Experimental model studies
CCN2 deletion: Mice used were CCN2
CCN2flox/floxROSA26-ERT/Cre, henceforth CCN2-KO mice, which are
time-conditional knockout (CCN2-KO) mice. They were developed as
previously described (Fontes et al., 2015) and used in all experimental
models. 13-14 weeks old mice were randomly divided in 2 groups:1) CCN2-KO group, in which tamoxifen were intraperitoneally
(IP) injected (0.1 ml of a 10mg/mL solution; C8267, Sigma-Aldrich) four
times in alternate days and 2) wild-type (WT) group, which were
IP injected with corn oil (0.1 ml) and used as control group. After
two-week washout period, CCN2 deletion was confirmed by polymerase chain
reaction (PCR) using CCN2-floxed Forward: 5’-
AATACCAATGCACTTGCCTGGATGG-3’ and CCN2-floxed Reverse:
5’-GAAACAGCAATTACTACAACGGGAGTGG-3’ primers. The amplified DNA was
resolved by size in agarose gel (1,5%) (Suppl Figure 1 ).
Description of experimental models: *For more
information see Suppl Figure 2
Angiotensin II (Ang II) infusion model . Systemic
infusion of Ang II was done using ALZET osmotic mini-pumps (Model 2002,
ALZET Alza Corp., Palo Alto, CA) at the dose of 1000 ng/kg/min (A9525,
Sigma Aldrich), as previously described (Daugherty et al., 2000;
Rodrigues-Diez et al., 2015). Four experimental mice groups were
studied: WT (corn oil); WT + Ang II (corn oil and Ang II); CCN2-KO
(tamoxifen); and CCN2-KO + Ang II (tamoxifen and Ang II). Several sets
of models were done to perform different studies, with at least 5 mice
per group. The end point of all experimental models was 15 days of Ang
II treatment, except one model used for magnetic resonance imaging (MRI)
and Multiple reaction monitoring (MRM) mass spectrometry (MS) studies,
which end-point was 5 days after starting Ang II infusion (not shown at
models’ scheme). To allow MRI studies, the mini-pumps stainless-steel
flow moderator was replaced by PEEK Tubing (DURECT Corporation) using
the ALZET MRI Compatibility Technical Tip.
Spironolactone treatment on Ang II infusion model: To
study the role of aldosterone, some CCN2-KO + Ang II mice were treated
with Spironolactone (SP) (S3378-1G, Sigma-Aldrich), a mineralocorticoid
receptor antagonist (Liu et al., 2013), dissolved in corn oil at the
dose of 50mg/kg/day by IP injection every 48 hours, starting one day
prior first tamoxifen injection. Four experimental mice groups were
studied in this case: WT (corn oil); CCN2-KO (tamoxifen); CCN2-KO + Ang
II (tamoxifen and Ang II) and CCN2-KO + Ang II + Spironolactone (SP)
(tamoxifen, Ang II and SP).