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Flow Stimulation of Endothelin‐1 Production by the Collecting Duct: Role of Nitric Oxide and Reactive Oxygen Species
Author(s) -
Kohan Donald E,
Wheatley Will
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.857.7
Subject(s) - chemistry , endothelin 1 , endocrinology , medicine , reabsorption , nitric oxide , autocrine signalling , stimulation , biophysics , biology , biochemistry , kidney , receptor
Collecting duct (CD)‐derived endothelin‐1 (ET‐1) is an important autocrine inhibitor of CD Na + and water reabsorption; CD‐specific ET‐1 deletion causes marked salt‐sensitive hypertension. Volume expansion increases CD ET‐1 production thereby facilitating elimination of the volume load. We have previously shown that increased luminal flow, as occurs in salt and/or water loading, stimulates CD ET‐1 production via Ca 2+ ‐dependent pathways; however, how this flow effect occurs is incompletely understood. To examine this, a mouse inner medullary CD cell line (IMCD3) was exposed to shear stress in flow chambers and ET‐1/GAPDH mRNA assessed (ET‐1 mRNA very accurately reflects ET‐1 protein and ET‐1 protein levels are too low to measure in this system). A shear stress of 9.8 dyne/cm 2 for 2 h was used based on previous studies (N=6–12 for all of the following data). Perfusion with HBSS + 0.25 mM L‐Arg (used in all subsequent studies) increased ET‐1 mRNA by 4‐fold compared to no flow (ET‐1 flow response). Exposure to 1 μM L‐NAME reduced the ET‐1 flow response to 67% compared to vehicle. Inhibition of NOS1 (1 μM SMTC or 200 μM TRIM) or NOS2 (0.1 nM 1400W) did not alter the ET‐1 flow response. NOS3 siRNA (no specific NOS3 antagonists exist) reduced NOS3 levels by 70%, however cells detached on exposure to flow (likely due to lower cell density needed for transfection). Inhibition of NO signaling pathways, including PKG (1 μM KT5823), guanylyl cyclase (10 μM ODQ) or NFkB (50 μg/ml NFkB inhibitory peptide), did not affect the ET‐1 flow response. These findings suggested NO may not be involved, but rather NOS (at least in part)‐generated reactive oxygen species (ROS) may play a role in the ET‐1 flow response. Inhibition of ROS with 5 mM tempol reduced the ET‐1 flow response to 67% vs. vehicle, no further inhibition was seen with tempol + L‐NAME. Superoxide dismutase (SOD) inhibition (50 μM MnTBAP or 100 U/ml PEG‐SOD) reduced the ET‐1 flow response to 65% of vehicle, while 1000 U/ml PEG‐catalase was without effect. Apocynin (100 μM) also reduced the ET‐1 flow response to 60% of vehicle, suggesting NAPDH oxidase (NOX) might be involved. Inhibition of NOX1/4 with 5 μM GKT137831 also reduced the ET‐1 flow response to 70% of vehicle. Taken together, these data suggest that both NOS and NOX contribute to flow stimulated superoxide‐mediated stimulation of CD ET‐1 production. Since ROS have been reported to augment ENaC activity, the current studies suggest that ROS‐stimulated ET‐1 may mitigate their Na + ‐retaining effects in the CD. Finally, to our knowledge, this is the first report of flow‐stimulated reactive oxygen species in the CD, as well as the first report of such flow‐stimulated ROS exerting a biologic effect in the CD. Support or Funding Information Supported by NIH R01 DK107454.