]. However, the roles of the cytoplasmicthioredoxin system in the renal function of hyperoxia-induced hypertension are not yet fully understood.In addition, previous investigations have shown that the stimulation of Na+-K+-ATPaseresulted from O2--dependent NO deficiency is via the cGMP-PKG dependent manner[1].Previous studies suggest thioredoxin system can reduce blood pressure by increasing NOlevels[2]. So we speculated Trx1may regulate renal Na+-K+-ATP activity in thecGMP-PKG dependent manner. Therefore, our study investigated the role of thioredoxin1(Trx1) in the impaired renal sodium excretion in D5F173L transgenic mice exhibitingdysfunction of dopamine D5receptor.Methods：PartⅠ:1. Detected the Blood pressure and Sodium Excretion of D5WT and D5F173Ltransgenic mice2. Determine O2-levels and select the gene mRNA expression3. Comparing the expression levels of Trx1of WT and D5F173L transgenic micePart Ⅱ:1．The co-localization of D5R and Trx1were observed by Immunofluorescence.2．Deleted the Trx1mRNA and protein levels of Trx1in mRPT cells transfected D5WTand D5F173L plasmid by QT-PCR and western-blot.3．Examined the effect of rTrx1protein on Na+-K+-ATPase activity of mRPT cellstransfeted D5WT and D5F173L plasid using ouabain assay.4．Detected the expression of cGMP in mRPT cells transfected with D5WT、D5F173Lplasid and treated with rTrx1protein by cGMP Elisa kit.Part Ⅲ:1．Extracted and identified the Primary RPT cells2．Comparing the Trx1expressing levels of the primary RPT cells of D5WT andD5F173L transgenic mice by western-bolt.3．Comparing the Na+-K+-ATPase activity of the primary RPT cells of D5WT and D5F173L transgenic mice using ouabain assay.ResultsPartⅠ:1．The blood pressure of D5F173L transgenic mice were significantly higher thanD5WT mice. Compare to D5WT mice, the function of renal sodium excretion is impaired inD5F173L transgenic mice.2．Compare to D5WT mice, the level of ROS was significantly higher in D5F173Ltransgenic mice. The Trx1mRNA expression level of D5F173L transgenic mice wassignificantly lower than D5WT transgenic mice.The expression levels of Trx1protein wasdownregulated remarkably in D5F173L transgenic mice than D5WT transgenic mice.PartⅡ:1．The D5R and Trx1showed extensive colocalizationin the cytoplasm of mRPT cells.The expressing levels of Trx1was obviously decreased in mRPT cells transfected D5F173Lplasmid.2．The hTrx1could significantly decrease the Na+-K+-ATPase activity in the mRPTcells transfected with D5F173L plasmid but not D5WT plasmid.The hTrx1could increasethe expression of cGMP in mRPT cells transfected with D5F173L plasid not D5WT.PartⅢ:The primary RPT cells were isolated and identified using the cytokeratin18antibody.We found that Trx1protein level was obviously decreased in the primary mouse RPT cellsof D5F173L transgenic mice than D5WT. Compare to the D5WT mice, the activity ofNa+-K+-ATPase was significantly increased in the primary RPT cells of D5F173L transgenicmice.Conclusion：Our present study investigated the role of thioredoxin1(Trx1) in the impaired renalsodium excretion in D5F173L transgenic mice exhibiting dysfunction of dopamine D5receptor.1．The mRNA and protein expression of Trx1was significantly decreased in the kidneyof D5F173L transgenic mice. 2．Trx1played an key role in BP regulation and renal sodium excretion in D5F173Ltransgenic mice exhibiting dysfunction of dopamine D5receptor in the cGMP-PKGdependent manner.