The diabetic state has been shown to increase total renal oxygen consumption (Q_O2). Since C- peptide has been reported to be renoprotective, we investigated the effect of C-peptide on Q_O2 in immortalized mouse proximal tubular cells and in a mouse model of oxidative stress; immortalized mouse proximal tubular cells over-expressing the NADPH oxidase subunit p22phox. All cells were maintained at 37°C and 5% CO₂, in DMEM/F12 medium containing 5% FBS. At a 50% sub-confluency, cell splitting was routinely performed with 2.5% trypsin. Q_O2 was measured in cells incubated with and without C-peptide (5 nM) in high (400 mg/dl for 48 h) and low glucose medium (100 mg/dl for 48 h). Q_O2 was determined using the BD Oxygen Biosensor System using a GENios multimode reader at 4-min intervals for 2 h at an excitation of 480 nm and emission of 630 nm. For semiquantitative analysis the maximum slope of fluorescence units/s was used and converted into arbitrary units. Q_O2 was adjusted for protein concentration. For all data, n=4. Hyperglycemia increased Q_O2 both in control cells (40%) and p22phox cells (30%). C-peptide induced an increase in control cell Q_O2 in low glucose (150%) and high glucose medium (100%) both. In p22phox cells, C-peptide reduced Q_O2 in low glucose (44%) as well as high glucose (54%) medium. In conclusion, C-peptide induced separate effects in the normal state vs. oxidative stress independently of glucose concentration. These results might explain the beneficial effects of C- peptide on the function of the diabetic kidney.