ET‐1 and Nitric Oxide Stimulate Trans‐Epithelial Electrical Resistance (TER) in Inner Medullary Collecting Duct Cells

Abstract

In the renal collecting duct, it is the epithelial Na channel (ENaC) that is the rate‐limiting step for Na reabsorption. We recently reported, that collecting duct nitric oxide synthase‐1 (NOS1) is necessary for endothelin‐1 (ET‐1) dependent inhibition of ENaC activity. Loss of either ET‐1 or NOS1 expression specifically in the collecting duct generates a salt‐sensitive blood pressure phenotype. In the kidney, it is the IMCD that expresses the highest NOS activity in the kidney, and produces the most ET‐1 throughout the body. Previous studies demonstrating ENaC regulation in the collecting duct have utilized patch‐clamp electro‐physiology in the cortical collecting duct, however the inner medullary collecting duct (IMCD) is technically difficult to patch clamp. Thus, the purpose of this study was to utilize TER, which is inversely related to ion transport, to determine the effect of ET‐1 and NOS inhibition on IMCD ENaC activity. TER was measured with a volt‐ohmmeter and STX2 electrode. Transwell cell culture inserts were used, allowing mouse or rat IMCD cells to polarize. Both rat primary IMCD and mouse mIMCD‐3 exhibited amiloride‐sensitive ENaC inhibition. Amiloride (1 uM) increased TER (decreased ENaC activity) in rat IMCD cells (+5.27 ± 0.63%) and mIMCD‐3 cells (+9.53 ± 1.33%) compared to vehicle controls (+0.29 ±0.19%). ET‐1 (100 nM) stimulated a significant increase in TER for rat IMCD (+2.66‐‐ ± 0.23%) and mouse mIMCD‐3 cells (+3.25 ± 0.62%−) compared to vehicle, indicating an inhibition of ion transport. NOS inhibition (5 mM L‐NAME) also stimulated a significant decrease in TER in rat IMCD (−3.50 ± 0.5%) and mouse mIMCD‐3 cells (−8.35 ± 1.20%). The decrease in resistance, and therefore increase in ion transport, with NOS inhibition could not be reversed with the addition of ET‐1, indicating that the NOS system contributes to the tonic inhibition of ion transport in the IMCD. In conclusion, these data support the hypothesis that the ET‐1 and NOS systems regulate ion transport, most likely ENaC, in the IMCD of rats and mice.Support or Funding InformationNIHP01HL95499 and The University of Alabama at Birmingham Preparation for Graduate and Medical Education Program (PARADiGM) R25 HL120883