Background: Our recent analysis of electron microscopy demonstrated a mitochondrial damage of septum tissue derived from a rat model of long term chronic renal failure (CRF). In order to further assess these findings, we tested the expression levels of mitochondrial pro-apoptotic, oxidative stress and permeability genes in addition to apoptosis analysis. To this end, we analyzed septum tissues from Sham operated control (Sham) and CRF rats, in addition to ex-vivo experiments, where sera from experimental animals were applied to H9C2 cardiomyoblast cell-line.
Methods: CRF Lewis rats underwent 5/6 nephrectomy, allowing long term CRF development. At time of necropsy, 8 months post CRF induction, sera and septum tissue obtained from Sham and CRF rats were collected. In the ex-vivo experiments, sera drawn from Sham or CRF animals were applied to H9C2 culture for 48h (ShamH9C2/CRFH9C2, respectively). RNA isolated from septum tissue of Sham/CRF animals as well as from ShamH9C2/CRFH9C2 cells, was used for real-time PCR analysis for pro-apoptotic (BID, RIPK3), oxidative stress (NOX4) and mitochondrial permeability marker (VDAC1, NCLX) gene expression. An apoptotic assay was also performed on ShamH9C2/CRFH9C2 using Annexin-V-FITC-PI and was analyzed by Flow Cytometery (FACS).
Results FACS analysis of ShamH9C2 and CRFH9C2 showed that sera from CRF animal increased apoptosis (3±0.2% to 61±0.5% Annexin-V positive/PI negative, respectively). RT-PCR analysis of CRFH9C2 revealed 3- and 1.8-fold increase of the pro-apoptotic genes, BID and RIPK3 respectively, compare to ShamH9C2. Both CRF tissue samples and CRFH9C2 cells demonstrated increase of NOX4 expression compare to sham (5.6- and 21-fold, respectively). Gene expression of VDAC1 and NCLX was upregulated in the CRF tissue (2.3- and 3.7-fold) and CRFH9C2 cells (1.6- and 11.7-fold, respectively).
Conclusion: Long term CRF per-se may induce apoptosis in cardiomyocytes; a process which may be mediated, at least in part, by the cardiac mitochondria.
