CNS sites activated by renal pelvic epithelial sodium channels (ENaCs) in response to hypertonic saline in awake rats

Abstract

In some patients, renal nerve denervation has been reported to be an effective treatment for essential hypertension. Considerable evidence suggests that afferent renal nerves (ARN) and sodium balance play important roles in the development and maintenance of high blood pressure. ARN are sensitive to sodium concentrations in the renal pelvis. To better understand the role of ARN, we infused isotonic or hypertonic NaCl (308 or 500 mOsm) into the left renal pelvis of conscious rats for two 2 hours while recording arterial pressure and heart rate. Subsequently, brain tissue was analyzed for immunohistochemical detection of the protein Fos, a marker for neuronal activation. Fos-immunoreactive neurons were identified in numerous sites in the forebrain and brainstem. These areas included the nucleus tractus solitarius (NTS), the lateral parabrachial nucleus, the paraventricular nucleus of the hypothalamus (PVH) and the supraoptic nucleus (SON). The most effective stimulus was 500 mOsm NaCl. Activation of these sites was attenuated or prevented by administration of benzamil (1 μM) or amiloride (10 μM) into the renal pelvis concomitantly with hypertonic saline. In anesthetized rats, infusion of hypertonic saline but not isotonic saline into the renal pelvis elevated ARN activity and this increase was attenuated by simultaneous infusion of benzamil or amiloride. We propose that renal pelvic epithelial sodium channels (ENaCs) play a role in activation of ARN and, via central visceral afferent circuits, this system modulates fluid volume and peripheral blood pressure. These pathways may contribute to the development of hypertension.

Introduction

Renal denervation has been shown to attenuate or delay the development of experimental hypertension in several animal models (for reviews, see Katholi, 1985, Wyss et al., 1992). Based on these and other observations, clinical trials were undertaken to treat resistant primary hypertension in humans by catheter-based radiofrequency ablation of renal nerves (Krum et al., 2009, Esler and Symplicity HTN-2 Investigators, 2010). Results from the first two clinical studies suggested that renal denervation was successful in reducing arterial pressure for at least three years (Esler et al., 2014, Krum et al., 2014) although a recent study failed to confirm these findings (Bhatt et al., 2014). It has long been posited that elevated central sympathetic drive is a common contributing factor in essential hypertension (Esler and Kaye, 1998, Schlaich et al., 2004, Joyner et al., 2008, Malpas, 2009). Increased muscle sympathetic nerve activity in hypertensive patients has been reported to be reduced by catheter-based renal denervation (Schlaich et al., 2013, Hering et al., 2014). The mechanism by which renal denervation interrupts the development of hypertension or might interfere with central sympathetic outflow has not been elucidated.

Several investigators have suggested that ARN may trigger excess sympathetic activity contributing to the development of hypertension (Katholi, 1985, Ciriello and de Oliveira, 2002, Veelken and Schmieder, 2014). Renal afferent nerves are located predominantly in the renal pelvis with the majority containing CGRP and/or Substance P (Ferguson and Bell, 1988, Marfurt and Echtenkamp, 1991, Kopp, 2015). Renal afferent fibers carry both mechano- and chemoreceptor-sensitive nerves (Dietz and Gilmore, 1991, DiBona and Kopp, 1997, Kopp, 2011). Thus, these fibers are sensitive to various stimuli, including ischemia, as well as changes in ionic composition of urine and hydrostatic pressure within the renal pelvis (Recordati et al., 1980, Kopp et al., 1998). In the rat, chemosensitive neurons have been identified that respond to changes in NaCl concentration, as well as other chemoreceptor stimuli (Recordati et al., 1980, Kopp, 2011). The signaling of these renal afferents has been shown to play an important role in body fluid homeostasis and cardiovascular regulation, as stimulation of these fibers leads to release of vasopressin as well as changes in arterial pressure (Simon et al., 1989).

Renal afferent fibers arise from sensory neurons in the dorsal root ganglia (DRG) of caudal thoracic and rostral lumbar spinal segments (Ciriello and Calaresu, 1983, Knuepfer et al., 1988, Weiss and Chowdhury, 1998). Electrical stimulation of the renal nerve activated c-fos in several sites in the brain including the nucleus of the solitary tract (NTS), the subfornical organ (SFO), the paraventricular nucleus of the hypothalamus (PVH), and the supraoptic nucleus (SON) (Day and Ciriello, 1985, Solano-Flores et al., 1997). Multisynaptic ARN projections to the NTS, PVH, SON and circumventricular organs have also been mapped using the transneuronal tracer pseudorabies virus (Weiss and Chowdhury, 1998, Huang and Weiss, 1999, Huang et al., 2002) but this technique does not discriminate between afferent and efferent projections. These data demonstrate the existence of central renal projections but do not discriminate between specific mechano- or chemoreceptor inputs from the kidney.

Epithelial sodium channels (ENaCs) exist in principal cells in the renal collecting duct and mediate the apical entry of sodium (Rossier, 2014). ENaCs have been identified immunohistochemically in urothelium covering the pelvis and ureters (Hager et al., 2001). ENaCs are important in ion transport and in maintaining osmotic gradients between the tubular fluid and the blood. ENaCs are regulated by aldosterone and also sensitive to a host of other paracrine and autocrine factors (Pearce et al., 2014). Mechanoreceptor transduction in the heart and kidney has been reported to be dependent on activation of ENaCs since afferent nerve activity is prevented by low concentrations of amiloride (Kopp et al., 1998, Ditting et al., 2003).Frelin et al. (1988) determined that ENaCs can be inhibited by lower doses of amiloride or benzamil without interfering with other Na dependent channels. Therefore, ENaCs play a role both in regulating sodium excretion and reabsorption and in pressure sensitive renal afferent pelvic nerves.

In the present study, we identified specific central sites activated in response to renal afferent stimulation by intrarenal pelvic infusion of isotonic or hypertonic sodium chloride solutions. Sites of activation were identified using immunohistochemical staining for the protein Fos, a product of the immediate early proto-oncogene c-fos. We verified activation of renal afferent nerves by recording sensory nerves in isoflurane-anesthetized rats using a similar pelvic infusion procedure. We also determined that ENaCs were responsible for both physiological and immunohistochemical responses to hypertonic saline using benzamil and amiloride. By identifying these central sodium-sensitive projections, we hoped to gain a better understanding of the role of renal afferents in the control of central sympathetic drive and the development of hypertension.