Figure 5

YHC, GKW and GRA (GA) are responsible for changes in AQP 2 regulation and diuresis effect in vitro and in vivo. The mIMCD-3 cells were planted in 6 well plate and cultured in RPMI1640 medium supplied with 10% fetal bovine serum. All doses were given during cell logarithmic phase, and the highest doses were restricted until cell proliferation was not affected. (A) Blotting image of AQP 2 expressed by mIMCD3 cells treated by GRA for 24 hours. Bar chart of fold changes is given, and concentrations are labeled below. (B) Blotting image of AQP 2 expressed by mIMCD3 cells treated by YHC, GKW singly or in combination with GRA, for 24 hours. When GRA was combined, the working concentration was 12 μM. The blots was cropped properly and the full length blots are presented in Supplementary Fig. S6. (C to D) Bar chart for AQP 2 expression with different treatment as with in (B) *P < 0.05 when compared with control group (none drug-treated), ^& P < 0.05 compared between groups with or without GRA treatment (n = 3). (E to G) Low, middle and high doses of YHC, GKW as well as special doses of HCTZ, GA and GA-YHC combination, GA-GKW combination were all submitted to diuresis mice model. Accumulated urine output was calculated from animal weight loss 0 to 4 hours after NS loading. The data were represented as DI. (H and I) representative blotting image and bar chart of renal total AQP 2 levels treated by different chemicals and their combination. Doses of each group are given below the image. *P < 0.05 when YHC-h or GKW-m groups compared with M group, ^$ P < 0.05 when YHC-m or GKW-l compared with M group, ^& P < 0.05 when compared between groups with or without GA treatment (n = 6). The blots was cropped properly and the full length blots are presented in Supplementary Fig. S6.