Streptozotocin-induced diabetes model
Male Sprague Dawley rats weighing 200 ± 50 g obtained from Harlan Sprague Dawley Inc. were used for this study. All of the animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences to perform as described in the paper. Rats were randomly divided into two groups: control and diabetic. Diabetes was induced by injecting overnight fasted rats with streptozotocin (STZ; 65 mg/kg, i.p.) dissolved in 0.5 ml saline, while the control rats were injected with 0.5 ml saline. Fasting blood glucose levels were measured 48 hours post-STZ injection and rats with blood glucose levels greater than 250 mg/dl were classified as "diabetic". Diabetes-induced changes were studied at 5 weeks post-STZ injection (n = 6).
Metabolic studies, organ harvest and blood collection
Animals were kept in metabolic cages and acclimatized overnight prior to urine collection. Urine was collected from each rat for 24 hr, and their 24 hr food and water intake were also measured. Animals were sacrificed under isoflurane anesthesia and their kidneys harvested. Blood was collected via intracardiac puncture and serum samples were stored at -80°C until used for creatinine measurement.
Creatinine clearance & urine albumin measurement
Serum creatinine (sCr) and urine creatinine (uCr) levels were measured by a modified Jaffe's method (Pointe Scientific, Canton, MI, USA) in a Cobas Mira clinical analyzer (Roche Diagnostics, Indianapolis, IN, USA). Creatinine clearance (CrCl) was then calculated from the formula: CrCl (ml/min/kg) = (uCr × uVolume × Body weight)/(sCr × 1440 min × 1000). Urine albumin levels were determined by an immunoturbidometric assay (Pointe Scientific, Canton, MI, USA) in a Cobas Mira clinical analyzer and expressed as mg/day/kg.
Periodic acid-Schiff (PAS) staining was performed on formalin fixed, paraffin embedded kidney sections to assess tubular integrity and renal morphology as previously described [12, 13]. Tissue sections were scored on a 0 to 4 scale by two pathologists blinded to the treatment groups according to the extent of tubular dilation and glomerular changes as follows: 0 = absent; 1 = mild; 2 = moderate; 3 = severe; 4 = very severe.
Measurement of respiratory complex activities
Renal mitochondria were isolated from whole kidneys by differential centrifugation in a sucrose-containing buffer as previously described [14, 15]. The activity of mitochondrial Complexes-I through IV as described by Birch-Machin et al.  with minor modifications as previously described .
-ATP-ase (Complex-V) activity was assayed by measuring the release of Pi from ATP by the method of Law et al.. Each sample was run in the presence and absence of oligomycin (10 μg/ml), and the FoF1-ATP-ase activity was expressed as the oligomycin-sensitive ATPase activity.
Blue Native Gel Electrophoresis (BN-PAGE)
Mitochondrial complexes were analyzed by performing BN-PAGE on the control and diabetic mitochondria as previously described . To confirm the identity of mitochondrial Complex-III following BN-PAGE, the native gel was directly transferred to a PVDF membrane after first dimension and probed for known Complex III subunits Core 2 and Rieske subunits as described below. In addition, immunoblot analysis for Rieske and Core 2 subunits was performed following two-dimensional BN-PAGE by performing BN-PAGE, excising the Complex III bands, followed by SDS-PAGE in the second dimension.
ATP levels were determined in the mitochondrial fractions of control and diabetic rat kidneys using a luciferase based bioluminescent assay kit (Sigma-Aldrich, St. Louis, MO, USA) in a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA, USA).
Immunoprecipitation of Complex-III
Complex-III was immunoprecipitated from control and diabetic rat renal mitochondria (2.5 mg) as described by Shilling et al.  using Complex-III immunocapture beads (MS301) from Mitosciences, USA. After SDS-PAGE, the gels were stained with Coomassie blue staining solution (0.1% bromophenol blue R-250 in 40% methanol and 10% acetic acid) for 30 minutes and then destained overnight in 7.5% acetic acid/5% methanol solution.
For immunoblotting, the gel was transferred to a PVDF membrane, and probed for Core 2 protein or the Rieske subunit using mouse monoclonal antibodies – MS304 or MS305 (Mitosciences) at 1:1,000 dilution overnight at 4°C. Blots were then probed with peroxidase labeled goat anti-mouse secondary antibody, and developed using an enhanced chemiluminescent substrate system.
Data analysis was performed using Origin 6.0. All values were expressed as mean ± SEM. Independent t-test (two-tailed) was used to compare the mean values between control and diabetic groups. Differences were considered statistically significant if the p values were less than 0.05.