Luminal Flow in the Connecting Tubule induces Afferent Arteriole Vasodilation

Abstract

Background: Renal autoregulatory mechanisms modulate renal blood flow. Connecting tubule glomerular feedback (CNTGF) is a vasodilator mechanism in the connecting tubule (CNT), triggered paracrinally when high sodium levels are detected via the epithelial sodium channel (ENaC). The primary activation factor of CNTGF-whether NaCl concentration, independent luminal flow, or the combined total sodium delivery-is still unclear. We hypothesized that increasing luminal flow in the CNT induces CNTGF via O2^- generation and ENaC activation.

Methods: Rabbit afferent arterioles (Af-Arts) with adjacent CNTs were microperfused ex-vivo with variable flow rates and sodium concentrations ranging from <1 mM to 80 mM and from 5 to 40 nL/min flow rates.

Results: Perfusion of the CNT with 5 mM NaCl and increasing flow rates from 5 to 10, 20, and 40 nL/min caused a flow rate-dependent dilation of the Af-Art (p<0.001). Adding the ENaC blocker benzamil inhibited flow-induced Af-Art dilation, indicating a CNTGF response. In contrast, perfusion of the CNT with <1 mM NaCl did not result in flow-induced CNTGF vasodilation (p>0.05). Multiple linear regression modeling (R²=0.51;p<0.001) demonstrated that tubular flow (β=0.163 ± 0.04;p<0.001) and sodium concentration (β=0.14 ± 0.03;p<0.001) are independent variables that induce afferent arteriole vasodilation. Tempol reduced flow-induced CNTGF, and L-NAME did not influence this effect.

Conclusion: Increased luminal flow in the CNT induces CNTGF activation via ENaC, partially due to flow-stimulated O2- production and independent of nitric oxide synthase (NOS) activity.

Keywords: Afferent Arterioles; Connecting Tubule Glomerular Feedback (CNTGF); Distal Sodium Delivery; Epithelial Sodium Channel (ENaC); Renal Blood Flow; Superoxide (O2−).

Figure 1.. Kidney tubule microperfusion

Male New Zealand white rabbits were anesthetized, and their kidneys were removed and sectioned along the corticomedullary axis. Superficial afferent arterioles (Af-Art) with attached connecting tubules (CNT) were microdissected under a stereomicroscope. The isolated complexes were transferred to a temperature-controlled perfusion chamber and viewed using an inverted microscope. Both the Af-Art and CNT were cannulated and perfused with glass pipettes. The CNT was perfused with variable NaCl concentrations and flow rates. Af-Art diameter was measured at three points, averaged, and recorded at 5-second intervals. Each protocol involved two consecutive dose-response curves. Measurements were analyzed using Metavue software.

Figure 2.. Luminal flow in the connecting tubule induces afferent arteriole dilation.

The connecting tubule (CNT) was perfused with 5 mM NaCl. Luminal flow rates of 10, 20, and 40 nL/min induced significant vasodilation of preconstricted afferent arteriole (Af-Art). Figure 2A shows no difference between the first (○) and second (•) dose-response curves (n = 7), indicating the reproducibility of vasodilation and independence from time (time control). Figure 2B demonstrates the effect of benzamil in the CNT on flow-induced Af-Art dilation. Increasing the CNT flow rate significantly dilated the Af-Art (○), but 0.1 μM benzamil (•) in the CNT completely inhibited this effect (n = 6), indicating the participation of the ENaC-dependent connecting tubule glomerular feedback. ***p < 0.001, benzamil vs. control. NE = norepinephrine; CNT = connecting tubule; Af-Art= Afferent arteriole.

Figure 3.. Effect of luminal flow on NaCl-induced vasodilation.

When the CNT was perfused with 5 mM NaCl, increasing the flow rate significantly dilated the afferent arteriole (Af-Art) (•). However, no dilation occurred when the CNT was perfused with 0 mM NaCl (○) (n = 6), indicating the dependence of sodium for the flow-induced vasodilation (Figure 3A). ***p < 0.001, 0 mM NaCl vs. 5 mM NaCl. Figure 3B plots Af-Art diameter against sodium delivery (NaV) at two luminal flow rates. The 40 nL/min slope was steeper than at 20 nL/min, indicating an independent effect of luminal flow, adjusted for NaV, on Af-Art diameter. NE = norepinephrine; CNT = connecting tubule; Af-Art= Afferent arteriole.

Figure 4.. Effect of O2− on flow-induced CNTGF.

Increasing the CNT luminal flow rate significantly dilated the Af-Art (○), but the addition of 100 μM tempol (•) in the CNT significantly attenuated this response (Figure 4A, n = 6, *p < 0.05; ***p < 0.001, tempol vs. control). Figure 4B shows that adding 100 μM L-NAME to the tempol infusion further attenuated vasodilation (•) compared to L-NAME alone, suggesting that the effect of O2^⁻ is independent of its action on NO (n = 6). ***p < 0.001, L-NAME vs. L-NAME + tempol. NE = norepinephrine; CNT = connecting tubule. NE = norepinephrine; CNT = connecting tubule; Af-Art= Afferent arteriole.