We noticed multiple cases when the difference in expression shown in our heat map correlated with previously reported data on deregulation of the gene

e cells harvested from MFN2f/f mice were transduced with Cre-expressing adenovirus. Compared to non-transduced or control empty virus transduced cells, CRE transduction reduced MFN2 expression by about 5075%. Furthermore, CRE-mediated MFN2 reduction but not empty virus caused MitoTracker Green-stained mitochondria to appear punctate indicating that mitochondria were fragmented in most MFN2- cells. In a blinded review, nearly 70% of mitochondria in CRE-treated cells appeared fragmented vs. 10 15% in either control. Breeding and Phenotype of MFN2 cKO mice Pax2-Cre efficiently deletes MFN2 in kidney epithelial cells MFN2, which is readily detectable in kidney from wild type neonatal mice, was undetectable in kidney homogenates of MFN2 cKO mice by immunoblot analysis or immunostaining of kidney sections. Kidneys harvested from Pax2-Cre+/ MFN2f/+ exhibited highly variable MFN2 expression. MFN2 cKO mice had defective mitochondrial fusion in renal epithelial cells as evidenced by the presence of punctate, fragmented mitochondria while mitochondria of control mice appeared filamentous. MFN2-deficiency increases susceptibility to apoptosis following metabolic stress In the absence of glucose in the cell culture medium, exposure of cells to metabolic inhibitors such as cyanide or rotenone causes apoptosis in proximal tubule cells. MFN2f/f cells were transduced with either an empty or CRE-containing adenovirus producing MFN2 replete or MFN2 deficient cells respectively. Subsequently, CTL and MFN2- cells were stress by three hours of cyanide exposure followed by six hours recovery in glucose containing medium. In the absence of stress, minimal apoptosis was detected in Hoechst stained ” cells and this was not influenced by MFN2 deficiency. During recovery from stress however, the number of apoptotic cells was far greater in MFN2 deficient cells compared to control. In at least five randomly selected fields, a blinded observer detected an 83% increase in apoptosis in MFN2 deficient cells compared to control. 2 January 2012 | Volume 7 | Issue 1 | e31074 MFN2 cKO mice have Naringin normal kidney histology and a low level of apoptosis Kidney sections obtained from four day old MFN2 cKO pups and control littermates were stained with hematoxylin and eosin. MFN2 in Renal Stress 3 January 2012 | Volume 7 | Issue 1 | e31074 MFN2 in Renal Stress MFN2 deficiency in renal proximal tubule cells does not alter the oxygen consumption profile or Bax 6A7 epitope exposure Despite normal kidney and tubule function, proximal tubule epithelial cells with reduced MFN2 content are more susceptible to stress. To determine whether this susceptibility is due to altered mitochondrial metabolism, oxygen consumption rate was measured in control and MFN2 deficient cells at baseline as well as in the presence of oligomycin, CCCP, or antimycin. The OCR profile was similar in non-treated, CTL, and MFN2- cells. The OCR related to baseline “8496905 ATP turnover rate and maximal mitochondrial ATP turnover rate were calculated in three separate experiments. Despite marked differences in morphology, both baseline ATP turnover and maximal mitochondrial ATP turnover OCR were indistinguishable in CTL and MFN2- cells. Within 10 minutes, exposure of CTL or MFN2- renal epithelial cells to sodium cyanide resulted in a similar, profound and sustained decrease in OCR. Bax is a critical pro-apoptotic protein responsible for outer mitochondria membrane injury after metabolic stress. To determine whether MFN2-deficie