Prostaglandin E2 mediates connecting tubule glomerular feedback

Abstract

Connecting tubule glomerular feedback (CTGF) is a mechanism in which Na reabsorption in the connecting tubule (CNT) causes afferent arteriole (Af-Art) dilation. CTGF is mediated by eicosanoids, including prostaglandins and epoxyeicosatrienoic acids; however, their exact nature and source remain unknown. We hypothesized that during CTGF, the CNT releases prostaglandin E2, which binds its type 4 receptor (EP4) and dilates the Af-Art. Rabbit Af-Arts with the adherent CNT intact were microdissected, perfused, and preconstricted with norepinephrine. CTGF was elicited by increasing luminal NaCl in the CNT from 10 to 80 mmol/L. We induced CTGF with or without the EP4 receptor blocker ONO-AE3-208 added to the bath in the presence of the epoxyeicosatrienoic acid synthesis inhibitor MS-PPOH. ONO-AE3-208 abolished CTGF (control, 9.4 ± 0.5; MS-PPOH+ONO-AE3-208, -0.6 ± 0.2 μm; P<0.001; n=6). To confirm these results, we used a different, specific EP4 blocker, L161982 (10(-5) mol/L), that also abolished CTGF (control, 8.5 ± 0.9; MS-PPOH+L161982, 0.8 ± 0.4 μm; P<0.001; n=6). To confirm that the eicosanoids that mediate CTGF are released from the CNT rather than the Af-Art, we first disrupted the Af-Art endothelium with an antibody and complement. Endothelial disruption did not affect CTGF (7.9 ± 0.9 versus 8.6 ± 0.6 μm; P=NS; n=7). We then added arachidonic acid to the lumen of the CNT while maintaining zero NaCl in the perfusate. Arachidonic acid caused dose-dependent dilation of the attached Af-Art (from 8.6 ± 1.2 to 15.3 ± 0.7 μm; P<0.001; n=6), and this effect was blocked by ONO-AE3-208 (10(-7) mol/L). We conclude that during CTGF, the CNT releases prostaglandin E2, which acts on EP4 on the Af-Art inducing endothelium-independent dilation.

Keywords: 11,12-epoxy-5,8,14-eicosatrienoic acid; arachidonic acid; arterioles; endothelium; microcirculation; prostaglandin E2; receptors, prostaglandin E.

Figure 1

Repeatedly increasing NaCl concentration in the CNT dilated preconstricted Af-Art in a similar manner, indicating that CTGF is stable and reproducible over time.

Figure 2

In the presence of the EET synthesis inhibitor MS-PPOH (10⁻⁶ mol/L), addition of the EP4 receptor blocker ONO-AE3-208 (10⁻⁷ mol/L) completely inhibited CTGF, suggesting that PGE₂ acts on EP4 receptor on the Af-Art. *** P < 0.001.

Figure 3

In the presence of the EET synthesis inhibitor MS-PPOH (10⁻⁶ mol/L), addition of the EP4 receptor blocker L161982 (10⁻⁵ mol/L) completely inhibited CTGF, suggesting that PGE₂ acts on EP4 receptor on the Af-Art. ** P < 0.01.

Figure 4

Removal of endothelium-dependent relaxation by perfusion of the Af-Art with an antibody (anti-von Willebrand factor) and complement did not alter CTGF. This suggests that the eicosanoids that mediate CTGF are released from the CNT, rather than the Af-Art endothelium. Acetylcholine failed to dilate preconstricted Af-Arts, suggesting successful endothelial removal with our antibody/complement method.

Figure 5

Addition of exogenous arachidonic acid (AA) to the CNT lumen in the absence of NaCl caused dose-dependent dilation of the attached Af-Art. *** P < 0.001 vs. preconstricted Af-Art diameter. The maximal vasodilation was similar to that achieved by CTGF induced with 80 mmol/L NaCl in the lumen of the CNT.

Figure 6

Addition of the EP4 blocker ONO-AE3-208 (10⁻⁷ mol/L) and the EET synthesis inhibitor MS-PPOH (10⁻⁶ mol/L) completely prevented the vasodilation induced by arachidonic acid (AA) added to the lumen of the CNT.