Up-regulation Of AQP2 In NRK Cells Treated With Shenqiwang Based On "V2R-cAMP-PKA" Pathway

Objective To study the effect of Shenqiwan on the expression of AQP2,and observe the changes of the correlation index of "V2R-cAMP-PKA-AQP2" pathway in NRK cells through the intervention of inhibitors and agonists,exploring the main role of Shenqiwan in the pathways,as well as preliminarily revealing the molecular mechanism of Shenqiwan in up-regulation of AQP2 in NRK cells.Methods 27 male SD rats were randomly divided into the control group and Shenqiwan group.The rats in the control were administered with normal saline,whereas the rats in the Shenqiwan group were administered Shenqiwan at 3.0g/kg(twice per day for 5days)dissolved in normal saline by gavage.After 12h fasting and the last administration for 1h,the blood samples were collected from heart after pentobarbital sodium(30mg/kg,i.p.)anesthesia,then centrifugation in 3000 rpm for medicated serum.In vitro cultured NRK cells and firstly divided into serum-free group,fetal bovine serum group and normal rat serum group,comparing the proliferation of NRK cells as well as the expression of V2R and AQP2 protein.Then NRK cells were divided into normal rat serum group,2.5%Sqw(3.0 g/kg)group,5%Sqw(3.0 g/kg)group,10%Sqw(3.0 g/kg)group and 20%Sqw(3.0 g/kg)group,in which Shenqiwan containing serum group was diluted with normal rat serum.Using H-89,SQ22536 and Tolvaptan inhibit PKA,cAMP and V2R from bottom to top,to explore whether V2R is the target of Shenqiwan upregulation of AQP2 expression,with 10%Shenqiwan containing serum as the optimal dose.To further explore whether Shenqiwan can act on other targets to regulate AQP2 expression except for V2R,we inhibit the activation of the "V2R-cAMP-PKA-AQP2" pathway with the cAMP inhibitor SQ22536 and add Shenqiwan containing serum or PKA agonist 8-Br-cAMP.Cell viability of NRK cells were analyzed using MTS,and distribution and expression of V2R and AQP2 were observed by cell immunofluorescence assay.The mRNA level of AQP2 was quantified by qPCR technique and the expression of AQP2,p-PKA,V2R protein in NRK cells were detected by Western blot.The content of cAMP in NRK cells was Detected by ELISA.Results(1)Different concentrations of Shenqiwan medicated serum significantly promote the proliferation of NRK cells.The mRNA and protein expression of V2R are up-regulated significantly in 10%and 20%medicated serum when compared with control group.The mRNA and protein expression of AQP2 significantly increased in 10%medicated serum and other groups show no difference.So 10%Shenqiwan medicated serum was adapted for the optimal dose.(2)Combine with ELISA,Western blot and MTS to screen the optimal condition for Tolvaptan,SQ22536 and H-89 and 8-Br-cAMP.The results shown that 25μM Tolvaptan and 25μM H-89 significantly decreased the expression of AQP2 in 3h.In addition,200μM SQ22536 has the same effect in the 6h.Phosphorylation of PKA was inhibited significantly when SQ22536 and H-89 in their optimum condition.80μM 8-Br-cAMP also can raise significantly the expression protein of AQP2 and phosphorylation of PKA when hatching 3h.(3)Medicated serum of Shenqiwan promotes the expression of AQP2 when compared with the normal rat serum group.Adding Tolvaptan,SQ22536 or H-89,the expression of mRNA and protein for AQP2 and phosphorylation of PKA were decreased significantly,indicating that V2R may be the main link of Shenqiwan up-regulation of AQP2 expression in NRK cells.(4)Both of the phosphorylation of PKA and AQP2 in SQ22536 group decreased significantly when compared with the normal rats serum group.However,the two indicators were significantly increased when 8-Br-cAMP or Shenqiwan medicated serum was added,indicating that PKA may be the other target for regulating AQP2 expression.Conclusion 10%Shenqiwan Medicated serum promotes the genetic level of AQP2 mRNA and the expression of AQP2 protein in NRK cells.Shenqiwan has an effect probably similar to AVP which activate the "V2 receptor-cAMP-PKA-AQP2" pathway to regulate the expression of AQP2 through the renal V2 receptor.Shenqiwan may promote the phosphorylation of the AQP2 through phosphorylating PKA when the upstream is suppressed.These results suggest that V2R and PKA are potential therapeutic target for Shenqiwan to ameliorate the disorder of water metabolism under kidney-yang deficiency syndrome.V2R and PKA may be important targets for Shenqiwan upregulation the expression of AQP2.