Role of H2O2 on the kinetics of low-affinity high-capacity Na+-dependent alanine transport in SHR proximal tubular epithelial cells

Research highlights: → H2O2 in excess is required for the presence of a low-affinity high-capacity component for the Na+-dependent [14C]-L-alanine uptake in SHR PTE cells only. → It is suggested that Na+ binding in renal ASCT2 may be regulated by ROS in SHR PTE cells. -- Abstract: The presence of high and low sodium affinity states for the Na+-dependent [14C]-L-alanine uptake in immortalized renal proximal tubular epithelial (PTE) cells was previously reported (Am. J. Physiol. 293 (2007) R538-R547). This study evaluated the role of H2O2 on the Na+-dependent [14C]-L-alanine uptake of ASCT2 in immortalized renal PTE cells from Wistar Kyoto rat (WKY) and spontaneously hypertensive rat (SHR). Na+ dependence of [14C]-L-alanine uptake was investigated replacing NaCl with an equimolar concentration of choline chloride in vehicle- and apocynin-treated cells. Na+ removal from the uptake solution abolished transport activity in both WKY and SHR PTE cells. Decreases in H2O2 levels in the extracellular medium significantly reduced Na+-Km and Vmax values of the low-affinity high-capacity component in SHR PTE cells, with no effect on the high-affinity low-capacity state of the Na+-dependent [14C]-L-alanine uptake. After removal of apocynin from the culture medium, H2O2 levels returned to basal values within 1 to 3 h in both WKY and SHR PTE cells and these were found stable for the next 24 h. Under these experimental conditions, the Na+-Km and Vmax of the high-affinity low-capacity state were unaffected and the low-affinity high-capacity component remained significantly decreased 1 day but not 4 days after apocynin removal. In conclusion, H2O2 in excess is required for the presence of a low-affinity high-capacity component for the Na+-dependent [14C]-L-alanine uptake in SHR PTE cells only. It is suggested that Na+ binding in renal ASCT2 may be regulated by ROS in SHR PTE cells.