Animals and Experimental Design
Thirty-six male Sprague-Dawley rats (Animal Laboratories of the Wuhan University, Wuhan, China) weighing between 180 and 220 g were housed in cages and maintained in a temperature-controlled room with a 12:12-hour light-dark cycle, with free access to tap water and standard rat chow for 2 or 4 weeks. For celiac mini-pump implantation (Alzet model 2002 or 2004; Alza, Mountain View, Calif., USA), rats were selected at random to be subjected to either human Ang II infusion (Sigma Chemical, St. Louis, Mo., USA) at 400 ng·kg–1·min–1 for 14 or 28 days (n = 6) or normal saline infusion for 14 or 28 days (n = 6) or to be used as normal controls (14 or 28 days, n = 6).
Systolic Blood Pressure Measurement
The systolic blood pressure was monitored by tail cuff plethysmography (China-Japan Friendship Hospital, Beijing, China) in conscious, trained, and preheated rats on days 0, 7, 14, 21, and 28.
Sample Collection and Preparation
Urine samples were collected throughout a 24-hour period to assay for proteinuria and albuminuria on days 0, 7, 14, 21, and 28. Blood and kidney samples were harvested on days 14 and 28. Trunk blood was collected into prechilled tubes, then centrifuged at 4,000 rpm for 30 min at 4°C. Plasma fractions were removed and assayed for creatinine.
After decapsulation, the kidneys were washed with ice-cooled saline, blotted dry, and weighed. The left kidney was cross-sectioned and fixed in 10% formalin in phosphate-buffered saline (PBS, pH 7.2) for histochemical and TUNEL studies (see below). The right renal cortex was separated and divided into three parts: the first one was embedded in Tissue-Tek OCT compound and snap frozen at −80°C without prior fixation for immunofluorescence staining, the second one was snap frozen in liquid nitrogen for Western blot and RT-PCR analyses, and the third one was fixed with 2.5% glutaraldehyde or with phosphate-buffered 3.5% paraformaldehyde plus 0.02% glutaraldehyde for transmission electron microscopic and immune transmission electron microscopic analyses, respectively.
Immunofluorescence Microscopic Analysis
Frozen sections (6 μm) were transferred to slides, washed with cold PBS, and processed as follows: Sections were fixed with chilled acetone for 10 min at −20°C and incubated with 0.1% Triton X-100 in PBS at 37°C for 15 min. After washing with PBS, the sections were then blocked with 1% bovine serum albumin for 30 min and stained with goat anti-human nephrin (1:50; Santa Cruz Biotechnology, Santa Cruz, Calif., USA) for 12 h at 4°C, followed by rhodamine-conjugated rabbit anti-goat IgG (1:100; Zhongshan, Beijing, China). As a control, some sections were stained with nonassociated antibody instead of anti-nephrin. The sections were examined by fluorescence microscopy (BX51 microscope; Olympus, Tokyo, Japan). The images were captured with a spot charge-coupled device camera (Olympus). All exposure settings were kept constant for each primary antibody.
DNA fragmentations in apoptotic cells were detected using the TUNEL assay. After paraffin dewaxing, sections (5 μm) were incubated with 3% H2O2 for 30 min followed by 0.1% Triton X-100 in PBS for 15 min at room temperature. Sections were washed and exposed to TdT buffer for 5 min and incubated in a moist chamber with a mixture of TdT and digoxigenin-11-dUTP in TdT buffer (R&D Systems, Minneapolis, Minn., USA) for 1 h at room temperature. They were then washed in PBS for 15 min. The sections were incubated for 30 min with streptavidin-biotin-peroxidase-conjugated anti-digoxigenin-11-dUTP antibody, and antibody-binding sites were visualized using diaminobenzidine. The slides were counterstained with hematoxylin. Negative controls included the omission of TdT; positive controls included the pretreatment of sections with 0.1 U/μl deoxynuclease-1 before TdT staining. Apoptotic podocytes from single cross-sections through the glomerulus were counted using the Weibel-Gomez method.
Transmission Electron Microscopic Analysis
Paraformaldehyde-glutaraldehyde-fixed 1-mm3 blocks of renal cortices were postfixed with 1% osmium in 0.1 M cacodylate buffer for 1 h, dehydrated in graded ethanols, embedded in Epon, sectioned, stained with uranyl acetate and lead citrate, and examined and photographed with a Hitachi H600 transmission electron microscope (Hitachi, Tokyo, Japan).
Immune Electron Microscopic Analysis
Paraformaldehyde-glutaraldehyde-fixed, 1-mm3 blocks of renal cortex were washed with PBS, dehydrated in graded ethanols, and embedded in Epon. Ultrathin sections were transferred to nickel grids and then blocked with 1% bovine serum albumin and 1% normal goat serum in PBS. Sections were incubated with the primary polyclonal goat anti-human nephrin antibody against the intracellular domain of nephrin (1:50; Santa Cruz Biotechnology) and then with the secondary gold-conjugated (10 nm) rabbit anti-goat secondary antibody (1:100; R&D Systems). The sections were postfixed with 1% glutaraldehyde, contrasted with uranyl acetate and lead citrate, then observed under an electron microscope and photographed for detailed analysis.
Reverse Transcription-Polymerase Chain Reaction
Total RNA was extracted with TRIzol reagent (Promega, Madison, Wisc., USA). Total RNA (5 μg) extracted from renal cortex was used to synthesize cDNA and served as a template for amplification of nephrin and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous standard. The forward and reverse primer sequences (Shenggong, Shanghai, China) were for nephrin 5′-AGCCTCTTGACCATCGCTAA and 5′-CCCAGTCAGCGTGAAGGTAG, respectively, and for GAPDH 5′-ACAGAGTACTTGCGCTCAGGAG and 5′-GTCACCCACACTGTGCCCATC, respectively. The PCR products were 302 and 542 bp, respectively. The amplification was performed under the following conditions: 94°C for 45 s (denaturation), 56°C for 45 s (annealing), and 72°C for 1 min (extension), with 35 cycles at 72°C for 10 min for final extension. PCR products were analyzed by electrophoresis and the intensity of the bands by means of AlphaEase FC image software (Alpha Innotech, San Leandro, Calif., USA).
Western Blotting Studies
The renal cortex sample was homogenized on ice and the proteins extracted using a lysis buffer, centrifuged at 12,000 g for 15 min at 4°C, and the resultant supernatant was collected. The protein concentration of the samples was determined with a BCA protein assay kit (Pierce Biotechnology, Rockford, Ill., USA). Western blotting analysis was performed as follows: Samples were separated on an 8% polyacrylamide gel and then semidry blotted on a PVDF membrane. The blotted membrane was incubated for 2 h at room temperature in blocking solution (5% blocking agent in 1% defatted milk) in Tris-buffered saline (TBS) with 0.02% Tween 20 (T) and then washed three times with washing solution. The blocked membrane was incubated overnight at 4°C with primary antibodies (1:50; Santa Cruz Biotechnology) in the TBS-T solution containing 1% bovine serum albumin and then with rabbit anti-goat secondary antibody (1:100; Zhongshan). The detection was performed according to the manufacturer's instructions using the ECL kit (Santa Cruz Biotechnology). The intensity of the bands was analyzed using Alpha Ease FC image software.
Statistical analysis was performed using SPSS software version 10.0 (SPSS, Chicago, Ill., USA). Values are presented as mean ± SD or as mean ± SEM. Testing between two groups was performed by Student's t test, and one-way analysis of variance was used for comparing three groups. Correlations were assessed by the Spearman rank correlation coeffiencent (r). p < 0.05 was considered statistically significant.