Mechanism Research Of Renal Denervation Suppresses The Progression Of Heart Failure By Targeting Renal Afferent Nerves And Regulating Central Sympathetic Outflow

Backgrounds: Heart failure(HF)has become the worldwide public health problem.Excessive activation of sympathetic nervous system plays a crucial role in the progression and deterioration of HF,and is related to the adverse cardiovascular outcomes of HF.Previous studies have demonstrated that catheter-based renal denervation(RDN)could reduce the renal and whole-body norepinephrine spillover.HF has been proposed as the potential indication of catheter-based RDN technique.Animal experiments have shown that RDN,by damaging the renal efferent sympathetic nerve,decreased renal sympathetic activity,improved renal blood flow and renal vascular resistance,suppressed the overactivation of renin-angiotensin system(RAS),increased sodium and water excretion,and finally improved the progression of HF.However,as an important component of renal nerves,the role of renal sensory afferent nerve in preventional effects of RDN onHF still remains largely unexplored.Objective: To explore the mechanisms of catheter-based RDN suppresses the progression of HF by targeting renal afferent nerves and regulating the central sympathetic outflow in canine,and finally provide direct animal evidences for the application of RDN in clinical practice of HF treatments.Methods: Twenty-four Chinese Kunming dogs were enrolled in this study and randomly assigned into 4 groups: Sham group(n=6),RDN group(n=6),RDN+HF group(n=6),and HF group(n=6).The dogs in RDN group received renal artery angiography and catheter-based RDN.The dogs in Sham group received renal artery angiography.For the dogs in RDN+HF group,besides the renal artery angiography and catheter-based RDN procedures,a high-rate cardiac pacemaker attached a ventricular endocardial pacing electrode were implanted with continuous right ventricular pacing at 250 beats/min for 4 weeks.Following renal artery angiography,the dogs in HF group also simultaneously received continuous right ventricular pacing at 250 beats/min for 4 weeks.All enrolled dogs received transthoracic echocardiographic examinations,measurement of plasma BNP and norepinephrine(NE)concentrations to assess the cardiac structure and function at baseline andreviewed 4 weeks after interventions.After completed the above 4 weeks’ follow-up items,all dogs were euthanized with an over-dose of sodium pentobarbital.The renal artery,kidney,brain tissue,left stellate ganglion,and heart were harvested immediately.Renal arteries were sectioned and stained with hematoxylin-eosin,Masson’s trichrome,and also the polyclonal antibodies to tyrosine hydroxylase(TH)and calcitonin gene related peptide(CGRP)to investigate the damage of RDN on renal efferent sympathetic and afferent sensory nerves.Enzyme linked immunosorbent assay(ELISA)was performed to determine the concentration of NE in plasma,kidney tissue,and also the ventricle.The m RNA and protein expressions of ACE,AT1 R,and ACE2 in hypothalamus were determined by Western blot analysis and RT-PCR,while the concentrations of Ang II and Ang(1-7)in hypothalamus were also detected by ELISA.The expressions of TH in left stellate ganglion were determined by Western blot analysis and imunohistochemical staining.Interventricular septum was sectioned and stained with Masson’s trichrome to assess the influence of RDN on cardiac fibrosis.Results: Left ventricular end-diastolic volume(LVEDV),left ventricular end-systolic volume(LVESV),and plasma BNP level of dogs in RDN+HF group and HF group were significantly increased comparing with that inSham group at 4 weeks follow up after interventions.Moreover,the increasing of LVEDV,LVESV,and plasma BNP in HF group was much more obvious than that in RDN+HF group.Left ventricular ejection fraction(LVEF)of dogs in RDN+HF group and HF group were significantly decreased comparing with that in Sham group at 4 weeks after interventions.The decreasing of LVEF in HF group was much more obvious than that in RDN+HF group.For dogs in Sham group and HF group,the renal nerve bundles were significantly positive for both renal efferent sympathetic nerves and also the renal sensory afferent nerves.However,for the renal nerve bundles damaged by RF energy in RDN group and RDN+HF group,the average optical density,no matter for renal efferent sympathetic nerve or renal sensory afferent nerve,was significantly decreased.Additionally,the level of renal cortical NE in RDN group and RDN+HF group was much lower than that in Sham group,while the renal cortical NE concentrations were higher in HF group than that in Sham group.Tissue Ang II in hypothalamus was significantly increased for the dogs in RDN+HF group and HF group,and decreased in RDN group,comparing with that in Sham group;Tissue Ang(1-7)in hypothalamus was significantly decreased in RDN+HF group and HF group,and increased in RDN group,comparing with that in Sham group.By RT-PCR and Western Blot technique,the m RNA and protein expressions of ACE and AT1 R inhypothalamus of dogs in RDN+HF group and HF group were significantly higher than that in Sham group.However,the m RNA and protein expression level of ACE and AT1 R in hypothalamus of dogs in RDN group were significantly lower than that in Sham group.Comparing with HF group,the application of catheter-based RDN in RDN+HF group significantly attenuated the increase of the m RNA and protein expressions of ACE and AT1 R in hypothalamus which were induced by HF.For the RAS component of ACE2,the m RNA and protein expressions in hypothalamus were down-regulated significantly in RDN+HF group and HF group,and slightly increased in RDN group,in comparison with that in Sham group.The application of RDN in RDN+HF group also significantly attenuated the down-regulation of the m RNA and protein expressions of ACE2 in hypothalamus of the dogs in HF group.Comparing with dogs in Sham group,the immunoreactivity or protein expression level of TH in LSG was significantly increased for the dogs in RDN+HF group and HF group,and decreased for the dogs in RDN group.Application of catheter-based RDN for dogs in RDN+HF group significantly attenuated the elevated expression of TH in LSG of dogs in HF group.Comparing with that in Sham group,the level of plasma NE in RDN group was significantly decreased.However,the plasma NE concentrations in RDN+HF group and HF group were significantly increased,while theincrease of plasma NE in RDN+HF group was partly attenuated by the application of catheter-based RDN technique comparing with that in HF group.Masson’s trichrome staining showed that perivascular cardiac fibrosis was significantly increased in RDN+HF group and HF group compared with the Sham group.The increasing trend was reduced in group of RDN+HF compared with HF group.No significantly difference was observed between RDN group and Sham group.Interstitial cardiac fibrosis showed a trend resembled the perivascular cardiac fibrosis.Additionally,compared to Sham group,the protein expression levels of TGF-β1 in RDN+HF and HF groups were significantly higher,but the HF group had a higher increasing trend than that in RDN+HF group.However,there were no significantly difference between RDN group and Sham group.Conclusions: In conclusion,besides the damage to renal efferent sympathetic nerves,catheter-based RDN technique could also simultaneously target renal afferent sensory nerves,decrease the sympathetic outflow to LSG and ventricle by downregulating the activity of Ang II/ACE/AT1 R axis and upregulating the activity of ACE2/Ang(1-7)axis in hypothalamus,suppress the progression of cardiac fibrosis and sympathetic nerve remodeling,andfinally contribute to attenuate the cardiac dysfunction induced by right ventricular rapid pacing.