2.4.2. Determinations of Renal Functions

Serum creatinine, urine creatinine, and urine microalbumin were analyzed using the AU480 Chemistry Analyzer (Beckman Coulter, Inc., Brea, CA, USA). Glomerular filtration rate (GFR) was estimated by calculation of the creatinine clearance using a standard clearance formula (the ratio of creatinine in urine/serum and the volume of urine produced).

#### 2.4.3. Determinations of Renal Oxidative Stress

The supernatant from kidney homogenate was used for oxidative stress assays. Malondialdehyde (MDA) was estimated using the TBARS assay kit (Cayman Chemical, Ann Arbor, MI, USA), glutathione (GSH) was assayed using the QuantiChrom™ Glutathione Assay Kit (Bioassay Systems, Hayward, CA, USA), and superoxide dismutase (SOD) and glutathione peroxidase (GPx) were determined using Calbiochem® Assay Kits (Merck Millipore, Darmstadt, Germany). All analyses were performed according to the manufacturer's instructions.

#### 2.4.4. Determinations of Mitochondrial Functions

Kidney mitochondria were isolated by differential centrifugation, and the mitochondrial proteins obtained were used to study mitochondrial function according to the protocols previously published [22]. Mitochondrial ROS production was determined using a fluorogenic dye dichlorofluorescin diacetate (DCFDA). ROS level was assessed from the fluorescent product and expressed in arbitrary units of fluorescent intensity. An increasing intensity indicates an increase in mitochondrial ROS production. Mitochondrial membrane potential change was assessed by staining mitochondria with a lipophilic cationic fluorescence JC-1 dye. The fluorescence intensity of JC-1 aggregate (red fluorescence) and monomer (green fluorescence) was measured, and a decrease in the red/green fluorescence intensity ratio implies the loss of membrane potential. Mitochondrial swelling was determined through changes in the absorbance of the mitochondrial suspension at 540 nm, where a decrease in absorbance intensity indicates the swelling of mitochondria.

#### *2.5. Histopathological Examinations*

Kidney tissues were routinely processed for light and electron microscopic examinations using the protocols previously described [13]. For the light microscopic study, a paraffin section of 4 μm was stained with hematoxylin and eosin (H&E) and examined by an unbiased pathologist using a Leica DM750 photomicroscope (Leica Microsystems, Heerbrugg, Switzerland). For electron microscopy, renal cortical sections embedded in Epon resin were stained with uranyl acetate and lead citrate, then examined using a JEM-2200 FS transmission electron microscope (JEOL, Tokyo, Japan).

The formalin-fixed paraffin-embedded kidney tissues were also used for CD34 immunofluorescence staining. Kidney sections (4 μm) were deparaffinized in xylene then rehydrated through an ethanol series. Antigen retrieval with 10 mM sodium citrate buffer (pH 6.0) was performed using a hot water bath for 10 min. The sections were permeabilized in PBST containing 1% BSA and 0.4% Triton X-100 for 10 min, followed by blocking with 5% BSA for 30 min at room temperature. Next, the slides were incubated with anti-CD34 antibodies (US Biological Life Sciences, Salem, MA, USA) for 2 h at room temperature then overnight at 4 ◦C. Fluorescence was observed under an inverted fluorescence microscope (Nikon, Eclipse Ts2, Nikon Instruments Inc., Melville, NY, USA).

#### *2.6. Western Blot Analysis*

Renal cortical tissues were homogenized in lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), and protein concentrations were measured using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). The protein sample was subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane, where it was blocked and probed with primary antibodies against peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), sirtuin 3 (SIRT3), acetylated superoxide dismutase 2 (Ac-SOD2), superoxide dismutase 2 (SOD2), and β-actin (Cell Signaling Technology, Danvers, MA, USA), followed by corresponding horseradish peroxidaseconjugated secondary antibodies (Millipore Sigma, Darmstadt, Germany). Protein band densities were detected by the ChemiDocTM Touch Imaging System (Bio-Rad Laboratories, Inc., Philadelphia, PA, USA) and quantified using ImageJ software (National Institute of Health, Bethesda, MD, USA).
