Increasing Na delivery to epithelial Na channels (ENaC) in the connecting

Increasing Na delivery to epithelial Na channels (ENaC) in the connecting tubule (CNT) causes AV-951 dilation of the afferent arteriole (Af-Art) a process we call CNT glomerular feedback (CTGF). (activation of AT1 AV-951 and that this effect requires activation of PKC and ENaC. Potentiation of CTGF by Ang II could help preserve glomerular filtration rate in the presence of renal vasoconstriction. tubuloglomerular opinions (TGF)1 2 In most nephrons a later segment of the distal nephron the connecting tubule (CNT) also contacts the Af-Art of the same nephron3-6. We have recently explained the presence of crosstalk between the CNT and the Af-Art which we called AV-951 CNT glomerular opinions (CTGF)7 8 Both TGF and CTGF are initiated by increases in Na concentration in the tubular lumen but TGF causes vasoconstriction and CTGF vasodilation. In the presence of an increased sodium weight in the distal nephron such as volume growth or high salt intake activation of TGF causes Af-Art constriction which tends to hinder sodium excretion by decreasing glomerular filtration rate (GFR). However additional mechanisms come into play whereby in volume expansion TGF is usually de-sensitized favoring sodium excretion9. Such re-setting mechanisms are not entirely comprehended but may include the newly explained CTGF. CTGF has an reverse effect to TGF in that in the presence of high distal sodium it tends to dilate the Af-Art. In the kidney a local tubular renin angiotensin system has been explained. Angiotensinogen secreted into the lumen of the proximal tubule can reach the distal nephron10. Renin expressed in the CNT and collecting duct11 can then convert angiotensinogen to angiotensin I. Angiotensin transforming enzyme present in the tubular fluid can generate angiotensin II (Ang II)12. Ang II acts via two different receptors AT1 and AT2 both types are expressed along the nephron13 14 Ang II is present in the tubular lumen where its concentration is higher than and regulated independently from systemic Ang II15. Shao the Na/K/2Cl cotransporter which initiates TGF19. Whether intratubular Ang II can also potentiate CTGF is THY1 not known but AT1 receptor activation can increase Na access the apical amiloride/benzamil-sensitive epithelial Na channels (ENaC) in collecting ducts20 and we have shown that Na access ENaC in the CNT initiates CTGF7. The signaling pathway(s) activated by Ang II in the CNT remains unclear but in other nephron segments Ang II activates the phospholipase C signaling pathway thereby elevating cytosolic Ca2+ and protein kinase C (PKC)21. PKC inhibitors suppress Ang II-induced Na+ transport and fluid reabsorption in proximal tubular cells22 23 In the present study we hypothesize that Ang II in the CNT lumen enhances CTGF via activation of AT1 receptors which require activation of PKC AV-951 and ENaC. To test this hypothesis while avoiding the confounding influence of the multiple systemic factors that regulate the renal microcirculation we simultaneously perfused a microdissected rabbit Af-Art and adherent CNT. Methods New Zealand White rabbits weighing 1.5-2 Kg (Myrtle’s rabbitry TN) were given standard chow AV-951 (Ralston Purina St. Louis MO) and tap water and anesthetized with ketamine (50 mg/kg i.m.) xylazine (10 mg/kg i.m.) and pentobarbital (25 mg/kg i.v.). All protocols were approved by Henry Ford Health System’s Institutional Animal Care and Use Committee and were conducted in accordance with the National Institutes of Health (NIH) Guideline for the Care and Use of Laboratory Animals. We used rabbits because their CNTs are well demarcated and microdissection of the CNT and attached Af-Art is easier than in rats or mice. To isolate and microperfuse the Af-Art and CNT we used methods much like those explained previously7 24 The kidneys were sliced along the corticomedullary axis and slices placed in ice-cold minimum essential medium (MEM; Gibco Laboratories Grand Island NY) made up of 5% bovine serum albumin (BSA; Sigma St. Louis MO). Using fine forceps a single superficial Af-Art with its glomerulus intact was dissected together with the adherent CNT. Using a micropipette the microdissected complex was transferred to a temperature-regulated perfusion chamber mounted on an inverted microscope with Hoffmann modulation. Both the Af-Art and CNT were cannulated with an array of concentric glass pipettes as explained previously25 26 This system allows us to exchange the perfusion answer in a few seconds.