The animal study was approved by the Ethics Committee of Chonnam National University Medical School. The experimental procedure conformed to the institutional guidelines for experimental animal care and use. Male Sprague-Dawley rats weighing 200 to 220 g were used.

Rats were unilaterally obstructed by ligation of the left proximal ureter for 7 days. Control rats were treated in the same way except that no ligature was made. The rats had free access to standard rat feed and tap water and were sacrificed by decapitation on day 7 after operation. The kidney was rapidly removed, dissected into three zones [cortex and outer stripe of outer medulla (cortex/OSOM), inner stripe of outer medulla (ISOM), and inner medulla (IM)], and processed for semiquantitative immunoblotting as described below.

The dissected cortex/OSOM, ISOM, and IM were homogenized in ice-cold isolation solution containing 0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, 8.5 µM leupeptin, and 1 mM phenylmethylsulfonyl fluoride, with pH 7.2. The homogenates were centrifuged at 1,000×g for 15 min at 4℃ to remove whole cells, nuclei, and mitochondria. The total protein concentration was measured (Pierce BCA protein assay reagent kit, Rockford, IL, USA). All samples were adjusted with isolation solution to reach the same final protein concentrations and solubilized at 65℃ for 15 min in SDS-containing sample buffer and then stored at -20℃. To confirm equal loading of protein, an initial gel was stained with Coomassie blue. SDS-PAGE was performed on 9% or 12% polyacrylamide gels. The proteins were transferred by gel electrophoresis (Bio-Rad Mini Protean II, Bio-Rad, Hercules, CA, USA) onto nitrocellulose membranes (Hybond ECL RPN3032D; Amersham Pharmacia Biotech, Little Chalfont, UK). The blots were subsequently blocked with 5% milk in phosphate-buffered saline with Tween 20 (PBST; 80 mM Na₂HPO₄, 20 mM NaH₂PO₄, 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 hour and incubated overnight at 4℃ with 1:1,000 dilutions of primary antibodies, followed by incubation with 1:1,500 dilutions of secondary anti-rabbit horseradish peroxidase-conjugated antibodies (P448; DAKO, Glostrup, Denmark). The labeling was visualized by use of an enhanced chemiluminescence system.

The antibodies used were as follows: affinity-purified anti-rabbit antibodies against phosphorylated phosphatidylinositol 3-kinase (p-PI3K; Cell Signaling Technology, Beverly, MA, USA), phosphorylated Akt (p-Akt; Cell Signlaing Technology), phosphorylated mTOR (p-mTOR; Cell Signlaing Technology), transforming growth factor-β1 (TGF-β1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (Cell Signaling Technology), and Bcl-2 (Cell Signaling Technology).

Results are expressed as means±SEMs. The statistical significance of differences between the groups was determined by using unpaired t-test, and it was defined as a p value less than 0.05.

On day 7 after the ureteral obstruction, the body weight of rats did not differ significantly between the two groups (238±5.8 g in controls vs. 240±4.5 g in UUO, p=0.79). However, the weight of the ureteral obstructed kidney was increased compared with the controls (1.2±0.1 g in controls vs. 2.3±0.2 g in UUO, p<0.05).

In the ureteral obstructed kidney, the expression of p-PI3K was significantly increased in the cortex/OSOM and IM, whereas it was not significantly changed in the ISOM (Fig. 1). The expression of p-Akt was also increased in the cortex/OSOM, ISOM, and IM (Fig. 2). The expression of p-mTOR was increased in the cortex/OSOM and ISOM (Fig. 3).

Following the UUO, the protein expression of TGF-β1 was increased in the cortex/OSOM, ISOM, and IM of the obstructed kidney (Fig. 4). The expression of Bax was also increased in the obstructed kidney compared with the control (Fig. 5). The protein expression of anti-apoptotic factor Bcl-2 was decreased in the obstructed kidney (Fig. 6).