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

Abstract

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.

Date of Award	2015
Original language	English
Awarding Institution	King's College London
Supervisor	Afshan Malik (Supervisor) & Peter Jones (Supervisor)