Identification of Renal Pelvic Sodium Channel Input to the Brain Stem and Hypothalamus in the Rat

Abstract

There is considerable evidence that elevated central sympathetic nerve activity contributes to the development of essential hypertension. It has been suggested that renal afferent nerves facilitate the increase in central sympathetic drive. Renal afferent projections to the CNS have been mapped using tract tracing techniques (e.g., pseudorabies virus) and electrical stimulation (c‐fos activation) demonstrating a number of regions that receive renal sensory input but not discriminating between chemo and mechanoreceptor afferents. Since renal sensory neurons are highly concentrated in the renal pelvis, we sought to identify the central projections of renal pelvic afferent nerves sensitive to hypertonic saline. We hypothesized that hypertonic saline would activate renal pelvic epithelial sodium channel (ENaC) projections to key autonomic areas of the CNS involved in blood pressure control.We implanted a triple lumen cannula into the left renal pelvis of rats and catheterized the femoral artery to measure arterial pressure and heart rate. After recovery, the left kidney continued to function voiding urine. Five days later, the renal pelvis was flushed in a push‐pull manner (40–60 μl/min) while recording renal pelvic pressure (third pelvic cannula) to avoid mechanoreceptor activation. We used either normotonic saline (308 mOsm/L), hypertonic saline (500 mOsm/L) or a mixture of hypertonic saline and amiloride (10 μM). We compared these results to instrumented sham rats without any infusion. After 2 hours, rats were deeply anesthetized and perfused transcardially with saline then 4% paraformaldehyde. Brains were removed and sectioned (50 μm) and stained immunohistochemically for c‐fos, a marker of increased neuronal activity. We observed labeled neurons within several regions of the CNS using ImageJ software. A number of regions known to regulate visceral input and/or sympathetic outflow in the brain were activated including the parabrachial nucleus (PBN), paraventricular hypothalamus (PVH), and nucleus tractus solitarius (NTS). The most consistent activation was observed in the PBN el‐inner (151 cells/mm2), parvocellular PVH (113 cells/mm2), and NTS (89.3 cells/mm2) compared to normal saline infusion (80.5, 58.4, and 61.4 cells/mm2 respectively) and sham treatment (34.8, 62.1, and 29.9 cells/mm2 respectively). The activation with hypertonic saline was attenuated by amiloride (16.1, 93.2, and 35.0 cells/mm2 respectively). Therefore, the results suggest that hypertonic saline activates c‐fos in the PBN el‐inner, parvocellular PVH, and NTS, in addition to other areas. Since the activation could be antagonized with an ENaC antagonist in the renal pelvis, we propose that the afferents have ENaC. We have identified specific renal afferent projections and developed a technique for determining selective projections from the renal pelvis. It remains to be determined whether these ENaC and specific renal sensory nerves are responsible for facilitating the development of hypertension.Support or Funding InformationSupported by St. Louis University and Washington University