Fig 1.
PNx-induced miR-29b-3p expression change in WT and α1+/- mice.
WT and α1+/- mice were subjected to sham or 5/6th partial nephrectomy (PNx) surgery and left ventricle tissue was collected at the end of 16th week for RNA extraction, RT-qPCR, and Western blot analyses. A): Western blot of Na/K-ATPase α1 subunit expression in left ventricle tissue from different group of mice. !! indicates p<0.01 α1+/- vs WT. B): Expression of miR-29b-3p measured by RT-qPCR in left ventricle tissue from experimental mice. Data was analyzed using Two-Way ANOVA with GraphPad software 7.0., N = 5 in each group * indicates p<0.05 PNx vs Sham. C): mRNA expression of miR-29b-3p targeted genes: collagen 1A1 (Col1a1), matrix metalloproteinase-2 (Mmp2), fibrillin 1 (Fbn1), and elastin (Eln). * indicates p<0.05 PNx vs Sham; ** indicates p<0.01 PNx vs Sham. !! indicates p<0.01 α1+/- vs WT.
Fig 2.
Activation of Src and NFκB in mice left ventricle tissue after PNx surgery.
A): Western blot shows Src activation indicated by phosphorylation at Tyrosine 418 (pSrc) in left ventricle tissue, normalized to total Src levels (cSrc). Data are normalized as percentage expression of WT Sham animals. B): Western blots for total NFκB p65 in heart left ventricle tissue homogenates from each group. C): Western blots of NFκB p65 in nuclear and cytosolic fractions of isolated from left ventricle tissue. Lamin B1 is used as a nuclear marker. Nuclear/cytosolic ratio of NFκB was calculated as indicator of NFκB activation. Data were presented as mean ± SEM from 5 mice in each group. Data were analyzed using Two-Way ANOVA followed by Pairwise comparisons using Tukey’s correction for multiple comparisons, N = 4 in each group. * indicates p<0.05 PNx vs sham; ** indicates p<0.01 PNx vs Sham; !! indicates p<0.01 α1+/- sham vs WT sham or α1+/- PNx vs WT PNx.
Fig 3.
Expression and translocation of NFκB in heart tissue after PNx surgery.
Left ventricle tissue slides (4 μm in thickness) from WT sham (N = 4), WT PNx (N = 5), α1+/- sham (N = 4), and α1+/- PNx (N = 4) animals were deparaffinzed and immunostained for NFκB p65. (A): Representative images from WT mice; (B): Representative images from α1+/- mice; (C): Quantification data. Colocalization of NFκB and DAPI was analyzed using ImageJ. The ratio of NFκB p65 positive nuclear and total nuclear was used for analysis by Two-Way ANOVA. ** indicates p<0.01, WT PNx vs WT sham. Scale bar is 25 μm.
Fig 4.
The effect of NFκB inhibitor on ouabain-induced miR-29b-3p regulation in cardiac fibroblasts isolated from WT and α1+/- mice.
Isolated primary cultures of mouse cardiac fibroblasts were pretreated with 1μM of the NFκB inhibitor BAY 11–7082 for 15 min followed by ouabain (10 or 100nM) treatment for 24h. Non-treated or ouabain treatment alone without BAY 11–7082 was used as control. After treatment, cell lysates were collected. Expression of miR-29b-3p was measured using RT-qPCR in RNA isolated from cells from WT animals (A) and α1 +/- animals (B). Fibroblasts were were obtained from 4 animals in each group. Data was analyzed using One-Way ANOVA with GraphPad software 7.0. * indicates p<0.05 versus non-treated controls. ! indicates p<0.05 Bay11-7082 plus ouabain vs ouabain alone.
Fig 5.
Injection of pNaKtide blocks Src activation in mice subjected to PNx surgery.
Tissue homogenate obtained from left ventricle tissue were used to probe phosphor-Src (pSrc) and total Src (cSrc) using Western blot. Data were obtained from 5 animals in each group and normalized as percentage expression of WT Sham and was analyzed using Two-Way ANOVA with GraphPad software 7.0. Sham: sham-operated animals, ShamP: sham-operated animals with pNaKtide injection. PNx: PNx-operated animals, PNxP: PNx-operated animals with pNaKtide injection.* indicates p<0.05 vs Sham; & indicates p<0.05, PNxP vs PNx; !! indicates p<0.01 α1+/- PNx vs WT PNx.
Fig 6.
Injection of pNaKtide diminishes PNx-induced decrease in miR-29b-3p expression.
Total RNA obtained from left ventricle tissue were used for RT-qPCR analyses. Expression of miR-29b-3p was presented as fold regulation relevant to WT sham animals. Data were obtained from 5 animals in each group and was analyzed using Two-Way ANOVA with GraphPad software 7.0. * indicates p<0.05 vs Sham; & indicates p<0.05 PNxP vs PNx.
Fig 7.
Injection of pNaKtide mitigates PNx-induced cardiac fibrosis.
IP injection of 25mg/kg pNaKtide was performed biweekly started at 12th week following PNx surgery and heart left ventricle tissue was collected at the end of 16th week. Cardiac fibrosis was evaluated using Trichrome staining. Data are presented as Mean ± SEM, N = 10 for Sham WT, 6 for ShamP WT, 7 for PNx WT, 9 for PNxP WT, 11 for Sham α1+/-, 5 for ShamP α1+/-, 11 for PNx α1+/-, 7 for PNxP α1+/. The upper panel are representative 20x Trichrome staining. Scale bar = 25μm. The lower panel is the quantification data of fibrosis (% area). Data was analyzed using Two-Way ANOVA with GraphPad software 7.0. ** indicates p<0.01 vs Sham; & indicates p<0.05 PNxP vs PNx; !! indicates p<0.01 α1+/- PNx vs WT PNx.
Fig 8.
Injection of pNaKtide attenuates PNx-induced cardiac hypertrophy.
A): Data of heart weight/body weight (HW/BW) ratio were obtained from 6–10 mice from each group. B): Myocytes cross sectional area were evaluated using wheat germ agglutinin (WGA) staining as described in Method section. Eight images were randomly taken from each tissue slide and 20 cells from each image were measured for their cross sectional area. Data is presented as Mean ± SEM and analyzed using Two-Way ANOVA with GraphPad software 7.0. * indicates p<0.05 vs Sham; ** indicates p<0.01 vs Sham; & indicates p<0.05 PNxP vs PNx.
Table 1.
Echocardiographic data in WT mice 16 weeks following PNx or Sham surgery.
Table 2.
Echocardiographic data in α1+/- mice 16 weeks following PNx or Sham surgery.