Overexpression of histamine H4 receptors in the kidney of diabetic rat

TL;DR: It is demonstrated, for the first time, that the H4 receptor is expressed in the kidney mainly by resident renal cells of the loop of Henlé and that this receptor is significantly overexpressed in diabetic animals, thus suggesting a possible role in the pathogenesis of diabetes-associated renal disease.

Abstract: Objective and design The renal expression of H1 and H2 receptors has previously been demonstrated, while that of the H4 receptor has been poorly investigated, and thus the aim of this research was to investigate the expression of the H4 receptor in the kidney of diabetic rats.

Summary

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Animals and protocols

• Male 8-week-old Wistar rats (Harlan–Italy, Udine, Italy) were provided with a Piccioni pellet diet (No. 48; Gessate Milanese, Italy) and water ad libitum.
• Animal care was in compliance with Italian regulations on the protection of animals used for experimental and other scientific purposes (D.M. 116/92).
• The experimental protocol was approved by the Turin University Ethics Committee for the animal use.

Diabetes induction

• Insulinopenic diabetes was induced by a single intravenous tail vein injection of streptozotocin (STZ; 50 mg/kg) diluted in citrate buffer 0.1 M (pH 4.0) in accordance to the literature (Wu and Huan, 2008).
• Six weeks after induction of diabetes, when experimental rats reached a blood glucose level > 300 mg/dl (severe hyperglycemia), rodents were weighed and placed in metabolism cages to collect 24 h urine.
• Histological sections, 5 µm thick, were cut from the paraffin-embedded kidney samples.
• Relative quantification of the products was performed using a 48-well StepOne™ Real Time System (Applied Biosystems).
• For all real-time PCR analyses, 18S mRNA was used to normalize RNA inputs.

Antibodies

• H4 receptor was detected by using the anti-hH4 receptor (374-390) antibodies produced and validated for detecting both human and rodent H4 receptors in the School of Biological and Biomedical Sciences, Durham University [21-27].
• The resultant conjugate was used to generate polyclonal antibodies in rabbits as previously described [29].
• The selectivity of the anti-h H4 receptor antibody was confirmed by blockade with the C-terminal peptide of the H4 receptor and a lack of cross reactivity with the human H3 receptor, the most related G-coupled receptor [26].
• In all cases, a major part of the staining elicited by the untreated antibody was abolished.
• Photomicrographs of the histological slides were randomly taken with a digital camera connected to a light microscope equipped with a ×40 objective (Leica DM750).

Immunofluorescence and confocal analysis

• After antigen retrieval and blocking, 5 µm kidney paraffin sections were incubated with primary anti-H4 receptor and anti-THP or -AQP1 antibodies, followed by incubation with Alexa Fluor-488 anti-goat or Alexa Fluor-594 anti-rabbit polyclonal antibodies.
• After counterstaining with DAPI, photomicrographs were obtained by a confocal laser-scanning microscope LSM-510 microscope equipped with a ×40 objective (Carl Zeiss, Oberkochen, Germany).

Image Analysis

• Images were processed by ImageJ 1.41 (NIH, USA) software and quantified using the Color Deconvolution image analysis tool as previously described [30-33].
• Briefly, H4 staining defined by diaminobenzidine were colour deconvoluted from counter-stained sections.
• A threshold was chosen for identifying H4 positive staining areas and used on all slides in the study without modification.
• The percentage area was calculated for H4 positive tissue.
• Values are means ± S.E.M. of the OD (in arbitrary units) measurements of individual rats (ten images/zone each) from the different experimental groups.

Data analysis

• Results are shown as means ± SEM and were analysed by Student's t-test or one-way ANOVA with Dunnett's multiple comparison and Student–Newman–Keuls tests were used to determine significant differences between means: p-values less than 0.05 were considered significant.
• Data analysis assumed normality, using Prism 4 software from Graphpad (CA, USA).

Pathophysiology and renal histology

• As shown in Table 1, in comparison to controls, body weight was reduced and blood glucose level was increased in diabetic rats.
• Consistently, these biochemical abnormalities were associated with clear changes in renal histology.
• This observation was confirmed using a quantitative image analysis, performed with the Color Deconvolution image analysis tool.
• The similar results validate the use of the antihH4 receptor (374-390) antibody to detect the H4 receptor in the rat kidney.
• To confirm and refine this result immunoflorescence staining and confocal analysis was performed with both THP, marker of the thick ascending limb of the loop of Henlé, and AQP-1, marker of both the proximal tubules, the thin descending limb of the loop of Henlé and also expressed on some walls of microvessels.

Discussion

• The authors results demonstrate for the first time that the H4 histamine receptor is mainly expressed by cells of the loop of Henlé, and that this protein is significantly upregulated in the kidneys of diabetic animals, hence suggesting a possible role of the H4 receptor in the pathogenesis of diabetes-associated renal disease.
• Herein, the authors have reported the immunohistochemical identification the H4 receptor in the kidney.
• Previously, it has been shown that diabetic animals have a higher intra-renal histaminergic level [6, 7].
• The authors findings, which demonstrate that the H4 receptor subtype is absent in the glomeruli, but present in the tubules, leads us to revisit a tubular effect for histamine.

Acknowledgments

• This work was supported by Royal College of Anaesthesia/BJA, COST Action BM0806 (STSM hosted by the University of Durham) and the University of Turin.
• The authors are grateful to Dr Sara Castiglia, Dr Mara Rogazzo and Dr Alice Alfonso (Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino) and Dr Stefania Bruno (Department of Internal Medicine, Centre for Molecular Biotechnology and Centre for Research in Experimental Medicine - CeRMS) for technical assistance.
• Part of this study was presented at the 41st Annual Meeting of European Histamine Research Society held jointly with COST Action BM0806, May 2-5 2012, Belfast – Northern Ireland, UK and at the 48th EASD Annual Meeting Berlin, Germany, October 1-5 2012.

Contribution statement

• ACR, PLC and RF contributed to the conception and design of this study and critically reviewed the intellectual content of this manuscript.
• GM, CG and GC contributed to analysis and interpretation of immunoistochemistry.
• AP and DB contributed to analysis and interpretation of kidney morphometry.
• MAK contributed to the anti-H4 receptor antibody supply and characterization.
• All authors contributed to the conception, design and drafting of this article and approved the final version of the article to be published.

Title and legends

• Figure 1. Renal morphology assessed by PAS staining.
• Data were analyzed by one-way analysis of variance and the Student–Newman–Keuls test.
• Representative micrographs of transverse kidney sections, immunolabelled with specific anti-H4 receptor antibody.
• Original magnification 40X. Figure 4. Quantitative assessment of H4 receptor distribution in the kidney.
• Results are the mean ± SEM of the OD (arbitrary units) of individual rats (ten images/zone) performed in duplicate.

Full text

10 August 2022
AperTO - Archivio Istituzionale Open Access dell'Università di Torino
Original Citation:
Overexpression of histamine H4 receptors in the kidney of diabetic rat
Published version:
DOI:10.1007/s00011-012-0587-7
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/2318/129125 since 2016-11-11T15:37:00Z

1
The final publication is available at Springer via http://dx.doi.org/doi: 10.1007/s00011-012-0587-7

2
Overexpression of histamine H
4
receptors in the kidney of diabetic rat
Rosa A.C.
1
, Grange C.
2
, Pini A.
3
, Katebe M.A.
4
, Benetti E.
1
, Collino M.
1
, Miglio G.
1
, Bani D.
3
, Camussi G.
2
, Chazot
P.L.
4
and Fantozzi R.
1
1
Dipartmento di Scienza e Tecnologia del Farmaco, Università di Torino, Turin, Italy
2
Department of Internal Medicine, Centre for Molecular Biotechnology and Centre for Research in Experimental
Medicine (CeRMS), Torino, Italy
3
Department of Anatomy, Histology and Forensic Medicine, Section of Histology, University of Florence, Florence,
Italy
4
School of Biological and Biomedical Sciences and Wolfson Research Institute, Durham University, Durham, UK
Running title: Histamine H
4
receptors kidney expression
Correspondence: Paul L Chazot, PhD,
School of Biological and Biomedical Sciences & Wolfson Research Institute,
Durham University,
South Road,
Durham DH1 3LE, UK
Phone: +44 191 33 41305
Fax: +44 191 334 1201
e-mail: paul.chazot@durham.ac.uk

3
Abstract
Objective and design: The renal expression of H
1
and H
2
receptors was previously demonstrated, while that of the H
4
receptor has been poorly investigated, thus the aim of this research was to investigate the expression of the H
4
receptor
in the kidney of diabetic rats.
Material or subjects: 24 8-week-old male Wistar rats
Treatment: Diabetes was induced in 12 rats by a single i.v. injection of streptozotocin, and animals were sacrificed 6
weeks later.
Methods: Kidneys were collected and processed for quantitative PCR or immunohistochemical analyses. To ascertain
the renal topology of the H
4
receptor, colocalization experiments were performed with a series of markers.
Results: H
4
receptor is expressed in healthy rats, although at a very low level, and is profoundly upregulated in diabetic
animals. Immunohistochemical analysis revealed the highest immune-positivity in the medulla. Colocalization
experiments revealed a close overlap in expression topology of the H
4
receptor and both Tamm-Horsfall glycoprotein
and aquaporin 1 was observed.
Conclusions:
The results demonstrate, for the first time, that the H
4
receptor is expressed in the kidney mainly by
resident renal cells of the loop of Henlé and that this receptor is significantly overexpressed in diabetic animals, thus
suggesting a possible role in the pathogenesis of diabetes-associated renal disease.
Keywords Histamine, histamine H
4
receptor, diabetes, kidney, loop of Henlé

4
Histamine is a biogenic amine that exerts many (patho)physiological effects through its interaction with four subtypes
of G-protein-coupled receptors, designated H
1-4
, which are differentially expressed in various tissues and cell types. It is
synthesized from L-histidine through the catalytic activity of the rate-limiting enzyme histidine decarboxylase (HDC).
Although HDC is principally expressed in mast cells, the most important source of histamine, it is well recognized that
a number of other cell types, such as tuberomammillary nucleus (TMN) neurons in the brain and parietal cells in the
stomach [1], also express this enzyme. Notably, HDC expression has been also reported in the kidney, where it was
found in the proximal tubule [2], thus in keeping with the demonstrated ability of the kidney to produce histamine.
In particular, in humans the renal histamine content markedly exceeds plasma levels (<10
-8
M in plasma vs ≈10
-6
M in
glomeruli) [3]. Indirect demonstrations sustain the hypothesis that histamine could be involved in renal pathophysiology
in humans. In fact, it was demonstrated in healthy subjects that loading doses of L-histidine led to an increase in
histamine concentration in urine, but not in blood [4], and that urinary and blood levels of histamine are elevated
following renal transplantation [5].
Some studies suggest that histamine may be involved in diabetes-related kidney disease. Studies performed in
streptozotocin (STZ) diabetic rats have shown, consistently with a generalized effect of diabetes on inducing an increase
in histamine, a greater content of histamine in the kidney of diabetic animals than that of controls [6, 7]. This increase
has been related to a significantly greater tissue HDC activity of diabetic rats than controls without a concomitant
decrease in histaminase activity [8], an enzyme that catalyzes the inactivation of histamine. Besides, it has been
suggested that renal histamine may represent one triggering stimulus for the functional microangiopathy in diabetes
mellitus, which may lead to the development of diabetic nephropathy. In fact, it has been reported, in rats, that
histamine reduces the ultrafiltration coefficient [9]. These data are in keeping with more recent studies aimed to assess
the role of mast cells in the kidney. It is well recognized that in the normal kidney, mast cells are constitutively present
at low numbers. However, their density and activation increase in the renal cortical tubulointerstitium and in the
periglomerular and perivascular area, but not in glomeruli, in a variety of human renal diseases, including diabetic
nephropathy [10-12].
Hitherto, the renal effects of histamine have been related to the activation of H
1
and H
2
receptor, both identified in the
glomeruli [3, 13], while the H
4
receptor expression in the kidney was poorly investigated. A very low level of H
4
receptor mRNA has been reported in the kidney of dog, monkey, rat, mouse, guinea pig and pig [14-18]. Despite the
demonstration of an increasing histamine release in diabetic kidney, there is no evidence for the involvement of H
4
receptor expression in the diabetes-related kidney disease. Thus, in order to gain further insight into the histamine signal
in the diabetic kidney, the aim of this pilot study was to investigate the expression of the H
4
receptors in the kidney of
diabetic rats with severe hyperglycemia.

Frequently asked questions

Q1. What was used as a marker of tubular injury?

Creatinine concentrations and creatinine clearance were used as indicators of impaired renal function, whereas N-Acetyl-β-glucosaminidase (NAG), measured in the urine by a colorimetric assay (Roche Diagnostics), was used as a marker of tubular injury [19]. 

Q2. What are the contributions in this paper?

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 

Q3. What are the future works in this paper?

However, the present pilot study was only observational, and future work will focus on the functional consequences of the elevated levels of H4 receptor and the respective regulatory mechanisms. Thus, these data suggest that a possible role of the H4 receptor in mediating the renal effect of histamine could not be ruled out. Moreover, the discovery of H1 and H2 receptors only in the glomerulus led researchers to discard the hypothesis of a potential role for histamine in the tubules. This cellular localization suggests a possible role of the H4 receptor distinct from the regulation of immune responses and inflammatory cell recruitment [ 43 ], leading us to hypothesize an active role of this receptor in modulating the loop of Henlé function ( s ), such as the transmembrane soluble transport processes. 

Q4. How many weeks after induction of diabetes?

Six weeks after induction of diabetes, when experimental rats reached a blood glucose level > 300 mg/dl (severe hyperglycemia), rodents were weighed and placed in metabolism cages to collect 24 h urine. 

Q5. What was the effect of PAS staining on kidneys?

Using PAS staining, histological analysis of kidney sections from the diabetic animals showed severe tubular alterations and moderate glomerular modifications. 

Q6. What was the hypothesis of a potential role for histamine in the tubules?

the discovery of H1 and H2 receptors only in the glomerulus led researchers to discard the hypothesis of a potential role for histamine in the tubules. 

Q7. What was the level of labelled cells in the medulla of diabetic rats?

In particular, while there was very weak H4 receptor-like immunoreactivity in control rats, a high level of labelled cells in both medulla and cortex of diabetic animals was observed. 

Q8. What is the effect of the kidney on histamine?

In fact, it was demonstrated in healthy subjects that loading doses of L-histidine led to an increase in histamine concentration in urine, but not in blood [4], and that urinary and blood levels of histamine are elevated following renal transplantation [5]. 

Q9. What is the role of the H4 receptor in the pathogenesis of diabetes?

the authors observed that all diabetic animals with overt hyperglycemia (6 weeks after induction of diabetes), although did not meet the criteria for diabetic nephropathy, showed renal damage and expressed profoundly higher levels of H4 receptors, thus suggesting a strong association between diabetes-associated renal damage and H4 receptor overexpression. 

Q10. What was the resulting higher histamine renal content?

The resulting higher histamine renal content was previously linked to the microvascular diabetes-associated changes in the glomerulus, where the expression of only H1 and H2 receptors was demonstrated [3, 13, 40]. 

Q11. What is the role of the H4 receptor in the renal effect of histamine?

This cellular localization suggests a possible role of the H4 receptor distinct from the regulation of immune responses and inflammatory cell recruitment [43], leading us to hypothesize an active role of this receptor in modulating the loop of Henlé function(s), such as the transmembrane soluble transport processes. 

Q12. What is the role of the H4 receptor in diabetes?

In particular, understanding which effects an activation of H4 receptor could exert on the loop of Henlé function is still to be resolved, and could represent the basis for proposing the renal H4 receptor as a new potential pharmacological target for treating diabetes-associated renal disease. 

Q13. What is the renal histamine content in humans?

In particular, in humans the renal histamine content markedly exceeds plasma levels (<10-8 M in plasma vs ≈10-6 M inglomeruli) [3]. 

Q14. What is the role of the H4 receptor in the renal effect of diabetes?

This compound was initially characterized as a selective H2 receptor agonist, but nowadays it has been found to act also on the H4 receptor subtype, but with a much higher affinity [41]. 

Q15. What is the effect of the antih4 receptor on the glomeruli of diabetic?

the glomeruli of diabetic rats displayed moderate capsular fibrosis and tuft-to-capsule adhesion, with capillary dilatation and mild mesangial matrix expansion (Figure 1c and 1d). 

Q16. What is the role of the H4 receptor in the renal effect?

Their findings, which demonstrate that the H4 receptor subtype is absent in the glomeruli, but present in the tubules, leads us to revisit a tubular effect for histamine. 

Q17. What did the authors do to confirm the expression of the H4 receptor in the kidney?

the constitutive, but very low H4 receptor gene expression, was confirmed in different species [14-18, 36]; however, all these studies did not provide for the topological evaluation and cell type identification of the receptor. 

Q18. What is the significance of the H4 receptor in the kidney?

although expression differences were observed among species [14, 15, 17, 18, 34], the similar tissue distribution in different mammals indicates conserved and comparable physiological roles of the H4 receptor across species.