The role of SLC4A4 in the pathogenesis of aspirin-induced Upper Gastrointestinal Injury

Aspirin, a non-steroidal anti-inflammatory drug widely prescribed as an analgesic and at low doses as anti-platelet drug for prevention of cardiovascular diseasesis often associated with serious adverse events, including upper gastrointestinal (GI) injuries caused by acid-back diffusion into the GI mucosa (1). The electrogenic sodium bicarbonate co-transporter (NBCe1) protein mediates transportation of bicarbonate ions and contributes to regulation of intracellular pH (2). It was hypothesised that impaired function of the solute carrier family, member 4 (SLC4A4),which encodes NBCe1 may have a role in the pathogenesis of gastric injury resulting from aspirin therapy. This study aimed to investigate this role of SLC4A4 and determine the molecular mechanism underpinning this hypothesis.

Initial analysis from a Genome Wide Association Study has identified genetic variants of SLC4A4 as putative risk-factors for aspirin-induced upper GI ulceration (185 cases, 217 controls). The study was approved by the Liverpool (Adults) Research Ethics Committee, and informed consent was obtained from eligible patients. Their NSAID use prior to endoscopy was also determined. Ahuman gastric adenocarcinoma cell line (AGS) was used as an in vitro model to study NBCe1 transporter activity and the mechanisms of cell death by aspirin. SLC4A4 transcript expression in AGS cells was determined using TaqMan RT-PCR. The effects of S0859, a sodium bicarbonate transport inhibitor, and aspirin on accumulation of bicarbonate ions in AGS cells were assessed using 14C-labelled NaHCO3. Statistical analysis was performed using Student’s t- test (unpaired, assuming unequal variance), or Mann-Whitney U test with two-sided p ≤ 0.05 defined as significant.

In H. Pylori negative aspirin-induced gastric ulceration, rs4521314, an intronic SNP within the SLC4A4 locus was associated with ulcer risk (p=7.5x10-5; heterozygous OR (95% CI) = 2.33 (1.44-3.80). RNA sequencing data (n= 10 cases, 10 controls) strongly suggests that carriage of the associated SLC4A4 variant correlates with significantly lower transcript levels of SLC4A4 in gastric biopsy taken from the antrum of aspirin tolerant controls (AA vs AC allele; p = 0.01). Further validation of this finding has been carried out in a larger cohort of healthy control patient gastric biopsies (n=93). A dose-dependent fall in cell viability was recorded in aspirin-exposed cells (p = 0.0024) between 0 and 50mM, but further investigation revealed that this does not occur via the caspase (3/7)-dependent pathway. Results obtained from transport studies showed that S0859 [100µM] significantly inhibited bicarbonate uptake by approximately 45% at 60 and 90 minutes (p = 0.02), compared to control but no significant differences were observed in the aspirin-treated cells. This suggests aspirin may not inhibit bicarbonate transport, though additional analysis with an SLC4A4 transfected cell line will be carried out to validate this.

Overall, data from our genetic studies are suggestive of an association between the SLC4A4 gene and complications of aspirin-induced upper GI injury. Future work on the functional consequence of the associated locus, and its role in the pathogenesis of aspirin-induced upper GI complications are required.

(1) Fromm D (1987). Clin Invest Med 10(3): 251–258.

(2) Dinour D et al. (2004). J Biol Chem 279: 52238–52246.