Renal Denervation Findings on Cardiac and Renal Fibrosis in Rats with Isoproterenol Induced Cardiomyopathy

Cardio-renal fibrosis plays key roles in heart failure and chronic kidney disease. We sought to determine the effects of renal denervation (RDN) on cardiac and renal fibrosis in rats with isoproterenol induced cardiomyopathy. Sixty male Sprague Dawley rats were randomly assigned to Control (n = 10) and isoproterenol (ISO)-induced cardiomyopathy group (n = 50). At week 5, 31 survival ISO-induced cardiomyopathy rats were randomized to RDN (n = 15) and Sham group (n = 16). Compared with Control group, ejection fraction was decreased, diastolic interventricular septal thickness and left atrial dimension were increased in ISO-induced cardiomyopathy group at 5 week. After 10 weeks, cardio-renal pathophysiologic results demonstrated that the collagen volume fraction of left atrio-ventricular and kidney tissues reduced significantly in RDN group compared with Sham group. Moreover the pro-fibrosis factors (TGF-β1, MMP2 and Collagen I), inflammatory cytokines (CRP and TNF-α), and collagen synthesis biomarkers (PICP, PINP and PIIINP) concentration significantly decreased in RDN group. Compared with Sham group, RDN group showed that release of noradrenaline and aldosterone were reduced, angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/angiotensin II type-1 receptor (AT1R) axis was downregulated. Meanwhile, angiotensin-converting enzyme 2 (ACE2)/angiotensin-1-7 (Ang-(1-7))/mas receptor (Mas-R) axis was upregulated. RDN inhibits cardio-renal fibrogenesis through multiple pathways, including reducing SNS over-activity, rebalancing RAAS axis.

Echocardiography. Cardiac structure and function were evaluated by echocardiography with Vevo2100-a high resolution imaging system (VisualSonics, Canada) with a MS-250, 16.0-21.0 MHZ imaging transducer at baseline, week 5 and 10. All of the rats were anesthetized by aether before the process of echocardiography work-up.
Histopathology. After perfusion with ice-cold PBS, the heart and kidney were cut and fixed in 4% phosphate buffered formalin for 48-72 h at 4 °C, then tissues were dehydrated and embedded in paraffin. Apex cordis was fast frozen by liquid nitrogen, then moved to − 80 °C. Masson's trichrome staining was performed to detect cardiac (including left atrium and ventricular) and kidney (including tubulointerstitial, renal perivascular and renal glomerular) fibrosis. Five fields of each sample were randomly selected and collagen volume fraction (CVF) was assessed by Image-Pro Plus 6.0.
ELISA. Blood samples were obtained by EDTA-tubes, and then centrifuged at 3000 rpm at 4 °C for 10 minutes to separate the plasma which stored at − 80 °C for later use. Plasma B-type natriuretic peptide (BNP), norepinephrine (NE), aldosterone (ALD), Ang II and Ang-(1-7), tumor necrosis factor α (TNF-α ), C-reactive protein (CRP), amino-terminal pro-peptides of types I and III collagen (PINP, PIIINP), and carboxyl-terminal pro-peptide of type I collagen (PICP) levels at week 10 were measured using enzyme linked immunosorbent assay (ELISA) kits, according to the manufacturer's instructions (Uscn Life Science Inc, Wuhan, China).
Statistics. Data are expressed as mean ± SEM and analyzed by SPSS 16.0 (SPSS Inc, Chicago, IL, USA). For two groups comparison, data were analyzed with two-tailed unpaired t tests. For multiple groups comparisons, data were performed using one-way ANOVA with LSD test. P < 0.05 was considered statistically significant.

Discussion
We firstly demonstrated that RDN has comprehensive anti-fibrosis effects on heart and kidneys. The main findings of our study were that: 1) RDN significantly decreased the hyperactivity of SNS and modulated the out-of-balanced RAAS axis, which related to the expression of TGF-β 1 (core mechanism for organ fibrosis) in tissue. 2) RDN lowered valid collagen synthesis biomarkers concentration (PICP, PINP, PIIINP) and reduced cardio-renal CVF. ISO-induced cardiomyopathy is a process from cardiac compensatory hypertrophy to decompensate heart failure, which is accompanied by dysfunction of kidney 18,19 . After continuous injection of ISO for 5 weeks, cardiac dysfunction and myocardial hypertrophy were verified by echocardiography. Meanwhile, LAD was significantly     enlarged. At week10, we observed a further progression of cardiac dysfunction, EF and IVSd significantly decreased and BNP level significantly increased, associated with increased plasma NE levels, fibrogenic factor expression, increased inflammatory cytokines production, out-of-balanced RAAS axis, and development of left atrioventricular and renal fibrosis, and all of these changes were reversed by RDN.
Fibrosis is a common pathway to organ injury and failure 1 . A 3% increase in the CVF of fibrous tissue is associated with a 50% increase in the risk of adverse cardiovascular events 20 . Thus, a need clearly exists to find a way to slow, arrest, or even reverse the progression of tissue fibrogenesis. We and others have shown that RDN could prevent the progressive ventricular fibrosis with accompanying reduction in SNS activity 17,21 . In line with previous findings, we found that RDN significantly reduced the CVF of LV, simultaneously, we also provided evidence that ISO-induced cardiomyopathy caused a fairly striking increase in CVF in the LA and kidney, and that those were prevented by RDN. However, what underlie the beneficial effects by RDN were poorly defined.
Our present study defines some possible mechanisms by which RDN affects cardio-renal fibrosis process. Firstly, RDN inhibits excessive activation of SNS and modulates RAAS axis. Previous studies showed that inhibitors of SNS, such as beta-blockers, could attenuate and reverse remodeling in hypertension, heart failure and myocardial infarction [22][23][24] . As in our prior study, we found that RDN could regulate the expression of β 1,2-receptors, which could contribute to improvements of fibrosis in myocardium 17 . What's more, several studies pointed toward a relevant role of increased sympathetic activation for structural and functional changes in the kidney 25 . In accordance with these studies, RDN clearly reduced NE level down to Control group levels and prevented the progressive LA, LV and kidney damage. The RAAS is classically defined as a coordinated hormonal cascade in the control of multiple organs, such as cardiovascular and renal functions, mainly through the actions of Ang II 26 . Data from prior studies suggest that the inhibitors of ACE/Ang II/AT 1 R axis have beneficial effects on remodeling. Long-term treatment with AT 1 R antagonists could significantly decrease cardiac fibrosis and improve cardiac function 27 . Moreover, combined ACEI and ARB treatments in chronic heart failure could further prevent cardiac fibrosis, compared with ACEI or ARB alone 28 . Ang-(1-7) is mainly formed from Ang II or Ang I by ACE2 29 , which is part of the RAAS, with the counteracting effects against the adverse actions of Ang II. Ang-(1-7) can induce anti-fibrosis, vasodilatation, anti-proliferation and anti-hypertrophy via binding to the Mas-R 30 . The activation of ACE2/Ang-(1-7)/Mas-R axis has an inhibitive effect on pathological collagen synthesis. ACE2 can inhibit pathological hypertrophy, cardiac remodeling, and improve cardiac function 31 . Lakshmanan AP et al. also reported that telmisartan (ARB) attenuated renal fibrosis and oxidative stress through alteration of Mas-R expression 32 . On the molecular level, Ang II and ALD are major stimuli for increasing inflammation cytokines and TGF-β 1, which promote fibroblast proliferation, migration, extracellular matrix remodeling and deposition of pro-collagen 33,34 . While, Ang-(1-7) decrease TGF-β 1 mediated signalling in fibroblasts 35 . A critical finding in prior studies is that Ang-(1-7) axis provide a renoprotective role in the kidney diseases, administration of either an Ang-(1-7) antagonist or an ACE2 inhibitor aggravated the process of CKD 36,37 , while Ang-(1-7) reversed the damage of glomerular in rats 38 . In ISO-induced cardiomyopathy rats, we found that the downregulated ACE2/Ang-(1-7)/Mas-R axis produced a RAAS imbalance leading to abnormal predominance of the ACE/Ang II/AT 1 R axis. Consequently, these rats presented cardio-renal fibrosis and inflammation. Interestingly, RDN downregulated ACE/Ang II/AT 1 R axis and upregulated ACE2/ Ang-(1-7)/Mas-R axis in our study. As we know, ALD can induce cardio-renal injury, inflammation, and fibrosis, long-term ALD exposure aggravates heart and kidney remodeling 33,39 . While, ALD antagonist can significantly attenuate remodeling of heart and kidney 40,41 . After 5 weeks therapy of RDN, the plasma levels of ALD was significantly decreased. Secondly, RDN significantly reduces pro-fibrosis factors and inflammation cytokines production. Abundant experimental evidence supports the notion that TGF-β 1 plays an important role in the pathogenesis of fibrosis 1,34 . Simultaneously, TGF-β 1 can upregulate mRNA level of MMP2 in cardiac fibroblasts, and the elevated protein expression of MMP2 could promote the activation of fibroblasts 42 , further increasing the collagen synthesis. Fumitaka Kuwahara et al. had reported that anti-TGF-β 1 neutralizing antibody can inhibit fibroblast activation and subsequently prevented collagen mRNA induction and myocardial fibrosis 43 . In our study, RDN significantly reduced the expression of TGF-β 1, MMP2 and Collagen I in LV tissue. Inflammatory cytokines, including TNF-α and CRP, are independently associated with increased cardiovascular and renal remodeling 44,45 . It has reported that TNF-α antagonism can attenuate the inflammation and cardiac fibrosis 46 . Trends toward decreased TNF-α and CRP after RDN were observed. Finally, RDN suppressed fiber synthesis. PINP, PIIINP and PICP are specific biomarkers for collagen synthesis 47 . Increased contents of PICP, PINP, and PIIINP in plasma suggest excessive collagen production and are highly associated with repair and fibrosis 48 . In recent experimental studies, RDN could remarkably depress cardiovascular ECM turnover and deposition, reflected by the three biomarkers 47 . In our study, these biomarkers sustained at an over-synthesis state in Sham group, RDN blocked this pathological state.
Hyperactivity of SNS and RAAS are common pathological processes in multiple organ remodeling. For example, overactivity of neurohumor becomes part of the disease process itself resulting in further worsening of cardio-renal function 49,50 . RDN reduce SNS activity, together with normalization of RAAS axis, might underlie the beneficial effects of RDN in regulating homeostasis, further reduce inflammatory cytokines and profibrogenic factors production, and then significantly decrease collagen synthesis.
In conclusions, RDN has the comprehensive suppression effects of cardiac and renal fibrosis in ISO-induced cardiomyopathy. RDN inhibits cardio-renal fibrogenesis through multiple pathways, including reduced SNS over-activity, rebalanced RAAS axis, and suppressed actions of TGF-β 1.