Transgenic Mice and Genotyping
We previously reported bitransgenic mice bearing the NPHS2
-rtTA (A) and TetO-LacZ
]. We crossbred A+/–
mice with homozygous Immortomouse (I+/+
;Charles River Laboratories, Wilmington, Mass., USA), harboring tsA58, thermosensitive SV40 large T antigen [7
]. Glomeruli were isolated from bitransgenic offspring with the A+/–
genotype. Animal care conformed to the National Institutes of Health Guidefor the Care and Use of Laboratory Animals and was approved by the National Institutes of Diabetes and Digestive and Kidney Diseases Animal Care and Use Committee.
For genotyping, PCR was done with Taq
PCR Master Mix Kit (Qiagen, Valencia, Calif., USA) for the A
gene as previously reported [6
]. PCR for the I
gene employed a forward primer, 5′-AGC GCT TGT GTC GCC ATT GTA TTC-3′, and a reverse primer, 5′-GTC ACA CCA CAG AAG TAA GGT TCC-3′, yielding a 1,000-bp product. PCR cycle parameters for the I
gene were 94°C for 4 min for 1 cycle, then 30 cycles of 94°C for 30 s, 58°C for 1 min, and 72°C 1 min 30 s, with a final extension at 72°C for 5 min.
Glomerular Isolation and Cloning
Glomeruli were isolated by sieving [8
]. Isolated glomeruli were resuspended in 5 ml of RPMI 1640 (BioSource, Rockville, Md., USA) with 100 units/ml of penicillin G sodium, 100 μg/ml of streptomycin sulfate (Invitrogen, Carlsbad, Calif., USA), 10% tetracycline-deficient fetal bovine serum (Clontech, Mountain View, Calif., USA) and 30 U/ml of mouse interferon-γ (Cell Sciences, Canton, Mass., USA). Glomeruli were placed in 2 wells (2.5 ml/well) in BD BioCoat collagen I 6-well plate (BD Biosciences, San Jose, Calif., USA), and cultured for 6–7 days at 37°C. Outgrowing cells were trypsinized, filtered with 40 μm cell strainer to remove glomeruli, and were cloned by limiting dilution.
Phenotyping of Clones
To examine podocyte marker expression, clones cultured both at 33 and 37°C with or without doxycyline (4 μg/ml) for 48 h, were analyzed by indirect immunofluorescent staining for WT1, synaptopodin and podocin, and by reverse transcriptase (RT)-PCR for podocin rtTA, nephrin and GAPDH mRNA. Cells cultured on BD BioCoat Collagen I glass coverslips or 100-mm culture dishes were fixed with 2% paraformaldehyde (Sigma, St. Louis, Mo., USA) and 4% sucrose for 5 min at room temperature, permeabilized for 10 min with 0.3% Triton X-100 in phosphate-buffered saline (PBS) without calcium or magnesium, and incubated for an hour in blocking solution (2% fetal bovine serum, 2% bovine serum albumin, 0.2% gelatin in PBS). Primary antibodies included 1:100 dilution of WT1 polyclonal antibody (C-19; Santa Cruz Biotechnology, Santa Cruz, Calif., USA), 1× hybridoma culture supernatant of synaptopodin monoclonal antibody (G1D4; Research Diagnostics, Flanders, N.J., USA), and 1:100 dilution of rabbit polyclonal mouse podocin antibody (gift from Dr. Peter Mundel). Alexa Fluor 488 goat anti-rabbit IgG (H+L) and Alexa Fluor 488 goat anti-mouse IgG (H+L; Invitrogen) were used at 1:1,500 dilution as secondary antibodies. Stained cells were mounted in 20% Mowiol 4-88 reagent (EMD Biosciences, La Jolla, Calif., USA) or ProLong Gold antifade reagent (Invitrogen), and were observed using Leica DMRXE microscope (Leica, Microsystems CMS GmbH, Wetzlar, Germany). Signals were captured as digital images with MicroPublisher 5.0 and QCapture software (Qimaging, Burnaby, B.C., Canada).
Total RNA was purified from each podocyte clone using the RNeasy mini kit and RNase-free DNase set (Qiagen). RNA concentration was measured by an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, Del., USA). RT-PCR was performed using the OneStep RT-PCR kit (Qiagen). Primers for podocin were as follows: forward primer 5′-GAA AGG AAG AGC ATT GCC CAA G-3′ and reverse primer 5′-TGT GGA CAG CGA CTG AAG AGT GTG-3′, yielding a 288-bp product. RT-PCR parameters are 50°C for 30 min for RT, 95°C for 15 min for inactivation of RT, then 40 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 30 s, followed by 72°C for 10 min. The primers for rtTA were: forward 5′-GAA CAA CGC CAA GTC ATT CCG-3′ and reverse 5′-TAC GCA GCC CAG TGT AAA GTG G-3′ yielding a 196-bp product. RT-PCR parameters were 50°C for 30 min for RT, 35 cycles of 95°C for 15 min for RT inactivation, then 35 cycles of 95°C for 30 s, 58.2°C for 30 s, and 72°C for 30 s, with 72°C for 10 min as a final extension. Nephrin forward and reverse primers were used as previously reported [9
], with PCR conditions 50°C for 30 min for RT, 95°C for 15 min for RT inactivation, then 35 cycles of 95°C for 30 s, 55.6°C for 30 s, 72°C for 30 s, with a final extension of 72°C for 10 min. GAPDH forward and reverse primers were used as previously reported [9
], and RT-PCR parameters were 50°C for 30 min for RT, 95°C for 15 min for RT inactivation, then 30 cycles of 95°C for 30 s, 58.2°C for 30 s, 72°C for 30 s, with final extension of 72°C for 10 min. Total RNA extracted from the kidneys of an A+/–
mouse was used as a positive control, and total RNA from the murine mesangial cell line, MES13, was used as a negative control.
Stable Transfection of the Reporter Gene Construct
The podocyte clone AI1-1P4G5 was selected based on high expression of WT-1 and synaptopodin for transfection from among 29 clones obtained. pBI-EGFP (gift of Dr. Bert Vogelstein) was linearized using Ahd1 (New England Biolabs, Beverly, Mass., USA). Podocytes (6 × 104/well) were plated on a collagen I-coated 6-well plate using the glomerular culture medium described above. On the following day, AI or AIT podocytes in each well were transfected with 4 μg of DNA consisting of pBI-EGFP or Gaussia luciferase-subcloned into pBI-EGFP and the Linear Hygromycin Marker plasmid (Clontech) with 10:1 ratio, or ptTS-Neo alone, using the FuGENE 6 Transfection Reagent (Roche Diagnostics). The ratio of Fugene 6:DNA was 3:2. Hygromycin B (Invitrogen) or Geneticin (Invitrogen) at a concentration of 50–100 μg/ml were added to the culture medium 48–72 h after transfection. Culture medium including 50–100 μg/ml of hygromycin B or Geneticin was changed every 4th day. Surviving colonies were selected using cloning cylinders and passaged to BD BioCoat collagen I 25-cm2 flask (Becton Dickinson Discovery Labware).
Culture and Phenotyping of AIG Podocytes
One of the AIG podocyte clones, AI1G10, was arbitrarily chosen out of 16 clones obtained. These cells were cultured on collagen I cell ware under the permissive condition. When cells reached ~75% confluence, 1 μg/ml of doxycycline was added. Cells were transferred to the non-permissive temperature when cells reached 80–90% confluence, and cultured for at least 7 days without medium change, then exposed to doxycycline (1 μg/ml). Cells were observed 48 h after doxycycline treatment with Axiovert 405M (Carl Zeiss MicroImaging, Thornwood, N.Y., USA). The EGFP signal was captured by Hamamatsu-C4742-95 (Hamamatsu, Hamamatsu, Japan), and processed using IP-lab 3.95 (BD Biosciences Bioimaging, Rockville, Md., USA) and Photoshop version 3.0 (Adobe Systems, San Jose, Calif., USA). In time course and concentration-ranging studies carried out under permissive conditions, AIG podocytes were treated with doxycycline (0.01–4 μg/ml) for different time intervals (0–72 h), and GFP expression was quantitated using the Acumen Explorer Laser Scanning Plate Cytometer (TTP LabTech, Cambridge, Mass., USA). Objects were excited with a 488-nm laser at 4 mW of power and emission was detected with the 500–520 nM emission photomultiplier tube (PMT) set at 550 V. The plate was scanned with 1 × 4 μm sampling and objects ranging in size from 25 to 250 μm were collected.
Comparison of AI and AIT Podocytes Leakiness and Inducibility
6 × 104 cells/well of AI podocytes or AIT podocytes were plated on collagen I-coated 6-well plates at the permissive temperature. On the following day, Gaussia luciferase-pBI-EGFP was transfected using FuGENE6, and cells were subsequently treated with 0 or 10 μg/ml of doxycycline. After 48 h, EGFP signal was captured and Gaussia luciferase activity in the supernatant was detected by Gaussia luciferase assay kit (New England Biolabs, Ipswich, Mass., USA).
Time- and Dose-Ranging Study in AITLG Podocytes
AITLG podocytes were plated on collagen I-coated 6-well plates for EGFP assay or 24-well plates for luciferase assay at permissive and non-permissive temperatures. When confluence reached 70–80%, doxycycline (0, 0.01, 0.03, 0.11, 0.33, 1 and 3 μg/ml) was added at the permissive temperature, and supernatant was collected after 48 h and stored at −20°C. For the time-ranging study at the permissive temperature, podocytes were treated with 1 μg/ml doxycycline and supernatant was collected at the 0-, 6-, 12-, 24-, 48-, 72- and 96-hour time points and stored at −20°C until tested. At the non-permissive temperature, AITLG podocytes were cultured for 5 days without medium change, and doxycycline (0, 0.11, 0.33, 1, and 3 μg/ml) was added for the concentration- and 1 μg/ml doxycycline for the time-ranging (0, 12, 24, 48, 72 and 96 h) study. EGFP was captured as described above after 48 h of 1 μg/ml of doxycycline treatment at non-permissive and permissive temperatures.
Treated with or without 4 μg/ml of doxycycline for 48 h, 33°C AITLG podocytes and 37°C AITLG podocytes were pelleted and stored at −80°C. Cells were lysed with 0.5% CHAPS buffer (20 mM Tris-HCl, 500 mM NaCl, 0.5% CHAPS) with protease inhibitor cocktail (Roche Diagnostics, Indianapolis, Ind., USA) and the lysate protein concentration was quantified with BCA protein assay kit (Pierce, Rockford, Ill., USA). Thirty micrograms of lysate per lane was loaded in NuPAGE 4–12% Bis-Tris Gel (Invitrogen) and electrophoresed under the reducing condition, and transferred to nitrocellulose membranes (Pierce). The membrane was immunoblotted with rabbit poly anti-podocin (1:300 dilution; gift from Dr. Peter Mundel) and Immunopure antibody donkey anti-rabbit peroxidase conjugated (1:10,000; Pierce) for podocin, and the signal was detected by SuperSignal West Dura Extended Duration Substrate (Pierce). After anti-podocin was stripped with Restore Western Blot Stripping Buffer (Pierce), the membrane was reprobed with mouse monoclonal anti-β-actin (1:10,000 dilution; Sigma) and Immunopure antibody goat anti-mouse peroxidase conjugated (1:50,000) (Pierce) for β-actin.
At passage 16, 6 × 104 AIG and AITLG podocytes were plated on collagen I-coated 6-well plates and cultured for 24, 48, 72, 96, and 120 h. Cells were trypsinized and cell numbers were counted.
Statistical analysis was performed using PRISM 4.0b (GraphPad Software, San Diego, Calif., USA). Data are presented as mean ± SD, unless otherwise described. Proliferation data were analyzed by two-way ANOVA with Bonferroni post-testing, using the variables, time and temperature. Unpaired t test was used for comparison of 2 groups. p < 0.05 was considered to be statistically significant.