Sodium‐coupled neutral amino acid transporter SNAT2 counteracts edema formation and reduces autophagy and ER stress in acute lung injury

Abstract

ObjectiveAcute respiratory distress syndrome (ARDS), the most frequent cause of death in intensive care, is characterized by an excessive inflammatory response, increased vascular permeability, edema formation, and impaired gas exchange. Recent studies have suggested deprivation of amino acids may aggravate lung injury while promoting apoptosis, ER stress, and autophagy. However, RCTs have failed to identify beneficial pharmaco‐nutritional interventions. This may point to a critical role of amino acid transporters in ARDS pathophysiology and its non‐human analogue acute lung injury (ALI). Sodium‐coupled neutral amino acid transporters (SNATs) mediate cellular uptake of amino acids along with Na+ and are known to be upregulated in response to amino acid starvation. Here, we probed for the role of SNATs in a murine model of HCl‐ and LPS‐induced ALI and in a pulmonary epithelial cell culture system.MethodsExpression of SNATs under basal conditions or after amino acid deprivation in epithelial cell lines (NCI‐H441/A549) and in isolated rat type‐I (ATI cells) was determined by PCR and Western blot.For measurement of L‐alanine transport, H441 cells were cultured on Transwell inserts in the presence or absence of amino acids and treated either with HgCl2, which inhibits SNATs, or siRNA (scramble or siSNAT2). Transport of L‐alanine from the upper to the lower compartment was analyzed by ELISA.For in vivo experiments, SNAT2 knockout (slc38a2−/−), heterozygous (slc38a2+/−) and wildtype (slc38a2+/+) mice were anesthetized and HCl or saline was instilled intratracheally. After 2 h of mechanical ventilation, lungs were collected for measurement of wet–to‐dry lung weight ratio (W/D) and protein extraction. In a second model of ALI, mice were anesthetized and LPS (0.4 mg/kg) was given intranasally. After 24 h, mice were euthanized and lungs were collected for protein extraction.ResultsIsolated ATI, H441, and A549 cells showed strong expression of SNAT2 after incubation in amino acid free medium.Under amino acid deprivation, H441 cells showed quantitative L‐alanine transport that was significantly decreased by treatment with HgCl2 or siRNA silencing of SNAT2.Homozygous SNAT2 knockout mice were found to be sublethal and newborn pups typically succumbed to cyanotic dyspnea. In HCl‐induced ALI, W/D was elevated in slc38a2−/− and slc38a2+/− mice as compared to wildtype. Expression of ER stress and autophagy markers was increased in whole protein lysates of heterozygous mice compared to control in both models of ALI.ConclusionSNAT2 counteracts lung edema formation in acid‐induced ALI, presumably by mediating Na+ uptake that drives alveolar fluid clearance. In LPS‐ and HCl‐induced lung injury, SNAT2 reduces ER stress and autophagy, putatively through its role as amino acid transporter. SNAT2 activation may, hence, provide a new multi‐pronged strategy to counteract ALI/ARDS.Support or Funding InformationSupported by a CIHR grant to Dr. W.M. Kuebler