Sprague-Dawley rats were randomly assigned to receive either normal saline or Ang II (400 ng·kg^–1·min^–1) by means of a mini-osmotic pump for variable time periods. Systolic blood pressure and urinary protein and albumin excretion rate measurements were carried out on days 7, 14, 21, and 28. The animals were sacrificed on days 14 and 28 and evaluated for serum creatinine, renal pathological changes, podocyte apoptosis, renal nephrin mRNA, and protein expression.

The Ang II-infused rats developed hypertension and proteinuria. On day 14, the Ang II-infused rats showed narrowing of the slit diaphragm, an increase in podocyte nephrin mRNA and protein expression, and alterations in its distribution along the foot processes. On day 28, the Ang II-infused rats demonstrated the presence of apoptotic podocytes and decreased nephrin mRNA and protein expression. There was a negative correlation between nephrin expression and the numbers of apoptotic podocytes (r = −0.63, p < 0.05).

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.

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.

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).

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.

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).

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.

There were no apoptotic podocytes in the rats infused with normal saline. By contrast, a significant increase in apoptotic podocytes per glomerular section was detected on day 28 in Ang II-infused rats, but not on day 14 (fig. ​(fig.44).

Nephrin mRNA and protein levels were elevated on day 14 in the renal cortex of Ang II-infused rats, as shown by RT-PCR and Western blotting (fig. ​(fig.5).5). To determine the changes of the nephrin distribution pattern, we performed immunofluorescence labeling of glomerular sections. The nephrin expression was distributed linearly along the glomerular basement membrane in control and normal saline-infused rats, whereas the pattern was changed to a dispersed and granular distribution on day 14 in Ang II-infused rats (fig. 6a, b). By contrast, nephrin mRNA and protein expression decreased markedly on day 28 in Ang II-infused rats (fig. ​(fig.5,5, ​,6c).6c). In order to determine the localization of nephrin on the podocyte slit diaphragms in detail, we performed immunogold labeling of nephrin on ultrathin sections. In glomeruli of control and normal saline-infused rats, the gold particles were located on the slit diaphragms or the foot process surfaces (fig. ​(fig.7a).7a). In Ang II-infused rats, however, the numbers of gold particles on the slit diaphragms were decreased markedly, and most of them were located in the cytoplasm and vesicles on day 14 (fig. 7b, d). In accordance with the findings from immunofluorescence staining, the total number of gold particles located either on slit diaphragms or in podocyte cytoplasm decreased dramatically on day 28 (fig. ​(fig.7c7c).