MR silencing in transgenic mice.

<p>Control (con) and transgenic (tg) mice of line B and D received Dox via food pellets for 14 days or, in some cases, were left untreated (w/o). (A) Whole kidneys were analyzed for MR mRNA expression by RT-QPCR. N = 14/22 (Dox, line B), N = 5/7 (w/o, line B), N = 13/20 (Dox, line D), N = 5/6 (w/o, line D). (B) Whole hearts were analyzed for MR mRNA expression by RT-QPCR. N = 9/17 (Dox, line B), N = 5/7 (w/o, line B), N = 13/14 (Dox, line D), N = 6/7 (w/o, line D). Gene expression in panels A and B was normalized to HPRT; mRNA levels in control mice were arbitrarily set to 1 and are depicted as mean ± SEM. (C) Protein lysates were prepared from kidney and heart of Dox-treated mice of line B and analyzed by western blot for the amount of MR protein. Detection of ERK served as a loading control. One representative analysis each is depicted in the left panel. The right panel shows a densitometric quantification of MR in kidney and heart of control (con) and transgenic (tg) mice after normalization to the amount of ERK. Protein levels are depicted in arbitrary units as mean ± SEM. N = 5/6. (D) Colon, hippocampus, liver, lung, stomach and muscle were isolated from Dox-treated mice of line B and analyzed for MR mRNA levels by RT-QPCR. Gene expression was normalized to HPRT and is depicted in arbitrary units as mean ± SEM. N = 4/7. Statistical analysis in all panels was performed by unpaired two-tailed Student’s t test. n.s.: non-significant; *: p <0.05; **: p <0.01; ***: p <0.001.</p

Gene expression analysis in the kidney of inducible MR knock-down mice.

<p>Control (con) and transgenic (tg) mice of line B (upper panel) and line D (lower panel) received Dox via food pellets for 14 days. Subsequently, whole kidneys were analyzed for mRNA expression of serum and glucocorticoid-regulated kinase 1 (Sgk-1), the potassium channel IK-1 and the subunits α, β and γ of the epithelial sodium channel ENaC by RT-QPCR. N = 16/24 (line B), N = 19/22 (line D). Gene expression was normalized to HPRT, and mRNA levels in control mice were arbitrarily set to 1. Values are depicted as mean ± SEM, statistical analysis was performed by unpaired two-tailed Student’s t test. n.s.: non-significant; *: p <0.05; **: p <0.01.</p

Gene expression analysis and collagen deposition in the heart of inducible MR knock-down mice.

<p>(A) Control (con) and transgenic (tg) mice of line B received Dox via food pellets for 14 days. In the upper panel, analysis of the heart for mRNA expression of serum and glucocorticoid-regulated kinase 1 (Sgk-1), the hormone ANP and the structural proteins MHCα and β by RT-QPCR is depicted. N = 10/17. In the lower panel, analysis of the heart for mRNA expression of Col3a1 (Collagen, type III, α1), fibronectin (FN-1) and the pro-inflammatory cytokines IL-1β and IL-6 by RT-QPCR is depicted. N = 3/4. Gene expression in panel A was normalized to HPRT; mRNA levels in control mice were arbitrarily set to 1 and are depicted as mean ± SEM. (B) Control (con) and transgenic (tg) mice of line B and D received Dox via food pellets for 28 days. Exemplary photographs of heart sections of one control and one transgenic mouse of line B, stained with picrosirius red are depicted in the upper panel. The red color corresponds to collagen mainly found around vessels. Size bar: 100 μm. The lower panel shows computer-aided quantification of the collagen staining area in control and transgenic mice of both line B and D. N = 5/5 (line B), N = 3/5 (line D). Values in panel B are depicted as mean ± SEM, statistical analysis was performed by unpaired two-tailed Student’s t test. n.s.: non-significant.</p

Physiological changes in inducible MR knock-down mice after TAC.

<p>(A) Control (con) and transgenic (tg) mice of line B received Dox via food pellets throughout the entire duration of the experiment. Two weeks after the beginning of Dox treatment TAC surgery was performed and another four weeks later the mice were sacrificed. MR mRNA levels in the heart were determined by RT-QPCR at the end of the experiment, renin plasma levels were analyzed by ELISA. The ventricular weight was measured for each animal and is depicted relative to the respective tibia lengths. The collagen staining area was determined after incubating paraffin sections from heart with picrosirius red followed by computer-aided quantification. (B) Analysis by echocardiography was performed one week before TAC (pre) as well as one (1w) and four weeks after TAC (4w). The diagrams show alterations of four parameters over time, namely the end-diastolic anterior wall thickness (AWThd), the left ventricular weight relative to the body weight (LVW/BW), the fractional area shortening (FAS) and the ejection fraction (EF). Values in all panels are depicted as mean ± SEM; N = 5/7. Statistical analysis was performed by unpaired two-tailed Student’s t test. n.s.: non-significant; *: p <0.05; **: p <0.01; ***: p <0.001.</p

Generation and characterization of inducible MR knock-down mice.

<p>(A) Operating principle of the lentiviral system used for inducible MR knock-down. The vector comprises one cassette consisting of the H1 promoter with tetracycline resistance operator sequences (tetO) and a MR-specific shRNA, and a second one encompassing the tet repressor (TetR) linked to eGFP by a T2A element under the control of the ubiquitin C promoter (Ub-p). In the absence of doxycycline (Dox), the TetR binds to tetO and blocks shRNA expression. After addition of Dox, the TetR is released thus enabling shRNA transcription. Under both conditions, eGFP is expressed constitutively. (B) Peripheral blood leukocytes of one control (con) and one transgenic (tg) mouse were analyzed for eGFP expression by flow cytometry. (C) Kidney, heart, colon, hippocampus, liver, lung, stomach and muscle samples obtained from control (con) and transgenic (tg) mice of line B and D were analyzed for eGFP mRNA expression by RT-QPCR. N = 3–7 (line B), N = 3–5 (line D). Gene expression was normalized to HPRT and is depicted in arbitrary units as mean ± SEM. (D) Kidney sections from one control (con) and one transgenic (tg) mouse of line B were analyzed by immunohistochemistry for eGFP expression. One representative example out of three is shown for each genotype. Size bar: 50 μm.</p