The Detection and Partial Localisation of Heteroplasmic Mutations in the Mitochondrial Genome of Patients with Diabetic Retinopathy.

Diabetic retinopathy (DR) is a common complication of diabetes and a major cause of acquired blindness in adults. Mitochondria are cellular organelles involved in energy production which contain mitochondrial DNA (mtDNA). We previously showed that levels of circulating mtDNA were dysregulated in DR patients, and there was some evidence of mtDNA damage. In the current project, our aim was to confirm the presence of, and determine the location and prevalence of, mtDNA mutation in DR. DNA isolated from peripheral blood from diabetes patients (n = 59) with and without DR was used to amplify specific mtDNA regions which were digested with surveyor nuclease S1 to determine the presence and location of heteroplasmic mtDNA mutations were present. An initial screen of the entire mtDNA genome of 6 DR patients detected a higher prevalence of mutations in amplicon P, covering nucleotides 14,443 to 1066 and spanning the control region. Further analysis of 42 subjects showed the presence of putative mutations in amplicon P in 36% (14/39) of DR subjects and in 10% (2/20) non-DR subjects. The prevalence of mutations in DR was not related to the severity of the disease. The detection of a high-prevalence of putative mtDNA mutations within a specific region of the mitochondrial genome supports the view that mtDNA damage contributes to DR. The exact location and functional impact of these mutations remains to be determined.

Altered Mitochondrial Function, Mitochondrial DNA and Reduced Metabolic Flexibility in Patients With Diabetic Nephropathy.

The purpose of this study was to determine if mitochondrial dysfunction plays a role in diabetic nephropathy (DN), a kidney disease which affects > 100 million people worldwide and is a leading cause of renal failure despite therapy. A cross-sectional study comparing DN with diabetes patients without kidney disease (DC) and healthy controls (HCs); and renal mesangial cells (HMCs) grown in normal and high glucose, was carried out. Patients with diabetes (DC) had increased circulating mitochondrial DNA (MtDNA), and HMCs increased their MtDNA within 24 h of hyperglycaemia. The increased MtDNA content in DCs and HMCs was not functional as transcription was unaltered/down-regulated, and MtDNA damage was present. MtDNA was increased in DC compared to HC, conversely, patients with DN had lower MtDNA than DC. Hyperglycaemic HMCs had fragmented mitochondria and TLR9 pathway activation, and in diabetic patients, mitophagy was reduced. Despite MtDNA content and integrity changing within 4 days, hyperglycaemic HMCs had a normal bio-energetic profile until 8 days, after which mitochondrial metabolism was progressively impaired. Peripheral blood mononuclear cells (PBMCs) from DN patients had reduced reserve capacity and maximal respiration, loss of metabolic flexibility and reduced Bioenergetic Health Index (BHI) compared to DC. Our data show that MtDNA changes precede bioenergetic dysfunction and that patients with DN have impaired mitochondrial metabolism compared to DC, leading us to propose that systemic mitochondrial dysfunction initiated by glucose induced MtDNA damage may be involved in the development of DN. Longitudinal studies are needed to define a potential cause-effect relationship between changes in MtDNA and bioenergetics in DN.