10 August 2022
AperTO - Archivio Istituzionale Open Access dell'Università di Torino
Original Citation:
H3 receptor renal expression in normal and diabetic rats
Published version:
DOI:10.1007/s00011-015-0808-y
Terms of use:
Open Access
(Article begins on next page)
Anyone can freely access the full text of works made available as "Open Access". Works made available
under a Creative Commons license can be used according to the terms and conditions of said license. Use
of all other works requires consent of the right holder (author or publisher) if not exempted from copyright
protection by the applicable law.
Availability:
This is the author's manuscript
This version is available http://hdl.handle.net/2318/158681 since 2016-11-11T15:28:57Z
The final publication is available at Springer via
http://www.springerlink.com/openurl.asp?genre=article&id=doi:10.1007/
s00011-015-0808-y
H
3
receptor renal expression in normal and diabetic rats.
Alessandro Pini
1*
, Paul L Chazot
2*
, Eleonora Veglia
3
, Aldo Moggio
3
, Arianna Carolina Rosa
3
*
Authors contributed equally to this work
1
Dipartimento di Medicina Sperimentale e Clinica Sezione di Anatomia e Istologia Università degli Studi di Firenze,
Largo Brambilla 3, 50134 Florence, Italy;
2
School of Biological and Biomedical Sciences and Wolfson Research
Institute, Durham University, South Road, Durham DH1 3LE, UK;
3
Dipartimento di Scienza e Tecnologia del
Farmaco, Università di Torino, Via P. giuria 9, 10125 Turin, Italy;
Corresponding authors Arianna Carolina Rosa, PhD
Dipartimento di Scienza e Tecnologia del Farmaco, University of Turin, Italy, Via P.
Giuria 9, 10125, Turin, Italy
Phone: +390116707955
Fax: +390116707688
e-mail: ariannacarolina.rosa@unito.it
Abstract
Introduction
In order to extend our previous observation of H
4
R upregulation in the kidney of diabetic rats, we evaluated in the same
specimens the presence of the H
3
R.
Materials and methods
Kidney specimens from 24 8-week-old male Wistar rats (12 non-diabetic and 12 diabetic animals) were processed for
both immunohistochemical and immunofluorescence analyses.
Results and conclusion
H
3
R is expressed in the apical membrane by collecting duct cells in the kidney of rats and it is significantly increased in
diabetic animals. These data support the hypothesis that H
3
R could also mediate non-neuronal histamine effects,
suggesting its involvement in fluid homeostasis.
Key Words histamine H
3
receptor, histamine, kidney, diabetes, collecting ducts
Introduction
Recently, our group demonstrated the presence of the histamine H
4
receptor (H
4
R) in resident renal cells of the loop of
Henlé and its profound upregulation in the kidney of diabetic rats[1]. This observation adds to independent evidence of
a role for histamine in renal (patho)physiology. In healthy subjects the administration of loading doses of L-histidine led
to an increase of histamine concentration in urine. In renal transplant patients the urinary and blood levels of histamine
are elevated. In the kidney of diabetic rats, histamine was increased compared with controls[2]. Basically, histamine has
been reported to regulate the renal microcirculation, to increase salt and water excretion[3-5], decrease the ultrafiltration
coefficient by reducing the total filtration surface area[4], and increase renin release[6].
The aim of this study was to extend our previous observation on H
4
R in healthy and diabetic rats evaluating in the same
specimens the renal expression of H
3
R.
Materials and Methods
Animals, protocols, diabetes induction, biochemical and histological parameters have been previously reported[1].
Conventional immunohistochemical procedures were employed as described. Immunoperoxidase staining for H
3
R was
performed on 5 µm kidney sections for formalin-fixed tissue from 24 male 8-week-old Wistar rats (12 non-diabetic and
12 diabetic animals). Renal sections were incubated overnight with anti-H
3
R (349–358) (2 µg/ml)[7], followed by a
three-layer streptavidin–biotin–peroxidase complex staining method. Photomicrographs were acquired randomly with a
digital camera connected to a light microscope equipped with a x40 objective (Leica DM750). Images were processed
by ImageJ 1.41 (NIH, USA) software and quantified using the Color Deconvolution image analysis tool. The per-
centage area was calculated for H
3
R-positive tissue. Values are mean ± SEM of the optical density (in arbitrary units)
measurements of individual rats (ten images/zone each) from the different experimental groups. For
immunofluorescence and confocal analysis, after antigen retrieval and blocking, kidney sections were incubated with
primary anti-H
3
R and anti-AQP2, antibody, followed by incubation with corresponding Alexa Fluor secondary
antibodies. After counterstaining with DAPI, photomicrographs were obtained by Apotome systems (Zeiss) at x40
objective. The percentage of positive cells for H
3
R and AQP2 was determine by ImageJ 1.41 software. Values are
expressed as mean ± SEM positive cell/collecting duct percentage of individual rats (ten images/zone each) from the
different experimental groups.
To confirm the absence of false staining, tissue was also screened in the absence of primary antibodies and following
pre-incubation with (349-358) peptide (data not shown). All sections were immunostained in a single session to
minimize artifactual differences.