Histamine type 1-receptor activation by low dose of histamine undermines human glomerular slit diaphragm integrity

TL;DR: The data demonstrate that histamine, via the H1R, modifies SD morphological and functional integrity, in part, by decreasing the expression of ZO-1 and P-cadherin.

About: This article is published in Pharmacological Research.The article was published on 2016-12-01 and is currently open access. It has received 11 citations till now. The article focuses on the topics: Histamine receptor & Histamine.

Summary

2.1 Materials

• All reagents and chemicals used were from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.
• (Rockford, IL, USA) and PVDF membrane from Millipore (Bradford, MA, USA).
• Visiglo™ HRP chemiluminescent substrate kit was purchased from Amresco llc. (Solon, OH, USA).

Cell media and reagents

• Histamine dihydrochloride (PubChem CID 5818), (+/-) chlorpheniramine maleate (PubChem CID 5281068), [ 3 H]mepyramine and difenhydramine (Pubmed CID 3100) were dissolved in dimethyl sulfoxide, and the final drug concentrations were obtained by dilution of stock solutions in the experimental buffers.
• The final concentration of the organic solvent was less than 0.1%, which had no effect on cell viability.

2.2 Cell cultures

• Podocytes were characterised for the positive expression of nephrin, podocin, and synaptopodin and for negative expression of von Willebrand factor, CD31, and smooth muscle cell actin.
• Cells were cultured in DMEM containing 4.5 mg/l glucose supplemented with 10% Fetal Calf Serum, penicillin/streptomycin (100 IU/ml), and l-glutamine and the cultures were maintained at 37 °C in a 95% air/5% CO 2 humidified incubator.

2.3 Permeability assay

• Podocyte monolayer permeability was determined as previously described [30, 31] .
• Human immortalised podocytes (40,000 cells well, 500 μl) were seeded on the top of HTS Transwell inserts (3 µm pore, 24-well plate) and cultured till confluence was achieved.
• Fold-change in expression with respect to the control mean was calculated for all samples.

2.4 Electron microscopy

• Ultrathin sections were stained with uranyl acetate and alkaline bismuth subnitrate and examined under a JEM 1010 electron microscope (Jeol, Tokyo, Japan) at 20 kV and 50 kV.
• Morphometrical analysis were performed on 50KV digitized images using ImageJ 1.41 software (http://rsbweb.nih.gov/ij; NIH, USA) in 20 regions of interest (ROI) for each sample.

2.5 RT-PCR

• Two µg/µl of total RNA extracted from podocytes by using RevertAid™ First Strand cDNA Synthesis Kit according to the manufacturer's instruction, were subjected to RT-PCR as previously described [26, 32] .
• The subsequent specific oligonucleotide sequences were used: hH1R forward 5' CATTCTGGGGGCCTGGTTTCTCT-3' and reverse 5'-CTTGGGGGTTTGGGATGGTGACT-3'; hH2R forward 5'-CCCGGCTCCGCAACCTGA-3' and reverse 5'-CTGATCCCGGGCGACCTTGA-3'; hH3R forward 5'-CTTCCTGCCCTAGCAGTT-3' and reverse 5'-GCAGAGAACAGCTTCGAGGTT-3' hH4R forward 5'-TGGAAGCGTGATCATCTCAG-3' and reverse 5'-ATATGGAGCCCAGCAAACAG-3'.
• PCR amplicons were resolved in an ethidium bromide-stained agarose gel (2.5%) by electrophoresis.
• GAPDH gene expression was used as an internal control.

2.6 Immunocytofluorescence and confocal analysis

• Podocytes plated on collagen-coated cover glasses were fixed with 4% paraformaldehyde for 10 minutes at room temperature.
• The negative control to exclude non-specific staining by the secondary antibody (where cells were incubated with secondary antibodies alone) is reported in Supplementary Material Fig. 1 .
• All the slides were examined at ×63 magnification using the SP5 Confocal Laser Scanning Microscope SMD .
• A variable number of optical section images in the z-dimension (z-spacing, 0.42µm) were collected ensuring that images throughout the 3D cellular structure, spanning multiple confocal planes, were fully captured.

2.7 Radioligand binding studies

• The incubation was stopped by rapid dilution with ice-cold assay buffer.
• Non-specific binding was determine using unlabelled 10 µM difenhydramine.
• The bound radioactivity was separated by filtration through GF/B Glass Fiber Filter Paper that had been treated with 0.3% polyethyleneimine (PEI).
• Filters were washed thrice with ice-cold assay buffer and the radioactivity retained on the filters was measured by liquid scintillation counting.
• Protein concentrations were determined according to Bradford methods, using bovine serum albumin as a standard.

2.8 Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET)

• The TR-FRET assay was used to evaluate both cAMP and IP 1 production by using the LANCE®.
• Ultra cAMP Detection Kit and the IP-One HTRF® assay kit, respectively, as previously described [26] and according to their manufacturer's instruction.
• The energy transfer was measured by the multiple plate reader Victor X4 (excitation: 620 nm, emission: 665 nm).
• TATA-binding protein (TBP) mRNA were used to normalise RNA inputs.
• Fold-change expression with respect to control was calculated for all samples.

2.11 Data analysis and statistical procedures

• Gene and protein expression data are expressed as fold-changes relative to the control (vehicle alone), which is represented as one-fold.
• For the permeability assay the mean of the control values have been calculated and all the individual control values and all the individual test values are expressed as fold-change relative to the control mean.
• Results are shown as mean ± SEM from 5 different experiments, and were analysed by Prism 4 software from Graphpad (CA, USA).
• In all the other cases, the non-parametric Kruskal-Wallis test was used.
• For concentration-response curves a four-parameter logistic equation was applied and the best-fit was obtained.

3.1 Histamine compromises SD integrity in human immortalised podocytes

• The authors tested whether the amine influences the transepithelial flow of FITC-albumin in a transwell assay of monolayer permeability.
• Notably, the 10 min pre-treatment of podocytes with chlorpheniramine maleate was effective in preventing the transepithelial flow of FITC-albumin, as demonstrated by its ability to partially prevent the effect exerted by histamine 0.1 nM (Fig. 1c ).
• The pre-treatment with chlorpheniramine maleate was able to ameliorate the histamine evoked-effects as intercellular spaces appeared narrower, although this was incomplete (Fig. 2a and b ).
• Chlorphenamine treatment alone did not affect the intercellular contact area.

Frequently asked questions

Q1. What is the role of the H4R in the genesis of proteinuria?

podocyte 46524dedifferentiation and mesenchymal transition could be a potential pathway leading to their 466 dysfunction, thereby playing a role in the genesis of proteinuria [59]. 

Q2. What are the contributions in this paper?

Veglia et al. this paper investigated the role of histamine in paracellular permeability of the human glomerular slit diaphragm. 

Q3. how was histamine shown to decrease the permeability of the nasal epithelial?

In particular, histamine was shown to significantly downregulate ZO-1 mRNA 65 expression in cultured human nasal epithelial cells [9]. 

Q4. How many immortalised human podocytes were obtained from the nephrectomy?

Human 140 immortalised podocytes (40,000 cells well, 500 μl) were seeded on the top of HTS Transwell inserts 141 (3 µm pore, 24-well plate) and cultured till confluence was achieved. 

Q5. How did histamine affect SD protein expression in human immortalized podocytes?

Histamine affects SD protein expression in human immortalized podocyte 343The effect evoked by histamine on SD associated proteins ZO-1 and P-cadherin was evaluated at 344 both gene and protein levels. 

Q6. What could be the reason for the lack of functional evidence?

The low level of localisation of the H4R at the cell membrane could be a possible 441 explanation for the lack of the functional evidence. 

Q7. What is the atypical high potency of histamine?

The atypical apparent high potency of histamine, may also reflect changes in local histamine 41322concentrations over time, H1R membrane expression changes and trafficking during the extended 414 exposure period (1-8h), or a large portion of spare receptors. 

Q8. what is the role of histamine in the development of renal aetiologie?

The authors provide for the first time, molecular 477 pharmacological evidence for a direct effect of histamine on human podocytes, suggestive of a 478 possible use of antihistamines as add-on therapy to counteract the onset and progression of both 479 albuminuria and glomeruosclerosis in different renal aetiologies. 

Q9. How many g/l of total RNA extracted from podocytes were subject?

1572.5 RT-PCR 158Two µg/µl of total RNA extracted from podocytes by using RevertAid™ First Strand cDNA 159 Synthesis Kit according to the manufacturer’s instruction, were subjected to RT-PCR as previously 160 described [26, 32]. 

Q10. What is the corresponding concentration of mepyramine in the podocytes?

315 The saturation isotherms revealed a Kd for [3H] mepyramine of 7.02 ± 1.76 nM, indicating the 316 presence of a single class of high affinity binding sites in podocyte membranes. 

Q11. What is the significance of the pre-treatment of human immortalized podocytes?

the 10 min pre-treatment 246 of podocytes with chlorpheniramine maleate was effective in preventing the transepithelial flow of 247 FITC-albumin, as demonstrated by its ability to partially prevent the effect exerted by histamine 0.1 248 nM (Fig. 1c). 

Q12. What is the theory that histamine affects the Kf?

until now histamine was assumed only to affect the Kf through the H1R and H2R 475 present on mesangial cells, in keeping with the theory that contraction of these cells leads to a 476 reduction in the glomerular capillary surface area. 

Q13. What is the data on histamine 87 receptors expression on renal parenchymal?

the data on histamine 87 receptors expression on renal parenchymal cells arise only from their recent observations of H1R, 88 H2R, H3R and H4R on tubular epithelial cells [24-26]. 

Q14. What is the pharmacological significance of the alternative forms of H1R?

Although these alternative forms of H1R have not been pharmacologically 417 characterized thus far, they could be more sensitive to histamine than the wild-type form. 

Q15. What is the sigmoidal increase of IP1?

cells exposed for 1 h to histamine 3 pM-10 nM showed a concentration-323 dependent decrease in TR-FRET signal, indicating a sigmoidal increase of IP1 (EC50 0.15 ± 0.03 324 nM). 

Q16. What could be the mechanism underlining the detrimental effect of histamine on the junction 454?

All these molecular events, the activation of H1R 452 leading to the increase in IP3 production and the co-incident reduction in ZO-1 and P-cadherin, may 453 represent the possible mechanism underlining the detrimental effect of histamine on junction 454 morphological and functional integrity as suggested by the comparable time- and concentration-455 response profiles.