The mechanisms underlying the development of diabetes-induced kidney damage are presently unclear. However, altered oxygen metabolism may be an important factor. Recently, mitochondria from diabetic rat kidneys displayed increased protein levels of Uncoupling Protein 2 (UCP-2) with subsequent increased oxygen consumption (JO₂) independent of oxidative phosphorylation. We therefore investigated the specific role of UCP-2 by in vivo gene silencing using short interference (si)-RNA. Control and streptozotocin-induced diabetic rats were administered either scrambled or UCP-2 siRNA (n=8 per group) and kidney cortex mitochondria were isolated 48h later. siRNA treatment resulted knockdown of UCP-2 protein expression in controls (-30%) and diabetics (- 54%). Mitochondria from untreated diabetics and both siRNA treated controls and diabetics displayed glutamate-stimulated JO₂ during ATP-synthase inhibition (oligomycin) (1.6±0.5, 1.5±0.5 and 3.8±0.5 nmol O₂/min/mg protein, respectively), whereas controls were unaffected by glutamate. The effect of siRNA on glutamate-stimulated JO₂ in diabetics was completely prevented by addition of ADP. These results suggest that the elevated JO₂ in siRNA treated diabetics is due to an alternative mechanism involving the adenine nucleotide transporter (ANT) which can cause mitochondria uncoupling and increase JO₂ independently of oxidative phosphorylation. Uncoupled ANT is inhibited by ADP, which locks ANT in c-conformation and thereby inhibits its uncoupling properties. Scrambled siRNA had no effect on any of the investigated parameters. By utilizing in vivo gene silencing, we were able to show an additional mechanism which results in mitochondria uncoupling and increased glutamate-stimulated JO₂. It may be speculated that mitochondria uncoupling is important for reducing mitochondria superoxide formation.