Immortalised RPTCs (passage <20, mycoplasma-free) from humans and rats were provided with authorisation by R. A. Felder of the University of Virginia [21, 22]. The cells, randomly grouped for treatments (simple randomisation), were cultured at 37°C in 95% air and 5% CO2 (vol./vol.) in DMEM containing Nutrient Mixture F-12 (DMEM/F12) (Invitrogen, Life Technologies, Grand Island, NY, USA) supplemented with 10% (wt/vol.) fetal bovine serum (Sigma-Aldrich, St Louis, MO, USA), 1% (wt/vol.) penicillin–streptomycin (Invitrogen) and epidermal growth factor (10 ng/ml) (Sigma-Aldrich).
Male C57Bl/6J mice (1 year old) from Jackson Laboratory (Bar Harbor, ME, USA) were housed in standard facilities. Mice were uninephrectomised 3 weeks before a 7 day renal subcapsular infusion of small interfering (si)RNA (SNX5-specific or non-silencing ‘mock’ siRNA at 3 μg/day, simple randomisation) into the remaining kidney, via an osmotic mini-pump (Alzet 1007D; Durect, Cupertino, CA, USA) [21, 22, 27]. The mice that were placed in metabolic cages for urine collection had free access to drinking water but food was withheld in some mice for 6–22 h prior to obtaining blood from the tail vein for measurement of glucose (AlphaTRAK 2 blood glucose monitoring system; Abbott, North Chicago, IL, USA) and insulin (ELISA; Crystal Chem, Downers Grove, IL, USA). The kidneys were harvested and homogenised for immunoblotting of SNX5 and IDE [21, 22]. In additional studies in mice, insulin sensitivity/resistance [28, 29] was tested by monitoring non-fasting blood glucose (AlphaTRAK 2) every 15 min for 60 min, following insulin administration (0.75 U/kg, s.c. injection) on day 7 of the renal-selective siRNA infusion and day 21 (i.e. 14 days after stopping the renal-selective siRNA infusion). In additional studies in mice fasted for 6 h, a glucose tolerance test was performed by monitoring the blood glucose level every 30 min for 120 min following glucose administration (1 g/kg of body weight, i.p. injection) [28, 29]. The quantity of glucose in the blood (i.e. AUC [28, 29]), was calculated from 0 to 60 min in the insulin injection study and from 0 to 120 min in the glucose tolerance study.
Male Wistar–Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs), 2 months old, from Japan SLC (Sendai, Japan), were housed in standard facilities. Eight of the WKY rats were uninephrectomised, as in the C57Bl/6 mice, and then given a 7 day renal cortical interstitial infusion of siRNA (Snx5-specific or mock siRNA at 3 μg/day) into the remaining kidney, via an osmotic mini-pump . Blood and kidney samples were collected after the rats were decapitated.
All animal studies were conducted in accordance with National Institutes of Health guidelines for the ethical treatment and handling of animals in research, and approved by the Institutional Animal Care and Use Committees of Georgetown University (Washington, DC, USA), University of Maryland (Baltimore, MD, USA) and Fukushima Medical University School of Medicine Animal Committee (Fukushima, Japan).
Total cell lysates and kidney homogenates were prepared on ice using RIPA lysis buffer, supplemented with a cocktail of protease and phosphatase inhibitors (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of protein (30 μg) were electrophoresed on a 10% (wt/vol.) SDS–polyacrylamide gel and electro-transferred onto nitrocellulose membranes. The following primary antibodies (diluted 1:500) were used: goat anti-SNX5 (Santa Cruz Biotechnology, Dallas, TX, USA); mouse anti-IDE (GeneTex, Irvine, CA, USA); rabbit anti-cathepsin D (Cell Signaling Technology, Danvers, MA, USA); mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, GeneTex) and rabbit anti-actin (Sigma-Aldrich). The specificity of the anti-SNX5 antibody was reported previously . A rabbit polyclonal anti-IDE antibody (described below) was also used to confirm the specificity of the mouse IDE antibody. The secondary antibodies (1:10,000) were obtained from Santa Cruz Biotechnology or LI-COR Biosciences (Lincoln, NE, USA). The band densities of the proteins were quantified using either Image J software (NIH, Bethesda, MD, USA) or the Odyssey CLx Imaging System (LI-COR Biosciences) and expressed as a percentage of the relevant control density.
Immunofluorescence confocal microscopy
hRPTCs were grown to 50–60% confluence on poly-d-lysine-coated coverslips and immunostained with goat polyclonal anti-SNX5 antibody (1:200; Santa Cruz Biotechnology), rabbit polyclonal anti-IDE antibody (1:200; Abcam, Cambridge, MA, USA) and then with the appropriate fluorophore-conjugated secondary antibodies (1:500; Molecular Probes, Grand Island, NY, USA).
Human kidney sections were obtained from a non-pathological section of a healthy kidney from a man (Imgenex/Novus Biologicals, Littleton, CO, USA). Kidney sections from WKY rats and C57Bl/6J mice were also studied. The glass slides containing the kidney sections were incubated in xylene for 1 h in a dry oven at 60°C for deparaffinisation. The incubated glass slides were washed three times (3 min each time) in 100%, 95% and 75% (vol./vol.) ethanol. The slides were boiled in sodium citrate buffer (pH 6.0), for 3 min for antigen retrieval and then incubated in 1% (wt/vol.) bovine serum albumin blocking buffer for 30 min at room temperature. Double-staining with anti-SNX5 and anti-IDE antibodies was performed. The primary antibodies were added onto the tissues and incubated at 4°C overnight. The secondary antibodies were subsequently added and incubated for 1 h at room temperature. The slides were mounted using ProLong Gold Antifade Mountant (Thermo Fisher Scientific) and imaged with a LSM 510 confocal microscope (Carl Zeiss, Oberkochen, Germany).
Immunoprecipitation was performed using an Immunoprecipitation Kit (Protein G; Roche Applied Science, Indianapolis, IN, USA). Uniform amounts (800 μg) of protein were immunoprecipitated with the primary antibody (2 μg) specific for the protein of interest for 2 h. Normal rat, mouse, and human IgGs (Santa Cruz Biotechnology) were used as negative controls. The samples were boiled for 5 min before western blotting.
RNA preparation and reverse transcriptase-quantitative real-time PCR
For reverse transcriptase-quantitative real-time PCR (RT-qPCR), the following reactions were all performed in a C1000 Thermal Cycler machine (Bio-Rad, Hercules, CA, USA). Total RNA was extracted using an RNeasy plus mini kit (Qiagen Sciences, Germantown, MD, USA) and equal amounts of RNA (1 μg) were loaded for cDNA synthesis using a Tetro cDNA Synthesis Kit (Bioline USA, Taunton, MA, USA). RT-qPCR was then performed, using SYBR Green PCR Master Mix (Qiagen Sciences) in a 7900HT Fast Real-Time PCR machine (Applied Biosystems, Life Technologies, Grand Island, NY, USA). Relative quantities of SNX5 and IDE were normalised by β-actin. Thermal cycler settings were as follows: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, 59°C for 1 min, 95°C for 15 s and 60°C for 15 s.
The gene-specific primer pairs were as follows: SNX5 forward (5′-ACGTTTCAGAGCCCAGAGTT-3′) and SNX5 reverse (5′-TCGAGGACCATCAAAGTCG-3′); IDE forward (5′-TTCCAAGAAGAACATCTTAAACAACT-3′) and IDE reverse (5′-ACCTTCATGCCCAATGAGAT-3′); β-actin forward (5′-ACCTGTACGCCAACACAGTG-3′) and β-actin reverse (5′-ACACGGAGTACTTGCGCTCA-3′).
SNX5-specific siRNA/small hairpin RNA transfection
Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) was used to transfect SNX5-specific siRNA (Santa Cruz Biotechnology) into hRPTCs. The cells were seeded into six-well plates, 1 day before the transfection. Serum-free medium was used once the cells reached 70–80% confluence. The cells were incubated in a mixture of 4 μl transfection reagent and 5 μl siRNA stock solution (10 μmol/l) at room temperature for 20–30 min. At about 6 h post-transfection, the same amount of complete medium was added into each well. The cells were harvested after 48 h for RNA quantification using RT-qPCR and after 72 h for total protein quantification by western blot.
FuGENE 6 transfection reagent (Promega, Madison, WI, USA) was used to transfect SNX5 small hairpin RNA (shRNA) into hRPTCs. The clones were isolated by adding puromycin 1 day after transfection and incubating for 14 days at 37°C. We tested several shRNAs for their ability to silence the expression of SNX5 and used the shRNA that gave the greatest downregulation of SNX5 expression.
InnoZyme Insulysin/IDE Immunocapture Activity Assay Kit (Calbiochem, EMD Millipore, Billerica, MA, USA) was used to measure IDE activity. hRPTCs were lysed with CytoBuster Protein Extraction Reagent (EMD Millipore).
Fluorescence energy transfer microscopy and data processing
The fluorophore pairs (Invitrogen) used for fluorescence energy transfer (FRET) imaging were Alexa Fluor 555 (acceptor dipole) conjugated with SNX5 antibody and Alexa Fluor 488 (donor dipole) conjugated with IDE. Seven images were acquired for each FRET analysis, using an Olympus FluoView FV300 confocal laser scanning microscope (Olympus Corporation of the Americas, Center Valley, PA, USA) equipped with a 60×/1.4 NA objective, Argon (488 nm), HeNe (543 nm) laser, emission filters of 515/50 nm and a 590 nm long pass filter.
All experimenters were blind to group assignment and outcome assessment. We included all outcomes for data report and analysis. Numerical data are reported as means ± SEM. Significant difference between two groups was determined by Student’s t test. Significant differences among groups were determined by one-way factorial ANOVA, followed by Student–Newman–Keuls post hoc test. p < 0.05 was considered significant.