1. Hypoxia-induced adrenomedullin production in the kidney
Daisuke Nagata, Yasunobu Hirata, Etsu Suzuki, Masao Kakoki, Hiroshi Hayakawa, Atsuo Goto, Toshihiko Ishimitsu, Naoto Minamino, Yukari Ono, Kenji Kangawa, Hisayuki Matsuo, Masao Omata 
Kidney International 
Volume 55, Issue 4, Pages (April 1999)
DOI: /j x
Copyright © 1999 International Society of Nephrology Terms and Conditions

2. Figure 1
Hypoxia-induced expression of adrenomedullin (AM) transcripts. The expression of AM transcripts was examined by Northern blot analyses. Twenty micrograms of total RNA was loaded in each lane. (A) Time course of the expression of AM transcripts in MDCK cells. Culture medium was replaced with low-serum medium 24hours before the experiment. The cells were placed in sealed hypoxic bags with a carbon dioxide generator for the indicated time. Twenty micrograms of total RNA were loaded in each lane. (B) The time course of the expression of AM transcripts in cultured rat VSMCs. The experiment was performed basically in the same way as in (A). The experiments were repeated three times for both (A) and (B), and arrows indicate the position of the 1.6kb AM transcripts. The lower panels show ethidium bromide staining of 18S ribosomal RNA as internal controls.
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions

3. Figure 2
(A) Amount of adrenomedullin (AM) peptide accumulated in cultured media of Madin Darby canine kidney (MDCK) cells and rat vascular smooth muscle cells (VSMCs) under normoxic and hypoxic conditions. Some cells were placed in the hypoxic bags for the indicated time, whereas others were used as the normoxic control. AM in the cultured media was measured by RIA. Symbols are: the amounts of AM in the cultured media from normoxic (•) and hypoxic (○) MDCK cells; the amounts of AM in the cultured media from normoxic (▪) and hypoxic (□) rat VSMCs. The values are the mean ±sem.*P < 0.01, **P < vs. normoxia. (B) cAMP accumulation under normoxic and hypoxic conditions in rat VSMCs. Some cells were incubated under hypoxic conditions with low serum medium containing 0.5mmol/liter 3-isobutyl-1-methylxanthine, whereas others were used as the normoxic control. Some cells were incubated with human AM (10-7 mol/liter) in the presence of 0.5mmol/liter 3-isobutyl-1-methylxanthine as the positive control. The values are the mean ±sem.*P < 0.01 vs. normoxia, **P < vs. hypoxia.
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions

4. Figure 3
Time course of TNF-α accumulation in MDCK cells (A) and rat VSMCs (B). The amount of this protein in the cells and in the culture media was evaluated by Western blot analyses. The cells were placed in sealed hypoxic bags for the indicated times. The lanes on the left represent the positive control, in which 20ng of human TNF-α was loaded in each lane.
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions

5. Figure 4
Stability of AM transcripts under hypoxic conditions. After changing the medium to low-serum medium, the expression of AM transcripts was induced by incubating the cells in the hypoxic bags for six hours (“control” in the figure). Some plates were returned to normoxia, whereas others were maintained in hypoxic bags for the indicated periods in the presence of 5 μg/ml of actinomycin D. Twenty micrograms of total RNA were loaded in each lane for Northern blot analyses. The amount of AM transcripts at each time point was compared with the “control” level, and the half-life in the both conditions was calculated. The arrow shows the position of the 1.6kb AM transcripts.
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions

6. Figure 5
Activity of AM promoter under hypoxic conditions. Rat VSMCs were transiently cotransfected with 2 μg of the AM promoter/luciferase plasmid and 1 μg of Seapansy Control Vector as the internal control using lipofection method. Transfected rat VSMCs were placed under hypoxic conditions for 12hours (hypoxia; ▪) or stimulated with cytokines cocktail containing interleukin-1β 50 U/ml, TNF-α 4000 U/ml, and lipopolysaccharide 50 μg/ml for 12hours (cytokine; ▪). Some transfected cells were maintained under normoxic conditions as a control (control; □). Dual luciferase assay was performed using a luminometer. The vertical axis indicates the relative luciferase activity compared with control. The values are the mean ±sem.*P < 0.01 vs. control.
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions