ReviewCNS sites activated by renal pelvic epithelial sodium channels (ENaCs) in response to hypertonic saline in awake rats
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.
Section snippets
Surgical preparation
Male Sprague-Dawley rats (n = 56, 220–300 g BW, Harlan Laboratories, Indianapolis, IN) were housed in individual cages on a 12–12 hour light-dark cycle. All recovery surgical procedures were performed using aseptic technique. All wounds were sutured and treated with Bactroban cream (GlaxoSmithKline, Research Triangle Park, NC) and with antibiotic therapy using enrofloxacin (Baytril, 10 mg/kg, sc, Bayer Corp, Pittsburgh, PA) and analgesic/anti-inflammatory agent carprofen (Rimadyl, 20 mg/kg, ip,
Anatomical mapping
Arterial pressure and heart rate were recorded during infusion of various concentrations of sodium chloride into the renal pelvis. There was no significant change in arterial pressure or heart rate in response to any of the stimuli (Table 1). We excluded eight rats with behavioral responses suggesting that intrapelvic pressure was elevated or with measured increases of intrapelvic pressure > 10 mm Hg and four rats due to inadequate perfusion. Results from the remaining 44 rats are described below.
Discussion
We provided evidence that specific central sites are activated by ARN stimulation in response to NaCl infusion, and thus identified structures that integrate sensory information from the kidneys. Two concentrations of NaCl (500 and 308 mOsm) were used under the assumption that only hypertonic NaCl would activate renal sodium sensitive receptors and thus cause increased ARN activity. However, there were gradations of activation within some sites activated that had some Fos after normal saline.
Acknowledgements
The authors thank Laura A. Willingham, Nicole Hoffman-Schepers and Xay Van Nguyen for their technical assistance and Dr. Rebecca L. Miller for assistance with the microscope and imaging programs. Funding for this work was provided by USPHS DA-017371 (MMK) and by St. Louis University.
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