Na delivery and ENaC mediate flow regulation of collecting duct endothelin-1 production

Abstract

Collecting duct (CD) endothelin-1 (ET-1) is an important autocrine inhibitor of Na and water transport. CD ET-1 production is stimulated by extracellular fluid volume expansion and tubule fluid flow, suggesting a mechanism coupling CD Na delivery and ET-1 synthesis. A mouse cortical CD cell line, mpkCCDc14, was subjected to static or flow conditions for 2 h at 2 dyn/cm(2), followed by determination of ET-1 mRNA content. Flow with 300 mosmol/l NaCl increased ET-1 mRNA to 65% above that observed under static conditions. Increasing perfusate osmolarity to 450 mosmol/l with NaCl or Na acetate increased ET-1 mRNA to ∼184% compared with no flow, which was not observed when osmolarity was increased using mannitol or urea. Reducing Na concentration to 150 mosmol/l while maintaining total osmolarity at 300 mosmol/l with urea or mannitol decreased the flow response. Inhibition of epithelial Na channel (ENaC) with amiloride or benzamil abolished the flow response, suggesting involvement of ENaC in flow-regulated ET-1 synthesis. Aldosterone almost doubled the flow response. Since Ca(2+) enhances CD ET-1 production, the involvement of plasma membrane and mitochondrial Na/Ca(2+) exchangers (NCX) was assessed. SEA0400 and KB-R7943, plasma membrane NCX inhibitors, did not affect the flow response. However, CGP37157, a mitochondrial NCX inhibitor, abolished the response. In summary, the current study indicates that increased Na delivery, leading to ENaC-mediated Na entry and mitochondrial NCX activity, is involved in flow-stimulated CD ET-1 synthesis. This constitutes the first report of either ENaC or mitochondrial NCX regulation of an autocrine factor in any biologic system.

Fig. 1.

Effect of increased media osmolarity, Na, or chloride concentration on flow-stimulated endothelin (ET)-1 mRNA content in mpkCCDc14 cells. Each data point represents the comparison of ET-1 mRNA in cells exposed to flow vs. cells not undergoing flow, with both flowed and stationary cells exposed to identical media osmolarity and electrolyte concentrations; n = 12 each data point. *P < 0.05 vs. cells not exposed to flow. **P < 0.05 vs. cells exposed to 300 mosmol/l NaCl.

Fig. 2.

Effect of reduced Na concentration on flow-stimulated ET-1 mRNA content in mpkCCDc14 cells. Each data point represents the comparison of ET-1 mRNA in cells exposed to flow vs. cells not undergoing flow, with both flowed and stationary cells exposed to identical media osmolarity and electrolyte concentrations; n = 12 each data point. *P < 0.05 vs. cells not exposed to flow.

Fig. 3.

Effect of epithelial Na channel (ENaC) inhibition on flow-stimulated ET-1 mRNA content in mpkCCDc14 cells. Cells were pretreated for 30 min with 1 μM amiloride or 0.2 μM benzamil. Each data point represents the comparison of ET-1 mRNA in cells exposed to flow vs. cells not undergoing flow, with both flowed and stationary cells exposed to identical media and ENaC inhibitors. All studies were done in Hanks' balanced salt solution (HBSS; 300 mosmol/l); n = 12 each data point. *P < 0.05 vs. cells not exposed to flow.

Fig. 4.

Effect of aldosterone ± amiloride on flow-stimulated ET-1 mRNA content in mpkCCDc14 cells. Cells were pretreated with 1 μM aldosterone for 2 days and pretreated with 1 μM amiloride for 30 min. Each data point represents the comparison of ET-1 mRNA in cells exposed to flow vs. cells not undergoing flow, with both flowed and stationary cells exposed to identical media and reagents. All studies were done in HBSS (300 mosmol/l); n = 12 each data point. *P < 0.05 vs. cells not exposed to flow. **P < 0.05 vs. aldosterone alone.

Fig. 5.

Effect of plasma membrane Na calcium exchanger inhibition on flow-stimulated ET-1 mRNA content in mpkCCDc14 cells under static (no flow) and flow conditions. Cells pretreated for 30 min with 3 μM SEA0400 (n = 7) or 10 μM KB-R7943 (n = 12). Each data point represents the comparison of ET-1 mRNA in cells exposed to flow vs. cells not undergoing flow, with both flowed and stationary cells exposed to identical media and reagents. All studies were done in HBSS (300 mosmol/l). *P < 0.05 vs. cells not exposed to flow.

Fig. 6.

Effect of mitochondrial Na calcium exchanger inhibition on flow-stimulated ET-1 mRNA content in mpkCCDc14 cells under static (no flow) and flow conditions. Cells were pretreated for 30 min with 1.2 μM CGP37157 (n = 12). Cells were pretreated with 100 μM nifedipine (n = 6) to control for an effect on L-type dihydropyridine Ca²⁺ channels. Each data point represents the comparison of ET-1 mRNA in cells exposed to flow vs. cells not undergoing flow, with both flowed and stationary cells exposed to identical media and reagents. All studies were done in HBSS (300 mosmol/l). *P < 0.05 vs. cells not exposed to flow.