SUPPLEMENTAL ONLINE DATA
Expression And Functional Role Of The Prorenin Receptor In The Human
Adrenocortical Zona Glomerulosa And In Primary Aldosteronism
Chiara Recarti, PhD1, 2;* Teresa Maria Seccia, MD, PhD1;* Brasilina Caroccia, PhD1;
Abril Gonzales-Campos, PhD1; Giulio Ceolotto, PhD1; Livia Lenzini, PhD1; Lucia Petrelli3,
Anna Sandra Belloni, BSc3; William E. Rainey, MD, PhD4; Juerg Nussberger, MD5 ;
and Gian Paolo Rossi, MD, FACC, FAHA1.
1Department
of Medicine-DIMED and 3Department of Molecular Medicine,
University of Padova, Italy;
2Current address CARIM, University of Maastricht, The Netherlands;
4 Molecular & Integrative Physiology, Ann Arbor, University of Michigan;
5 Department of Internal Medicine - Division of Angiology and Hypertension
University of Lausanne, Switzerland.
*These authors have contributed equally to this manuscript
Abbreviated title: Prorenin receptor in the adrenal gland
Key words: Prorenin receptor, renin, adrenal, primary aldosteronism
Conflict of interest and financial disclosure to be disclosed: none.
_____________________________________________________________
Corresponding author:
Prof. Gian Paolo Rossi, MD. FACC, FAHA
Dept. Medicine – DIMED, Internal Medicine 4
University Hospital
Via Giustiniani, 2
35126 Padova, Italy
Phone: 39-049-821-2279 or 7821
Fax: 39-049-821-7873
E-mail: gianpaolo.rossi@unipd.it
EXPANDED MATERIALS AND METHODS
RNA isolation and gene expression studies
Total RNA was extracted from tissues and cell lines of interest using RNeasy Mini Kit (Qiagen, Milan Italy)
and a standardized protocol. The quality of the RNA was checked in a Bioanalyzer (Agilent 2100) using the
RNA6000 Nano assay (Agilent Technologies, Santa Clara, CA).
One µg total RNA was then reverse
transcribed with Iscript (Bio-Rad, Milan, Italy) in a final volume of 20 µL.
The PRR mRNA was measured using Droplet Digital PCR [1, 2]. TaqMan™ reaction mix containing 20 L
sample cDNA was partitioned into aqueous 20,000 nanoliter-sized droplets in oil via the QX200 Droplet
Generator (Bio-rad Laboratories, Segrate, Italy). Droplets were then transferred to a 96-well PCR plate for
thermocycling in the Bio-Rad C1000. After PCR, droplets from each sample were streamed in single file
through the QX100 Droplet Reader. The PCR-positive and PCR-negative droplets were counted to provide
absolute quantification of target DNA in digital form.
Droplet Digital PCR data were analyzed with
QuantaSoft analysis software (Bio-Rad), and the quantification of the target molecule was presented as the
number of copies per μg RNA.
As a confirmatory approach the PRR mRNA was also measured using real time RT-PCR and Universal Probe
Library in the LightCycler 480 Instrument (Roche, Monza, Italy). PRR expression was calculated relative to
porphobilinogen deaminase (PBGD), used as an internal control.
PRR expression at protein level
Immunohistochemistry
Four µm thick serial sections from paraffin blocks of rat and human normal adrenal gland were processed
for immunohistochemistry. The sections were dewaxed with decreasing concentration of ethanol and
rehydrated with distilled water.
Antigen was retrieved by incubation with MS-Unmasker (Diapath,
Martinengo; Italy) 1:10 in distilled water at 96°C for 30 minutes. Endogenous peroxidase was inhibited by 5
minutes incubation with 0.5% hydrogen peroxide. Blocking of non-specific sites was obtained by 30 min
incubation with PBS, 0.2% BSA, 0.2% Triton, 1:50 rabbit serum. Sections were incubated overnight at 4°C
with a goat polyclonal antibody specific for ATP6IP2 (ab5959; Abcam, Cambridge, UK) diluted 1:100 in PBS,
0.2% BSA, 0.2% Triton. Antigen was detected by incubation with a secondary antibody labelled with
horseradish peroxidase (diluted 1:200 in PBS, 0.2% BSA, 0.2% Triton) and diaminobenzidine (DAKO,
Glostrup, Denmark). After development the reaction was stopped by adding water, the sections were
dehydrated and mounted. Negative controls were processed in the same way but by omitting the primary
antibody.
Immunocytochemistry
Immunoseparated ZG CD56 positive cells were seeded on coverslips and fixed, after adhesion, with 4% PFA
for 30 minutes at 4°C. Endogenous peroxidase was inhibited by 5 minutes incubation with 3% hydrogen
peroxide. Blocking of non-specific sites was obtained by 30 minutes incubation with PBS, 5% milk, 0.5%
Triton. Sections were incubated for 1 hour at room temperature with a goat polyclonal antibody specific for
ATP6IP2 (ab5959 Abcam). Antigen was detected by incubation with a secondary antibody labelled with
horseradish peroxidase and diaminobenzidine (DAKO, Glostrup, Denmark).
Negative controls were
processed in the same way but with omission of the primary antibody.
PRR localization
Confocal microscopy
HAC15 cells and immunoseparated ZG CD56 positive cells were fixed with 4% PFA for 30 minutes at 4°C.
Cells were incubated with primary antibodies against CD56 (Biolegend, San Diego, CA) and PRR (ab5959;
Abcam, Cambridge, UK) and secondary antibodies anti-mouse IgG Alexa Fluor 488 (Invitrogen, Carlsbad, CA)
and anti-goat Alexa Fluor 594 (Invitrogen). The fluorescence was detected using the confocal system Leica
TCS SP5 with a 488 nm filter and the images were acquired using the software LAS AF.
Cells membrane and cytosol separation and immunoblotting for PRR
HAC15 proteins were extracted by incubation with Lysis buffer (Euroclone, Pero, Italy) and scraped.
Samples were sonicated and supernatants were collected after centrifugation. Sample supernatants were
ultracentrifuged for 1 hour at 100,000xg at 4°C. Supernatants were collected (cytosol fraction) and pellets
(membrane fraction) were resuspended with lysis buffer. Protein quantification was performed with BCA
kit (Thermo Pierce, Rockford, IL), and then 50 μg proteins were loaded in a 10% acrylamide gel and SDSPAGE. Non-specific sites were blocked by overnight membrane incubation at 4°C with T-PBS, 5% milk.
Membranes were incubated for 1 hour at room temperature with a goat polyclonal antibody specific for
ATP6IP2 (ab5959; Abcam, Cambridge, UK). Antigen was detected by 1 hour room temperature incubation
with the secondary antibody Donkey anti-Goat (sc2020; Santa Cruz; dallas, TX) and ECL kit (Thermo
Scientific, Milan, Italy). Images were acquired with Versadoc (Biorad, Segrate, Italy).
Electron Microscopy Immuno-Gold
For electron microscopy-immuno gold studies, cells monolayer (CD56 positive cells and HAC15) were fixed
in 3% paraformaldehyde–1% glutaraldehyde in 0.1 M sodium cacodylate buffer with CaCl2, tannic acid
(Fluka, Sigma-Aldrich, Milan, Italy) 0.5% in maleate buffer and P-phenylenediamine (Fluka, Sigma-Aldrich,
Milan, Italy) for 2 hours,[3] dehydrated, and then embedded in an epoxy resin.
Sixty nm ultrathin sections were cut with a Reichert-Jung Super Nova ultramicrotome and collected on 400mesh nickel grids. The epoxy resin was removed by exposing the sections to 0.2% (w/v) NaOH in 35% (v/v)
aqueous ethanol, rinsing in 35% ethanol and in bi-distillate water. Nickel grids, were incubated for 15 min
at 90°C in order to unmask antigen and washed in PBS. Ultrathin sections were pre-incubated in Blocking
buffer (0.2% normal goat serum, 2% BSA, 0.2% Tween-20 in PBS) for 60 minutes at room temperature, and
then incubated with the primary goat polyclonal antibody anti-ProRenin Receptor (ab5959; Abcam,
Cambridge, UK; 1:50 dilution) overnight at 4°C. After repeated PBS washing, ultrathins sections were
incubated for 60 min at room temperature with 18 nm colloidal gold-labelled antiGoat (Jackson
Immunoresearch, West Grove, PA)(1:15 dilution) in blocking buffer. After washing in PBS and bi-distilled
water, grids were counterstained with lead hydroxide.
Negative controls were obtained performing the same protocol but omitting the primary antibody. The
samples were examined with a Hitachi H-300 electron microscope.
Functional Studies
Cell culture and stimulation
Cells were stimulated 30 min at 37°C with 100 nM Angiotensin II (Sigma, Milan, Italy), 50 nM Prorenin
(Cayman, Ann Arbor, MI), 50 nM Renin (Cayman) with or without 30 min preincubation of the cells and coincubation with 5 µM Irbesartan (Bristol Myers Squibb, Princeton, NJ) or 30 min preincubation of the
stimuli and co-incubation with 5-50 µM Aliskiren (Novartis, Basel, CH). Seven independent experiments
were performed for HAC15 cells.
Immunoblotting and analysis of ERK 1/2 phosphorylation
Proteins of HAC15 cells that were stimulated following the protocol described above were extracted after
addition of Lysis buffer (Euroclone, Pero, Italy) and scraping. Samples were sonicated and supernatants
were collected after centrifugation. Protein quantification was performed with BCA kit (Thermo Pierce),
and then 50 μg of proteins were loaded in a 10% acrylamide gel and SDS-PAGE. Non-specific sites were
blocked by overnight membrane incubation at 4°C with T-PBS, 5% milk. Membranes were incubated for 1h
at room temperature with an antibody specific for ERK phosphorylated form or total ERK (Cell Signalling,
Milan, Italy). Antigen was detected after 1h room temperature incubation with the secondary antibody
(Amersham GE Healthcare, Milan, Italy) and ECL kit (Thermo Scientific, Milan, Italy). Images were acquired
and analyzed with Versadoc (Biorad, Segrate, Italy).
Results
PRR gene expression in the human adrenal gland
Absolute quantification of PRR gene showed similar PRR levels in APAs and the normal human adrenal
cortex (Table 1). High levels were also found in HAC15 cell line.
Real time RT-PCR confirmed the marked expression of PRR in the normal adrenocortical tissue, in APA and
cell lines.
References
1. Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ et al. High-throughput
droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 2011; 83:8604-8610.
2. Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ et al. Absolute quantification by
droplet digital PCR versus analog real-time PCR. Nat Methods 2013; 10:1003-1005.
3. Berryman MA, Porter WR, Rodewald RD, Hubbard AL. Effects of tannic acid on antigenicity and
membrane contrast in ultrastructural immunocytochemistry. J Histochem Cytochem 1992; 40:845-857.
Table 1.
Normal adrenal
tissue
APA
HAC15 cell line
*mean (95% CI)
Digital Droplet PCR
Real Time PCR
PRR copies/ug*
PRR-to-PBGD ratio
24650
(24275-27585)
29550
(19185-39914)
54500
(44250-56500)
15
12.3
5.0