Role for reactive oxygen species in flow-stimulated inner medullary collecting duct endothelin-1 production.

Inner medullary collecting duct (IMCD)-derived endothelin-1 (ET-1) is stimulated by volume expansion, in part through augmented luminal flow, whereupon it can elicit natriuresis and diuresis. Since flow can alter nitric oxide (NO) and reactive oxygen species (ROS), both of which can affect collecting duct salt transport, we asked whether NO and/or ROS mediate flow-stimulated IMCD ET-1. Mouse IMCD3 cells were exposed to flow, and ET-1/GAPDH mRNA was assessed. A shear stress of 10 dyn/cm2 for 1 h increased ET-1 mRNA by fourfold compared with no flow (ET-1 flow response). Global NO synthase (NOS) inhibition [NG-nitro-l-arginine methyl ester (l-NAME)] reduced the ET-1 flow response; however, pharmacological inhibition of NOS1 or NOS2, inhibition of NOS3 siRNA, inhibition of arginase inhibition, removal of media l-Arg, or inhibition of NO-dependent signaling pathways (PKG, guanylyl cyclase, or NF-κB) did not affect the ET-1 flow response. Tempol reduced the ET-1 flow response; no further inhibition occurred with l-NAME. Superoxide dismutase, but not catalase, reduced the ET-1 flow response. Inhibition of NAPDH oxidase (NOX) (apocynin), pharmacological inhibition of NOX1/4, or NOX4 siRNA reduced the ET-1 flow response. Finally, flow increased IMCD3 ROS production and this was inhibited by apocynin, NOX1/4 inhibition, and, to a small extent, by l-NAME. Taken together, these data suggest that NOX4-derived ROS in general, and possibly superoxide in particular, are involved in flow-stimulated IMCD ET-1 production. To our knowledge, this is the first report of flow-stimulated ROS production by the CD, as well as the first report of such flow-stimulated CD ROS exerting a biological effect.