Preserved regulation of renal perfusion pressure by small and intermediate
conductance KCa channels in hypertensive mice with or without renal failure
Ludovic Waeckel1,2, Florence Bertin1, Nicolas Clavreul1, Thibaut Damery1, Ralf Köhler3,4,
Jérôme Paysant1, Patricia Sansilvestri-Morel1, Serge Simonet1, Christine VayssettesCourchay1, Heike Wulff5, Tony J. Verbeuren1 and Michel Félétou1
1
Institut de Recherches Servier, 92150 Suresnes, France;
2
Centre de Recherche des
Cordeliers, Inserm UMRS 872, Paris, France; 3Cardiovascular and Renal Research, IMM,
University of Southern and Denmark, Odense, Denmark, 4Aragon Institute of Health
Sciences-I+CS and ARAID, Zaragoza, Spain, 5Department of Pharmacology, University of
California, Davis, CA 95616, USA.
Legends of supplementary figures
Supplementary figure 1. Gene expression in the murine models studied
mRNA expression of the human angiotensinogen gene, the human renin gene and the murine
renin gene in wild type (BL), single transgenic mice overexpressing either the human
angiotensinogen gene (A) or the human pro-renin gene (R) and double transgenic mice (AR)
under control diet or after a 4-week of salt plus L-NAME diet (BLSL and ARSL).
RT-PCR confirmed the expression of the human renin and angiotensinogen gene in the
kidneys of the appropriate mice strains. The double gene expression (associated with an
increase in circulating angiotensin I level, see table 1), did not alter the expression of
individual transgenes when compared to mice subjected to mono transgene overexpression,
while producing a very significant decrease of the endogenous murine renin gene. This
observation is in agreement with earlier reports showing that the human transgenes, in
contrast to the murine endogenous gene, are not under the angiotensin II-dependent negative
feedback regulation [1,2,3]. In mice, high salt diet and L-NAME treatment (or eNOS
1
deficiency) have been shown independently to decrease renal renin gene expression [2,4,5].
The salt plus L-NAME diet partially but significantly decreased the expression of the murine
renin gene but also of the two human transgenes, suggesting that the salt regulation of the
transgenes was preserved [2].
Values are expressed as mean  SEM. The number of animals studied or from which tissues
were taken = 7-12. The * signs indicates a statistically significant effect of the salt plus LNAME diet and the # sign a statistically significant effect of the double transgene (ANOVA1
followed by a Bonferonni post-hoc test, P<0.05).
Supplementary figure 2. Concentration-response curves of methoxamine-induced increase in
RPP (0.1 to 100 nmol) in isolated kidneys of wild type (BL) and double transgenic mice (AR)
on control diet or after 4 weeks on a diet with salt plus L-NAME (BLSL and ARSL). Effects
of the cyclooxygenase inhibitor indomethacin (3 µM), the NO-synthase inhibitor L-nitroarginine (L-NA, 10 µM) and their combination.
Values are expressed as mean  SEM. The number of animals from which tissues were taken
= 6-13. The * signs indicates a statistically significant difference between the control and the
of L-NA + indomethacin groups (ANOVA2 followed by a Bonferonni post-hoc test, P<0.05).
Supplementary figure 3. Concentration-response curves of sodium nitroprusside-induced
vasodilatation (0.01 to 10 nmol) in methoxamine-vasoconstricted isolated murine kidneys of
wild-type (BL) and double transgenic mice (AR) on control diet or after 4 weeks on a diet
with salt plus L-NAME (BLSL and ARSL, respectively).
Top panel: Experiments were performed in the presence of the cyclooxygenase inhibitor
indomethacin (3 µM) plus the NO-synthase inhibitor L-nitro-arginine (10 µM).
2
Lower panel: Experiments were performed in the presence of a combination of the
cyclooxygenase inhibitor indomethacin plus the NO-synthase inhibitor L-nitro-arginine plus
the KCa3.1 blocker, TRAM-34 (1 µM) plus the KCa2.3 blocker, apamin (0.5 µM).
The endothelium-independent responses to the NO donor, sodium nitroprusside were similar
in the kidneys from the 4 group of mice indicating that the smooth muscle sensitivity toward
NO is not affected by transgenes overexpression and/or diet. Additionally, the presence of
TRAM-34 and apamin did not influence sodium nitroprusside-induced vasodilatation,
indicating that the inhibitory effects observed, versus either methacholine or the channel
activator SKA-31, were specific.
Values are expressed as mean  SEM. The number of animals from which tissues were taken
= 5.
References
1
Keeton TK, Campbell WB (1980) The pharmacologic alteration of renin release.
Pharmacol Rev 32: 81-227.
2
Thompson MW, Smith SB, Sigmund CD (1996) Regulation of human renin mRNA
expression and protein release in transgenic mice. Hypertension 28: 290-296.
3
Keen HL, Sigmund CD (2001) Paradoxical regulation of short promoter human renin
transgene by angiotensin II. Hypertension 37: 403–407.
4
Schricker K, Kurtz A (1996) Blockade of nitric oxide formation inhibits the
stimulation of the renin system by a low salt intake. Pflugers Arch 432: 187-191.
5
Wagner C, Gödecke A, Ford M, Schnermann J, Schrader J, Kurtz A (2000) Regulation
of renin gene expression in kidneys of eNOS- and nNOS-deficient mice. Pflugers Arch 439:
567-572.
3