Hyperglycaemia-induced mitochondrial DNA changes and mitochondrial dysfunction in diabetic nephropathy

• Main Author: Czajka, Anna Natalia
• Other Authors: Malik, Afshan Navid ; Jones, Peter Martin
• Published: King's College London (University of London) 2015
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.762289

Background: The mechanisms involved in the development of diabetic nephropathy (DN), which affects more than 30% of patients with diabetes worldwide, are not fully understood. DN is believed to result from hyperglycaemia-induced pathways in the kidney. Hypothesis: Hyperglycaemia/high glucose causes early changes in mitochondrial DNA (MtDNA), possibly contributing to mitochondrial dysfunction. Methods: Human renal immortalised and primary cultured mesangial cells (HMCL, HMCs) and transformed tubular epithelial cells (HK-2) were cultured in 5mM (NG) and 25mM (HG) glucose and in 5mM glucose plus 20mM mannitol. Organs and whole blood samples were collected from streptozotocin-induced (STZ), prohibitin 2 knockout (β-PhB2-/-) and leptin deficient (Lepob/ob) diabetic mice. MtDNA content was measured in cultured renal cells, mouse organs and circulating cells using qPCR. The cellular bioenergetics of HMCs and HK-2 cells was measured using XFe96 Seahorse analyser. Genes involved in mitochondrial life cycle and in the TLR-9 pathway in HMCs were measured using real-time qPCR. Reactive oxygen species (ROS) production and cell viability were assessed in HMCs using fluorescence and luminescent assays and hyperglycaemia-induced MtDNA damage using elongase PCR. Mitochondrial morphology and protein content were assessed by MitoTracker staining and Western blot respectively. Results: Increased MtDNA levels in circulation in STZ-induced and β-PhB2-/- diabetic mice (P < 0.05) was observed. In the STZ-induced mouse kidneys, MtDNA was reduced after 4 weeks diabetes (P < 0.05); a similar trend was observed in the kidneys of the ob/ob mice (P = 0.08). Growth of HMCs in HG resulted in 3-fold higher MtDNA and 2-fold higher TFAM (P < 0.05), no significant changes were observed in HK-2 cells. The expression of two mitochondrial genome encoded mRNAs were reduced (P < 0.05) in parallel with increased MtDNA damage, cellular ROS and apoptosis (P < 0.05) in HMCs exposed to HG. Mitochondrial length and degree of branching were reduced in HMCs cultured in HG (P < 0.01, P < 0.001). NF-κB and MYD88 expression were up-regulated in HMCs exposed to HG (P < 0.05). Hyperglycaemia caused a decrease in basal, maximal and ATP-linked respiration (P < 0.001) in the HMCs. Although no alteration in the MtDNA content was observed in HK-2 cells exposed to HG, bioenergetic profile of HK-2 cells was affected by hyperglycaemia with reduced basal, ATP-linked and maximal respiration (P < 0.01). Conclusion: These data show that hyperglycaemia can directly increase MtDNA in cultured renal and circulating cells in mouse models of diabetes. Hyperglycaemia-induced damage to MtDNA caused a dysregulation between MtDNA levels and mitochondrial transcription, suggesting the increased MtDNA may not be functional. Induction of the TLR-9 pathway suggests a potential inflammatory role of the damaged MtDNA. Therefore, the in-vitro and in-vivo data suggest altered MtDNA content may be a biomarker of an adaptive mechanism of failing mitochondrial function under stress conditions. Such changes may be the foundation of the damage seen in patients with DN and needs further investigation.