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    • A key finding of our study is

    2018-11-13

    A key finding of our study is the demonstration that exposure to HG leads to a rapid increase in mesangial cell MtDNA, accompanied by unchanging/decreased transcription of mitochondrial encoded mRNAs and little change in nuclear encoded mitochondrial mRNAs in renal cells. The amount of MtDNA is known to be directly proportional to the amount of mitochondrial encoded mRNAs (Hock and Kralli, 2009; Williams, 1986) however in our study increased MtDNA levels in patients (Fig 1A) are not accompanied by increased mitochondrial encoded mRNAs (Fig. 1B–D), we see the same trend in cells, where the mitochondrial encoded mRNAs are reduced (Fig. 4C–E) whereas the MtDNA in increased (Fig. 4A). As the function of MtDNA is to encode mitochondrial mRNAs, we deduce that the increased MtDNA in not functional. The only exception is the glucose induced up-regulation of TFAM mRNA, which has been previously reported (Choi et al., 2004). Our observation is suggestive of a disconnect between the increase in MtDNA and mitochondrial transcription/translation. A similar disconnect was reported in a study investigating the mechanisms of insulin resistance in muscle, with increased MtDNA but not increased mitochondrial content in myotubes (Aguer et al., 2013). Increased MtDNA in cultured Paclitaxel was reported in response to hydrogen peroxide induced oxidative stress (Lee and Wei, 2005). HG induced ROS may inhibit mitochondrial biogenesis, as has been shown in other systems (Ballinger et al., 2000) and this view is consistent with reduced/unaltered mitochondrial transcription and translation which we observe. The increase in MtDNA in these conditions could be a compensatory response to the impairment of transcription. We measured cellular respiration in HMCs and found that oxidative metabolism can cope for several days of high stress conditions and continues to provide energy. Cells started to display altered morphology within a few hours of exposure to HG with mitochondrial fragmentation, which may be part of the initiation of an adaptive biogenesis programme to increase MtDNA content. Whilst glucose induced MtDNA increase was evident within 24h, cells showed no detectable perturbation in their metabolic profile at this time point. Some changes were seen after 8days, however it was only after 12days of culture that significantly reduced ECAR and OCR were observed. To our knowledge this is the first report of the metabolic response of HMCs, although previously a meeting report on mouse mesangial cells showed similar profiles to ours (Chacko et al., 2010a). Our data agree with a study using rat retinal endothelial cells where growth in HG for 6days led to reduced basal and maximal respiration, however unlike our study where ECAR was decreased, they found increased ECAR (Trudeau et al., 2010). The reduced ECAR in our cells is suggestive of an energy deficit and may be a consequence of a ROS induced glycolytic block (Colussi et al., 2000). This idea is supported by our finding of HG induced ROS in our cells. The importance of mitochondrial ROS in oxidative stress in the kidney is well established, and mitochondrial targeted antioxidants ameliorate certain markers of renal damage (Chacko et al., 2010b). Here we show that the increased ROS is accompanied by increased MtDNA damage and NF-κB and MYD88 mRNAs. Like bacterial DNA, MtDNA is un-methylated and can initiate immune responses via the intracellular Toll like receptor (TLR) 9 (Higginbotham et al., 2002). The inflammatory properties of MtDNA (Collins et al., 2004; Zhang et al., 2010) and disruption of the normal degradation of MtDNA in the cytosol of cardiomyocytes was shown to cause TLR9-mediated inflammation leading to a heart failure in a mouse model (Oka et al., 2012). It is well established that chronic inflammation is a key mediator in diabetic complications but the underlying mechanisms have not been elucidated (Navarro-Gonzalez et al., 2011). Our data in HMCs of increased MtDNA content, ROS, MtDNA damage, and up-regulation of MYD88 and NF-κB support the idea of MtDNA induced activation of the inflammatory TLR9 pathway in these cells and are suggestive of a novel mechanism leading to chronic inflammation in DN.