Identification of arginine-vasopressin receptor 1a (Avpr1a/AVPR1A) as a novel candidate gene for chronic visceral pain

Abstract

Chronic abdominal pain in the absence of ongoing disease is the hallmark of disorders of gut-brain interaction (DGBIs), including irritable bowel syndrome (IBS). While the etiology of DGBIs remains poorly understood, there is evidence that both genetic and environmental factors play a role. In this study, we report the identification and validation of Avpr1a as a novel candidate gene for visceral hypersensitivity (VH), a primary peripheral mechanism underlying abdominal pain in DGBI/IBS. Comparing two C57BL/6 (BL/6) substrains (C57BL/6NTac and C57BL/6J) revealed differential susceptibility to the development of chronic VH following intrarectal zymosan (ZYM) instillation, a validated preclinical model for post-inflammatory IBS. Using whole genome sequencing, we identified a SNP differentiating the two strains in the 5' intergenic region upstream of Avpr1a, encoding the protein arginine-vasopressin receptor 1A (AVPR1A). We used behavioral, histological, and molecular approaches to identify distal colon-specific gene expression differences and neuronal hyperresponsiveness covarying with Avpr1a genotype and VH susceptibility. While the two BL/6 substrains did not differ across other gastrointestinal (GI) phenotypes (e.g., GI motility), VH-susceptible BL/6NTac mice had higher colonic Avpr1a mRNA and protein expression. Moreover, neurons of the enteric nervous system were hyperresponsive to the AVPR1A agonist AVP, suggesting a role for enteric neurons in the pathology underlying VH. These results parallel our findings that patients' colonic Avpr1a mRNA expression was higher in patients with higher pain ratings. Taken together, these findings implicate differential regulation of Avpr1a as a novel mechanism of VH-susceptibility as well as a potential therapeutic target specific to VH.

Keywords: disorders of gut-brain interaction; enteric nervous system; genetics; irritable bowel syndrome; visceral hypersensitivity.

Figure 1.. C57BL/6 mice develop ZYM-induced VH.

Visceromotor (EMG) responses were measured during colorectal balloon distension pre- and post-intracolonic zymosan (ZYM) instillation for each strain. The graph shows the following groups: C57BL/6J-Naïve (open teal square), C57BL/6J-ZYM (teal square), C57BL/6NTac-Naïve (open orange circle), and C57BL/6NTac (orange circle) mice. Each strain was normalized to its own naïve (baseline) group. Only VMR to 60 mm Hg distention pressure confirmed a significant main effect of condition [F (3, 38)] = 3.493, p < 0.05).

Figure 2.. Workflow for identifying pain candidate genes from whole genome sequencing data.

In brief, we performed a systematic review and collected a list of genes related to visceral pain, nociception, and/or pain assays. From this list, we used whole genome sequencing data published in Mortazavi, Ren [23] to compare the location of our genes of interest to locations of possible differential SNPs between BL/6J and BL/6NTac. It is important to note Archive Ensembl was used to match and compare whole genome sequencing data to the appropriate gene loci. To the right of the general flow is an example workflow used to identify an intergenic SNP within the 5’ region of Avpr1a.

Figure 3.

(A) Colonic Avpr1a mRNA levels were measured in SAL or ZYM-treated BL/6J and BL/6NTac mice. A 2 (strain) x 2 (treatment) ANOVA confirmed a significant Strain x Treatment interaction (F (1, 10) = 5.851, p = 0.036). No other significant main effects or interactions were observed (all F < 2.097, all p> 0.05). (B) AVPR1A protein levels were measured in ZYM-treated BL/6NTac and BL/6J mice. No significant main effects or interactions on the expression of Avpr1a in either thoracolumbar or lumbosacral colon-specific primary sensory afferents were detected in a similar 2 × 2 ANOVA (all F [(1, 14)] < 0.831, all p > 0.05).

Figure 4.. Colorectal spinal afferent responses to colorectal stretch measured by single-fiber recordings with an ex vivo colorectum-pelvic nerve preparation.

In ZYM-treated BL/6NTac and BL/6J mice, the stimulus-response function (SRF) was measured before, after AVP application to the receptive field, and subsequent washout. (A) A 2 (Strain) x 2 (Stretch Direction) ANOVA revealed a significant main effect of Strain (F (1, 15) = 30.810, p < 0.001) and a Strain x Stretch Direction interaction (F (1, 15) = 4.787, p < 0.05) on AUC.). (B) Similarly, 2 × 2 ANOVA for response during peak stretch force application confirmed a significant main effect of Strain (1, 15) = 57.203, p < 0.001), confirming a greater response magnitude in BL/6NTac mice treated with ZYM compared to BL/6J mice treated with ZYM. No other significant main effects or interactions were observed (all p< 0.05).

Figure 5.

(A) AVPR1A mRNA levels from pediatric IBS patients’ colonic biopsies with low or high levels of reported pain compared to pain-free controls. IBS/FAP (n = 19) vs. Functional Nausea/painless rectal bleeding as controls (n = 3). T statistic = 1.462, p value = 0.159, MEANS: IBS/FAP=2.58 (SEM=0.334) vs control = 1.262 (SEM=0.545). This could be due to a low number of controls and difficulty finding appropriate controls given that preventative colonoscopy is not recommended until adulthood, and these are all adolescents. (B) AVPR1A mRNA levels from adult patients with active UC who report pain compared to those reporting no pain and pain-free controls. n = 6–9 per group. A 2 × 2 ANOVA showed a main effect of pain status but not disease status. Active/NP = 0.51 ((SEM = 0.162), Active/P = 2.23 (0.467), Inactive/P = 1.56 (0.339), Inactive/NP = 1.11 (0.302). * Indicate significant ANOVA, p < 0.05.

Figure 6.. Representative images of immunohistochemistry stains of distal colon cross-sections from BL/6NTac mice instilled with ZYM.

Sections were stained with DAPI nuclear stain (blue), AVPR1A (red), PGP9.5 for neurons (green), and merge of all three to show co-localization of AVPR1A and neuronal cell bodies (yellow), n=5 BL/6NTac-ZYM.

Figure 7.

A) In vitro Ca²⁺ imaging of ENS neurons revealed individual neurons from VH mice (Mean = 0.5342, SEM = 0.0671) exhibited significantly greater Ca²⁺ influx (ΔF) in response to AVP compared to SAL controls (Mean = 0.2282, SEM = 0.0240). p<0.001. B) ENS neurons revealed individual neurons from VH mice (Mean = 0.2155, STEM= 0.0243) exhibited greater Ca²⁺ influx (DF) in response to capsaicin compared to SAL controls (Mean = 0.0228, SEM = 0.0212). The % of neurons responding to AVP (Person Chi-Square p = 0.051) and capsaicin (Pearson Chi-Square p = 0.569) were not statistically significant (data not shown).