The roles of NADPH-oxidase and nNOS for the increased oxidative stress and the oxygen consumption in the diabetic kidney

Abstract

Background: Sustained hyperglycaemia induces increased renal oxygen consumption resulting in reduced oxygen availability in the diabetic kidney. We investigated the roles of the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase and the neuronal nitric oxide synthase (nNOS) for the increased oxygen consumption in streptozotocin-diabetic rats.

Methods: Oxygen consumption was measured in isolated proximal tubular cells (PTC) from streptozotocin-induced diabetic rats (n = 7-9 per group) with and without chronic treatment with apocynin, a NADPH-oxidase inhibitor, or S-methyl-L-thiocitrulline (SMTC), a selective nNOS inhibitor, or a combination of the two and the results were compared to normoglycaemic controls (n = 10). Oxidative stress was estimated from thiobarbituric acid reactive substances and protein expression measured by Western blot.

Results: Proximal tubular cells from untreated diabetic rats had increased oxygen consumption compared to controls (40.6 +/- 7.9 versus 10.9 +/- 2.0 nmol/mg protein/min). All treatments reduced the diabetes-induced increase in oxygen consumption (apocynin 10.5 +/- 1.7, SMTC 19.7 +/- 3.0 and apocynin + SMTC 21.6 +/- 3.6 nmol/mg protein/min). Neither apocynin nor SMTC had any effect on the oxygen consumption in cells pre-incubated with ouabain, an inhibitor of active electrolyte transport. Oxidative stress was elevated in the diabetic kidney and inhibited by all treatments. The increased oxygen consumption by diabetic proximal tubular cells correlated with increased protein expressions of p47(phox) and nNOS and the treatments prevented these increases.

Conclusions: Diabetes induces oxidative stress, which increases oxygen consumption in proximal tubular cells. Inhibition of either NADPH-oxidase or nNOS prevented the increased oxygen consumption. The effect of blocking both these enzymes was less than additive suggesting overlapping pathways which warrant further studies.

Figure 1

Oxygen consumption by isolated proximal tubular cells from normoglycemic controls and diabetic rats with and without the different chronic and acute treatments. * denotes P<0.05 vs. baseline within the same group, # denotes P<0.05 vs. corresponding group of untreated control, and † denotes P<0.05 vs. corresponding group of untreated diabetics. All values are the means ± SEM.

Figure 2

Protein expression and a representative blot of p47^phox in kidney cortex lysate (A) and in the membrane fraction (B) as well as nNOS in kidney cortex and a representative blot (C) of control and diabetic rats with and without the different chronic treatments. The bands at the molecular weights of 47 kDa for p47^phox and 160 kDa for nNOS were quantified by densitometry from 8 rats in each group. * denotes P<0.05 vs. untreated control group and # denotes P<0.05 vs. untreated diabetic group. All values are means ± SEM.

Figure 3

Lipid peroxidation, estimated as TBARS, in plasma (A), kidney cortex (B) and 24h urinary TBARS excretion (C) in normoglycemic controls and diabetic rats with and without the different chronic treatments. * denotes P<0.05 vs. untreated control group and # denotes P<0.05 vs. untreated diabetic group. All values are means ± SEM.

Figure 4

Urinary excretion of nitrate/nitrite in normoglycemic controls and diabetic rats with and without the different chronic treatments. * denotes P<0.05 vs. untreated control group and # denotes P<0.05 vs. untreated diabetic group. All values are means ± SEM.