Renal Nerve Stimulation Identifies Autonomic Innervation Of Kidney During Renal Denervation In Canine

Background:Hypertension remains a global public health problem and a major cardiovascular risk factor,accounting for increased morbidity and mortality.Despite significant advances in lifestyle modifications and antihypertensive medications,a large number of patients fail to achieve recommended blood pressure(BP)goals.The pathophysiological role of the sympathetic nervous system in the pathogenesis and progression of hypertension is well established.In the last decade,Catheter-based renal denervation(RDN)followed the principle of regulating sympathetic activity by disrupting renal nerve fibers located in the adventitia of renal arteries,became available as a treatment option for hypertension.Although RDN was once controversial due to conflicting efficacy results from the SYMPLICITY trials,the latest results of a modest but consistent antihypertensive effect shown by SPYRAL HTN trials and RADIANCE HTN trials are encouraging and bring additional light to this situation.Even with the large number of ablations(43.8/per patient in SPYRAL OFF-Med study),more than 20% of patients were observed as either non-responders or had elevated BP.How to verify optimal ablation sites,assess successful renal nerve denervation during the procedure and identify responsive hypertensive populations are urgently needed.RNS may provide a meaningful method for identifying renal autonomic innervations and guiding RDN.The physiological responses of renal nerve fibers to RNS were intricate: stimulations could lead to not only increase and no changes in BP,but also decrease in a few cases.The recorded BP responses may depend on the integrated response of efferent sympathetic,afferent sensory and parasympathetic fibers.Objective:To assess the correlation between BP responses to RNS and renal autonomic innervations.To provide the evidence that RNS is an effective method to identify the sympathetic nerve-enriched area during RDN procedure and to improve the efficacy of RDN.Methods:The experiment was divided into two parts:Part one: Anatomically eligible dogs were randomly assigned into 3 groups: strong-response sites ablation(SRA)group,weak-response sites ablation(WRA)group,and RNS-control group(RSC).The plasma norepinephrine was assayed by Enzyme-Linked Immunosorbent Assay(ELISA)kits.The local norepinephrine in kidney was assayed by high-performance liquid chromatography(HPLC).The expression of tyrosine hydroxylase(TH)in kidney was measured by Western Blot.The distance from the luminal surface of the renal arteries to each nerve,the area and number of the nerves were measured with Image-Pro Plus 6.0.Part two: The radiofrequency current was delivered at 2 watts for 10 seconds,2 watts for 20 seconds,2 watts for 30 seconds,3 watts for 20 seconds,3 watts for 30 seconds and 4 watts for 30 seconds in each renal artery to determine the appropriate ablation parameters for low-energy radiofrequency ablation.Then,available renal arteries were randomly assigned into 3 groups: elevated response(ER)group,reduced response(RR)group,and non-response(NR)group.Tyrosine hydroxylase(TH)was used to label efferent nerves.In addition,calcitonin gene related peptide(CGRP)and neuronal nitric oxide synthase(nNOS)were used for the recognition of afferent nerves and parasympathetic nerves.Results:Part one: ablation at strong-response sites(SRS)showed a superior systolic BP-lowering effect than at weak-response sites(WRS)(P=0.002),as well as lower levels of tyrosine hydroxylase(TH)and norepinephrine(NE)in kidney and a greater reduction in plasma NE(P=0.004 for TH,P=0.002 for both renal and plasma NE).SRS showed a greater total area and mean number of renal nerves than WRS(P=0.012 for total area and P<0.001 for mean number).Systolic BP-elevation response to RNS before RDN and blunted systolic BP-elevation to RNS after RDN were correlated with systolic BP changes at 4 weeks follow up(R=0.649;P=0.012 and R=0.643;P=0.013).Changes of plasma NE and renal NE levels at 4 weeks were also correlated with systolic BP changes at 4 weeks(R=0.837,P<0.001 and R=0.927,P<0.001).Part two: Low-energy radiofrequency ablation with 3 Watts,20 s and 3 Watts,30 s were the appropriate ablation parameters(LERA,defined as inducing injury of the arterial wall without damaging nerve fibers).No statistical difference was found in the proportion of efferent sympathetic nerve among ERS,RRS and NRS(P=0.251).The proportion of sensory afferent nerve was higher at ERS than that at RRS and NRS(P=0.012 和P=0.004).There was no difference in the proportion of sensory afferent nerve between RRS and NRS(P=0.798).The proportion of parasympathetic nerve at RRS was the highest(RRS vs.ERS,P=0.017;RRS vs.NRS,P=0.023),and no statistical difference was found between ERS and NRS(P=0.723).Conclusion:The main findings were as follows: 1.Ablation at SRS showed a superior BP-lowering effect than at WRS,as well as a lower level of TH and NE in kidney and a greater reduction in plasma NE.2.SRS showed a greater total area and mean number of renal nerves than WRS.3.SBP-elevation response to RNS before RDN and blunted SBP-elevation to RNS after RDN were correlated with the SBP changes at 4 weeks follow up.4.The proportion of sensory afferent nerve was higher at ERS and the proportion of parasympathetic nerve at RRS was the highest.In summary,renal nerve stimulation could identify autonomic innervation of kidney during renal denervation.RNS is an effective method to identify the sympathetic nerve-enriched area during RDN procedure and to improve the efficacy of RDN.