Sonja Heinlein

Tonic Postganglionic Sympathetic Inhibition Induced by Afferent Renal Nerves

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 receptor–dependant 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*10−7 M; 10 &mgr;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 65.4±13.0 mV*s (baseline) to 12.8±3.2 mV*s (minimum; P<0.01). 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: 120.3±19.4 mV*s; P<0.01). 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.

Norepinephrine reduces ω-conotoxin-sensitive Ca2+ currents in renal afferent neurons in rats

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.

Complex reinnervation pattern after unilateral renal denervation in rats

Renal denervation (DNX) is a treatment for resistant arterial hypertension. Efferent sympathetic nerves regrow, but reinnervation by renal afferent nerves has only recently been shown in the renal pelvis of rats after unilateral DNX. We examined intrarenal perivascular afferent and sympathetic efferent nerves after unilateral surgical DNX. Tyrosine hydroxylase (TH), CGRP, and smooth muscle actin were identified in kidney sections from 12 Sprague-Dawley rats, to distinguish afferents, efferents, and vasculature. DNX kidneys and nondenervated kidneys were examined 1, 4, and 12 wk after DNX. Tissue levels of CGRP and norepinephrine (NE) were measured with ELISA and mass spectrometry, respectively. DNX decreased TH and CGRP labeling by 90% and 95%, respectively (P < 0.05) within 1 wk. After 12 wk TH and CGRP labeling returned to baseline with a shift toward afferent innervation (P < 0.05). Nondenervated kidneys showed a doubling of both labels within 12 wk (P < 0.05). CGRP content decreased by 72% [3.2 ± 0.3 vs. 0.9 ± 0.2 ng/gkidney; P < 0.05] and NA by 78% [1.1 ± 0.1 vs. 0.2 ± 0.1 pmol/mgkidney; P < 0.05] 1 wk after DNX. After 12 wk, CGRP, but not NE, content in DNX kidneys was fully recovered, with no changes in the nondenervated kidneys. The use of phenol in the DNX procedure did not influence this result. We found morphological reinnervation and transmitter recovery of afferents within 12 wk after DNX. Despite morphological evidence of sympathetic regrowth, NE content did not fully recover. These results suggest a long-term net surplus of afferent influence on the DNX kidney may be contributing to the blood pressure lowering effect of DNX.

MPS 11-09 Renal Afferent Peptidergic Neurons in the Control of the Autonomous Nervous System – What are Primary Stimuli?

Objective: Renal afferent nerve fibers comprise a complicated neuro-paracrine regulatory system influencing the autonomous nervous system. We tested the hypothesis that the activity of renal afferent neuronal units is mainly altered by inflammation in cardiovascualr disease and hypertension. Design and Method: As inflammatory model, normotensive renal inflammation (RI) was induced by i.v. injection of 1.75 mg/kg BW OX-7 antibody to rats, hypertension by unilateral clipping of one renal artery (2k1cl model) in rats 3 weeks prior to experiments. Retrograde labelling (DiI) identified renal afferent neurons among dorsal root ganglion (DRG) neurons (Th11-L2). Patch clamp recordings characterized neurons as highly active or “tonic”, i.e. sustained action potential (AP) firing versus low active or “phasic”, i.e. <5 APs according to their firing response to current injections. Electrophysiological parameters were determined in all neurons investigated, proteinuria and renal morphology assessed for all kidneys. Results: In nephritis, the number of neurons with a tonic response pattern decreased significantly (43,4% vs. 64,8%, p < 0.05) as compared to controls. Interestingly. in renovascular hypertension (2k 1cl model, n = 92) only neurons with projections to the clipped kidney showed a significant decrease in tonic firing pattern 30.6% [23/75], whereas the non-clipped kidney showed an unaffected amount of tonically firing units (67.5% [50/74], p < 0.05). There was no increase in blood pressure (BP) in RI animals. RI rats exhibited albuminuria (61 ± 6 &mgr;g/24 h), interstitial infiltration of macrophages (26 ± 4 cells/high-power field) and glomeruli (3.7 ± 0.6 cells/glomerular cross-section) suggesting active renal inflammation. In contrast, in 2K 1cl animals BP was increased to 180/100 mmHg, but no signs of renal inflammation occurred. Conclusions: Contrasting our hypothesis neither inflammation (nor high blood pressure) may be primarily responsible for the decreased activity pattern of afferent renal neuron in our experiments. Rather decreased perfusion occurring in a clipped kidney with renal stenosis and likely also present in nephritis might be important.

OS 29-03 INCREASED NEUROKININ RELEASE FROM AFFERENT RENAL NERVES IS ACCOMPANIED BY DECREASED AFFERENT ELECTRIC ACTIVITY

Objective: Afferent renal nerves exhibit a dual function. They influence intrarenal immunological processes by release of neurokinins like CGRP and control central sympathetic outflow via afferent electrical activity. The former seems to be important in renal inflammation whereas the sympathetic modulation by afferent electrical activity is not fully understood in cardiovascular disease and hypertension. Hence, we hypothesized that augmented effects of CGRP in renal inflammation occur with increased afferent renal nerve activity. Design and Method: As inflammatory model, normotensive renal inflammation (RI) was induced by i.v. injection of 1.75 mg/kg BW OX-7 antibody to rats to avoid confounding blood pressure effects by choosing a hypertensive model in the first place. Animals were investigated neurophysiologically and pathomorphologically using standard techniques 6 days after RI induction. Results: Blood pressure (BP) was normal, hence confounding BP effects were unlikely. RI rats exhibited albuminuria (61 ± 6 &mgr;g/24 h), infiltration of macrophages in the interstitium (26 ± 4 cells/hp field) and glomeruli (3.7 ± 0.6 cells/glomerular X-section). Pretreatment with the CGRP antagonist rCGRP 8-37 (15 nmol/kg) significantly reduced proteinuria and macrophage infiltration suggesting increased activity of CGRP. In an in-vitro assay, renal RI tissue exhibited increased CGRP release (14 ± 3 pg/ml vs. controls 6.8 ± 2.8 pg/ml; p < 0.05, n = 8). RI tissue from renally denervated rats showed no CGRP. Afferent renal nerve activity (ARNA; spikes/sec) was unexpectedly lower in RI compared to controls (8.0 ± 1.8 vs. 27.4 ± 4.1 Hz, n = 6, p < 0.05). On the other hand, the burst-frequency renal sympathetic nerve activity (RSNA) in RI was significantly higher than in controls (14.7 ± 0.9 vs. 11.5 ± 0.9 bursts/sec; n = 6, p < 0.05). Conclusions: In contrast to our hypothesis increased release of neurogenic CGRP aggravating RI occurred with decreased ARNA. Increased RSNA pointed to a reduced tonic inhibition by ARNA. The latter mechanism might worsen sympathetic dysregulation and renal damage as in hypertension.

Impaired excitability of renal afferent innervation after exposure to the inflammatory chemokine CXCL1

Recently, we showed that renal afferent neurons exhibit a unique firing pattern, i.e., predominantly sustained firing, upon stimulation. Pathological conditions such as renal inflammation likely alter excitability of renal afferent neurons. Here, we tested whether the proinflammatory chemokine CXCL1 alters the firing pattern of renal afferent neurons. Rat dorsal root ganglion neurons (Th11-L2), retrogradely labeled with dicarbocyanine dye, were incubated with CXCL1 (20 h) or vehicle before patch-clamp recording. The firing pattern of neurons was characterized as tonic, i.e., sustained action potential (AP) firing, or phasic, i.e., <5 APs following current injection. Of the labeled renal afferents treated with vehicle, 58.9% exhibited a tonic firing pattern vs. 7.8%, in unlabeled, nonrenal neurons (P < 0.05). However, after exposure to CXCL1, significantly more phasic neurons were found among labeled renal neurons; hence the occurrence of tonic neurons with sustained firing upon electrical stimulation decreased (35.6 vs. 58.9%, P < 0.05). The firing frequency among tonic neurons was not statistically different between control and CXCL1-treated neurons. However, the lower firing frequency of phasic neurons was even further decreased with CXCL1 exposure [control: 1 AP/600 ms (1-2) vs. CXCL1: 1 AP/600 ms (1-1); P < 0.05; median (25th-75th percentile)]. Hence, CXCL1 shifted the firing pattern of renal afferents from a predominantly tonic to a more phasic firing pattern, suggesting that CXCL1 reduced the sensitivity of renal afferent units upon stimulation.

458 DOES A KIDNEY-SPECIFIC EXPRESSION OF VOLTAGE GATED SODIUM CHANNELS LEAD TO AN ORGAN SPECIFIC FIRING ACTIVITY IN SENSORY RENAL INNERVATION

Introduction and Aims: Thermal ablation of renal innervation is effective in the treatment of hypertension. However, the underlying mechanisms are poorly understood. Renal afferent neurons exhibited predominantly sustained (“tonic”) firing throughout current injection, whereas non-renal neurons answered mostly with a “phasic” response. We hypothesized that a kidney specific expression of voltage-gated sodium-channels, which initiate the action potential, is the underlying cause. Methods: Dorsal root ganglion (DRG) neurons with renal afferents (Th11-L2) from Sprague Dawley rats were recorded in current clamp technique and characterized as tonic or phasic. Then, single cells were investigated in voltage-clamp mode. Potassium and calcium channel blockers were added to the external solution, eventually the sodium channel blocker Tetrodotoxin (TTX) to further characterize voltage-gated sodium currents. Results: 66 DRG neurons (n = 40 phasic, n = 26 tonic) were investigated. Tonically firing cells showed significantly a lower firing threshold (-21,75 ± 1,43 mV vs.-29,33 ± 1,63 mV*), a lower overshoot (46,79 mV [38,63 - 54,75] vs. 56,74 mV [53,6–60,96]) and longer action potential duration (4,61 ms[4,15 – 5,85] vs. 3,35 ms[2,12–5,67]).Tonic cells showed a significantly smaller fraction of TTX-sensitive Na+- currents (60,45 ± 12,94pA/pF vs. 140,44 ± 30,88pA/pF). (*p < 0,05). Concluison: Renal afferent neurons exhibited to a significant higher extent a tonic firing pattern upon electrical stimulation. Furthermore this observation was correlated with an altered expression of TTX-sensitive and TTX-resistant channels on neurons with renal afferents, pointing to an organ-specific pattern of innervation. Channel expression and consequently excitability is likely to be altered in e.g. hypertension and needs further investigation