Abstract-Other than efferent sympathetic innervation, the kidney has peptidergic afferent fibers expressing TRPV1 receptors and releasing substance P. We tested the hypothesis that stimulation of afferent renal nerve activity with the TRPV1 agonist capsaicin inhibits efferent renal sympathetic nerve activity tonically by a neurokinin 1 receptordependant mechanism. Anesthetized Sprague-Dawley rats were instrumented as follows: (1) arterial and venous catheters for recording of blood pressure and heart rate and drug administration; (2) left-sided renal arterial catheter for selective intrarenal administration of the TRPV1 agonist capsaicin (3.3, 6.6, 10, 33*10 Ϫ7 M; 10 L; after 15, 30, 45, and 60 minutes, respectively) to stimulate afferent renal nerve activity; (3) right-sided bipolar electrode for continuous renal sympathetic nerve recording; and (4) specialized renal pelvic and renal artery catheters to separate pelvic from intrarenal afferent activity. Before and after intrarenal capsaicin application, increasing intravenous doses of the neurokinin 1 receptor blocker RP67580 were given. Intrarenal capsaicin decreased integrated renal sympathetic activity from 65.4Ϯ13.0 mV*s (baseline) to 12.8Ϯ3.2 mV*s (minimum; PϽ0.01). This sustained renal sympathetic inhibition reached its minimum within 70 minutes and was not directly linked to the transient electric afferent response to be expected with intrarenal capsaicin. Suppressed renal sympathetic activity transiently but completely recovered after intravenous administration of the neurokinin 1 blocker (maximum: 120.3Ϯ19.4 mV*s; PϽ0.01). Intrarenal afferent activity could be unequivocally separated from pelvic afferent activity. For the first time we provide direct evidence that afferent intrarenal nerves provide a tonically acting sympathoinhibitory system, which seems to be rather mediated by neurokinin release acting via neurokinin 1 receptor pathways rather than by electric afferent effects on central sympathetic outflow. Key Words: renal nerve Ⅲ afferent Ⅲ efferent Ⅲ TRPV1 Ⅲ NK 1 -receptor Ⅲ tonic inhibition T he kidney has a very complex sympathetic efferent and peptidergic afferent innervation 1 that recently became of increased interest as renal nerve ablation was introduced into the treatment of severely hypertensive patients. 2 However, especially the role of the afferent renal innervation in hypertension is still far from being fully understood. 3 We know that afferent renal nerve traffic is able to suppress the contralateral renal nerve activity by a sympathodepressory renorenal reflex that is altered in hypertension. 4 So far afferent nerve fibers involved in this reflex were said to be mainly projecting from the renal pelvis to the first neuron in the dorsal root ganglion, 5 although afferent nerve fibers are also found intrarenally in close vicinity to efferent sympathetic nerve fibers. 6 Furthermore, it is very likely that afferent nerve fibers are able to secrete transmitters, specifically substance P (SP) and calcitonin gene-related peptide (CGRP), ...

Sympathetic efferent and peptidergic afferent renal nerves likely influence hypertensive and inflammatory kidney disease. Our recent investigation with confocal microscopy revealed that in the kidney sympathetic nerve endings are colocalized with afferent nerve fibers (Ditting T, Tiegs G, Rodionova K, Reeh PW, Neuhuber W, Freisinger W, Veelken R. Am J Physiol Renal Physiol 297: F1427-F1434, 2009; Veelken R, Vogel EM, Hilgers K, Amman K, Hartner A, Sass G, Neuhuber W, Tiegs G. J Am Soc Nephrol 19: 1371-1378, 2008). However, it is not known whether renal afferent nerves are influenced by sympathetic nerve activity. We tested the hypothesis that norepinephrine (NE) influences voltage-gated Ca(2+) channel currents in cultured renal dorsal root ganglion (DRG) neurons, i.e., the first-order neuron of the renal afferent pathway. DRG neurons (T11-L2) retrogradely labeled from the kidney and subsequently cultured, were investigated by whole-cell patch clamp. Voltage-gated calcium channels (VGCC) were investigated by voltage ramps (-100 to +80 mV, 300 ms, every 20 s). NE and appropriate adrenergic receptor antagonists were administered by microperfusion. NE (20 μM) reduced VGCC-mediated currents by 10.4 ± 3.0% (P < 0.01). This reduction was abolished by the α-adrenoreceptor inhibitor phentolamine and the α(2)-adrenoceptor antagonist yohimbine. The β-adrenoreceptor antagonist propranolol and the α(1)-adrenoceptor antagonist prazosin had no effect. The inhibitory effect of NE was abolished when N-type currents were blocked by ω-conotoxin GVIA, but was unaffected by other specific Ca(2+) channel inhibitors (ω-agatoxin IVA; nimodipine). Confocal microscopy revealed sympathetic innervation of DRGs and confirmed colocalization of afferent and efferent fibers within in the kidney. Hence NE released from intrarenal sympathetic nerve endings, or sympathetic fibers within the DRGs, or even circulating catecholamines, may influence the activity of peptidergic afferent nerve fibers through N-type Ca(2+) channels via an α(2)-adrenoceptor-dependent mechanism. However, the exact site and the functional role of this interaction remains to be elucidated.

Interventional renal ablation is a promising treatment procedure for therapy-resistant arterial hypertension. However, the underlying mechanisms are not completely understood: The efferent sympathetic renal nerves are known to play a key role in salt retention and renin release, but the role of the afferent nerves is not yet clearly defined, although strong evidence exists for a sympathoinhibitory function. It is widely accepted that there is some re-innervation of efferent sympathetic nerves after the denervation procedure, but re-innervation of afferent nerves is still doubted. Hence, we wanted to test the hypothesis that besides a sympathetic re-innervation a considerable afferent re-innervation occurs after renal denervation in rats. 50μm kidney slices from 12 male SD rats were stained for thyrosin hydroxylase (TH), calctonine gene related peptide (CGRP) and smooth muscle actin (SMA). Kidneys were examined 1, 4 and 12 weeks after left sided surgical renal denervation. The right innervated kidney served as control. Image stacks were generated using a confocal laser scanning microscope (0.5μm z-axis steps). Analysis of nerve density was done by 4 blinded investigators in 183 image stacks. Staining for TH (i.e. efferent) and CGRP (i.e. afferent) was visually scored (0-3). Stacks were visualized by Fiji Image J software. At week 1 both efferent [TH+] and afferent [CGRP+] fiber density was clearly reduced but was still detectable ([TH+]: right 2.43±0.10 vs. left 1.47±0.11; [CGRP+]: right 1.96±0.17 vs. left 0.86±0.12; P<0.001, each). After 4 weeks a clear-cut increase in nerve densities could be detected in the denervated kidneys, which further increased until week 12 ([TH+]: right 2.67±0.07 vs. left 2.35±0.12, P<0.03; [CGRP+]: right 2.06±0.16 vs. left 1.82±0.17, P=ns). Our study clearly indicates for the first time that there is not only a relevant sympathetic re-innervation but also a re-innervation of afferent nerves in the kidney. The afferent re-innervation process even seems to be more complete compared to sympathetic nerve fiber re-growth. Further studies have to be done to prove the functional relevance of our findings.