Osmolar regulation of endothelin-1 production by the inner medullary collecting duct.

AIMS: Endothelin-1 (ET-1) is an autocrine inhibitor of collecting duct (CD) Na(+) and water reabsorption. CD ET-1 production is increased by a high salt diet and is important in promoting a natriuretic response. The mechanisms by which a high salt diet enhances CD ET-1 are being uncovered. In particular, elevated tubule fluid flow, as occurs in salt loading, enhances CD ET-1 synthesis. Tubule fluid solute content and interstitial osmolality can also be altered by a high salt diet, however their effect on CD ET-1 alone, or in combination with flow, is poorly understood. MAIN METHODS: ET-1 mRNA production by a mouse inner medullary CD cell line (mIMCD3) in response to changing flow and/or osmolality was assessed. KEY FINDINGS: Flow or hyperosmolality (using NaCl, mannitol or urea) individually caused an ~2-fold increase in ET-1 mRNA, while flow and hyperosmolality together increased ET-1 mRNA by ~14 fold. The hyperosmolality effect alone and the synergistic effect of flow + hyperosmolality was inhibited by chelation of intracellular Ca(2+), however were not altered by blockade of downstream Ca(2+)-signaling pathways (calcineurin or NFATc), inhibition of cellular Ca(2+) entry channels (purinergic receptors or polycystin-2), or blockade of the epithelial Na(+) channel. Inhibition of NFAT5 with rottlerin or NFAT5 siRNA greatly reduced the stimulatory effect of osmolality alone and osmolality + flow on mIMCD3 ET-1 mRNA levels. SIGNIFICANCE: Both flow and osmolality individually and synergistically stimulate mIMCD3 ET-1 mRNA content. These findings may be relevant to explaining high salt diet induction of CD ET-1 production.

Flow regulation of endothelin-1 production in the inner medullary collecting duct.

Collecting duct-derived endothelin (ET)-1 is an autocrine inhibitor of Na(+) and water reabsorption; its deficiency causes hypertension and water retention. Extracellular fluid volume expansion increases collecting duct ET-1, thereby promoting natriuresis and diuresis; however, how this coupling between volume expansion and collecting duct ET-1 occurs is incompletely understood. One possibility is that volume expansion increases tubular fluid flow. To investigate this, cultured IMCD3 cells were subjected to static or flow conditions. Exposure to a shear stress of 2 dyn/cm(2) for 2 h increased ET-1 mRNA content by ∼2.3-fold. Absence of perfusate Ca(2+), chelation of intracellular Ca(2+), or inhibition of Ca(2+) signaling (calmodulin, Ca(2+)/calmodulin-dependent kinase, calcineurin, PKC, or phospholipase C) prevented the flow response. Evaluation of possible flow-activated Ca(2+) entry pathways revealed no role for transient receptor potential (TRP)C3, TRPC6, and TRPV4; however, cells with TRPP2 (polycystin-2) knockdown had no ET-1 flow response. Flow increased intracellular Ca(2+) was blunted in TRPP2 knockdown cells. Nonspecific blockade of P2 receptors, as well as specific inhibition of P2X7 and P2Y2 receptors, prevented the ET-1 flow response. The ET-1 flow response was not affected by inhibition of either epithelial Na(+) channels or the mitochondrial Na(+)/Ca(2+) exchanger. Taken together, these findings provide evidence that in IMCD3 cells, flow, via polycystin-2 and P2 receptors, engages Ca(2+)-dependent signaling pathways that stimulate ET-1 synthesis.

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

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.