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

Abstract

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.

Keywords: Collecting duct; Endothelin; Flow; Osmolality.

Figure 1

Effect of hyperosmolality (mannitol) on stationary and flow stimulated ET-1 mRNA content in mIMCD3 cells. n=10-12. *p<0.05 vs. no flow control same conditions; **p<0.05 vs. no flow control 300 mOsm: #p<0.05 vs. flow 300 mOsm.

Figure 2

Effect of hyperosmolality with mannitol, urea or NaCl on stationary and flow-stimulated ET-1 mRNA content in mIMCD3 cells. n=10-12. *p<0.05 vs. no flow control same conditions; **p<0.05 vs. no flow control 300 mOsm: #p<0.05 vs. flow 300 mOsm.

Figure 3

Effect of inhibition of ENaC (0.2 µM benzamil) on osmolality (mannitol) and flow stimulated ET-1 mRNA content in mIMCD3 cells. n=10-12. *p<0.05 vs. no flow control same conditions; **p<0.05 vs. no flow control 300 mOsm: #p<0.05 vs. flow 300 mOsm.

Figure 4

Effect of chelation of intracellular calcium (50 µM BAPTA-AM) on osmolality (mannitol) and flow stimulated ET-1 mRNA content in mIMCD3 cells. n=10-12. *p<0.05 vs. no flow control same conditions; **p<0.05 vs. no flow control 300 mOsm: #p<0.05 vs. flow 300 mOsm, $p<0.05 vs. flow 450 mOsm.

Figure 5

Effect of inhibition of purinergic receptors (30 µM PPADS) on osmolality (mannitol) and flow stimulated ET-1 mRNA content in mIMCD3 cells. n=10-12. *p<0.05 vs. no flow control same conditions; **p<0.05 vs. no flow control 300 mOsm: #p<0.05 vs. flow 300 mOsm.

Figure 6

Effect of inhibition of NFATc inhibitor peptide (0.2 µM VIVIT peptide) on osmolality (mannitol) and flow stimulated ET-1 mRNA content in mIMCD3 cells. n=10-12. *p<0.05 vs. no flow control same conditions; **p<0.05 vs. no flow control 300 mOsm: #p<0.05 vs. flow 300 mOsm.

Figure 7

Effect of Pkd2 gene (encodes polycystin-2) deficiency on osmolality (mannitol) and flow stimulated ET-1 mRNA content in mIMCD3 cells. n=10-12. *p<0.05 vs. no flow control same conditions; **p<0.05 vs. no flow control 300 mOsm: #p<0.05 vs. flow 300 mOsm, $p<0.05 vs. flow 450 mOsm.

Figure 8

Effect of calcineurin inhibition (3 µg/ml cyclosporine A) on osmolality (mannitol) and flow stimulated ET-1 mRNA content in mIMCD3 cells. n=12. *p<0.05 vs. no flow control same conditions; **p<0.05 vs. no flow control 300 mOsm: #p<0.05 vs. flow 300 mOsm.

Figure 9

Effect of rottlerin (10 µM) on osmolality (mannitol) and flow stimulated ET-1 mRNA content in mIMCD3 cells. n=10-12. *p<0.05 vs. no flow control same conditions; **p<0.05 vs. no flow control 300 mOsm: #p<0.05 vs. flow 300 mOsm, $p<0.05 vs. flow 450 mOsm.

Figure 10

Effect of NFAT5 siRNA on osmolality (mannitol) and flow stimulated ET-1 mRNA content in mIMCD3 cells. n=12. *p<0.05 vs. no flow control same conditions; **p<0.05 vs. no flow control 300 mOsm: #p<0.05 vs. flow 300 mOsm, $p<0.05 vs. flow 450 mOsm.