| 1. Background:Cardiorenal Syndrome (CRS) refers to heart/kidney related diseases, that causing acute or chronic dysfunction in heart/kidney. Cardio and renal functions are closely related in many ways, so that having cardio and kidney related diseases at the same time leads to high mortality and makes treatment even harder1,2. According to the 2010 ADKI (Acute Dialysis Quality Initiative) consensus, there are five subtypes in CRS3。The physiopathology for CRS is not clear so far, while research has been focusing on the cardio-renal interaction in recent years. Various CRS have some common characters in pathological mechanism, and usually divided into hemodynamic mechanisms and non-hemodynamic mechanisms4. The key factor in hemodynamic mechanism is the water-sodium retention and overload. While non-hemodynamic mechanisms include Renin-Angiotensin-Aldosterone System (RAAS) and sympathetic nervous system activation, oxidative stress, inflammation, and so forth.The majority of CRS animal model is established with operations on heart and/or kidney, which reflect the acute process. There is a lack of chronic CRS animal model that explains the physiopathology very well. The Deoxycorticosterone Acetate-Salt mice model mimics the salt-senstitive hypertension. In our previous studies, the structural and functional changes in kidney were observed. In this study, we aim to observe the cardio damages and explore the possibilities to use DOCA-Salt mice model as a chronic CRS animal model.Adenosine receptor type 1 (A1AR) is engaged in tubuloglomerular feedbacks and promotes atrial natriuretic peptide (ANP) release in the heart. In the non-hemodynamic mechanisms, A1AR inactivates RAAS by inhibiting renin secretion and regulates inflammation and apoptosis. So far, there are no studies on the role of A1AR in cardiorenal damages in DOCA-Salt mice. In our previous studies, we have found that A1AR protects kidney through hemodynamic mechanisms. We aim to study the function of A1AR in cardio damages thorough hemodynamic and non-hemodynamic mechanisms.2. Objectives:A. Observe heart damage in DOCA-Salt mice and explore the possibilities to use DOCA-Salt mice model as a chronic CRS animal model.B. Explore the role of A1AR in water-sodium retension in DOCA-Salt mice.C. Explore the role of A1AR in cardiac hypertrophy, microvessel density changes, inflammation, and fibrosis in DOCA-Salt mice.3. Methods and Results:A. Heart damage in DOCA-Salt mice Establish DOCA-Salt mice model in C57BL/6J mice by removal of left kidney, implanting deoxycorticosterone acetate long-releasing pills and feeding with high salt diet. Measure blood pressure and heart rate on a weekly-basis using the tail cuff method. Heart tissue slices are stained with HE, Van Gieson, and CD34 and CD68 immunohistochemistry staining. Analysis of Transforming Growth Factor-β1(TGFβ1), Collagenl, Chemokine C-C motif ligand 2 (CCL2) mRNA expression levels are conducted using realtime PCR.In DOCA-Salt mice:1) Heart rate decreased significantly (Week 0 vs. Week 8,685.8 ± 25.4 vs.526.6± 103.9 bpm, P<0.01), mean arterial pressure was significantly increased (Week 0 vs. Week 8,74.6±4.4 vs.88.5 ± 6.5 mmHg, P<0.01).2) Heart weight index increased significantly in week 8 (7.05 ± 0.83 vs.5.16± 0.20mg/g,P=0.007);3) Microvessel density increased significantly in week 4 ([1.01 ± 0.003]% vs. [0.59 ± 0.001]%, P=0.030), microvessel density did not change significantly compared to the control group in week 8;4) The macrophage infiltration and mRNA expression of CCL2 was not different between DOCA-Salt group and control group5) Focal interstitial fibrosis observed in week 4, TGFβ1 and Collagenl mRNA expression didn’t change between DOCA-Salt group and control group.B. Water salt load in DOCA-Salt mice24h Urine specimens were collected by metabolic cages on 0,4th, and 8th week.Cardiac tissue CD73, A1AR, Corin, ANP mRNA expression levels analyzed by realtime PCR. A1AR protein expression levels analyzed by Western Blot.In DOCA-Salt mice:1) 24h urine volume (Week 0 vs. Week 4 vs. Week 8,1173±464 vs.3606±2359 vs. 7635±3017μl, P<0.05) and 24h urinary sodium excretion (Week 0 vs. Week 4 vs. Week 8,133.3±39.5 vs.798.4±719.4 vs.1788±854.0μmol/d, P<0.05) increased significantly, urine osmolality decreased significantly (Week 0 vs. Week4, 1801±642 vs.910±213mOsm/kg H2O,P<0.05; Week 0 vs. Week8,1801±642 vs. 860±233mOsm/kg H2O, P<0.05).2) Corin and ANP mRNA expression in myocardial tissue significantly increased in week 4 (31.9-fold, P=0.005 and 180.7-fold, P=0.030).3) Cardiac tissue CD73 mRNA expression in DOCA-Salt mice increased significantly since week 4 (Week 4:18.79-fold, P=0.001; Week8:8.28-fold, P=0.036)), A1AR mRNA expression increased significantly in week 8 (12.12-fold, P=0.013), A1AR protein level didn’t change much compared to the control group in either week 4 or 8.C. A1AR role in heart damage in DOCA-Salt miceEstablish DOCA-Salt model in A1AR-knockout mice, using the same methods as above.In A1AR-/- DOCA-Salt mice:1) Heart rate decreased (Week 4 vs. Week 0,636±64.2 vs.696.7±35.2, P<0.05), mean arterial pressure did not change significantly.2) 24h urine volume increased significantly compared to wildtype DOCA-Salt group (7003±3742 vs.3606±2359μl, P<0.05).24h urinary sodium excretion increased compared to control group (7003±3742 vs.3606±2359μl, P<0.05). Urine osmolality decreased significantly compared to control group (702±165 vs.1394±397 mOsm/kg, P<0.05). ANP mRNA expression in myocardial tissue increased significantly (12.65-fold, P=0.026).3) Heart weight index and left ventricular wall thickness didn’t change significantly;4) Myocardial microvessel density didn’t increase significantly;5) The macrophage infiltration and mRNA expression of CCL2 was not different between DOCA-Salt group and control group6) Focal interstitial observed for 1 case (out of 6), TGFβ1 and Collagenl mRNA expression didn’t change between DOCA-Salt group and control group.4. Conclusions:In this study,A. DOCA-Salt mice had increased blood pressure and decreased heart rate. In week 4, the microvessel density was increased and focal interstitial fibrosis were observed. In week 8, the heart weight index was increased. With the renal damages we observed in previous studies, the DOCA-Salt mice has concomitant cardio and renal damages and may have the potential to serve as a chronic CRS animal model.B. The blood pressure elevation in DOCA-Salt mice reflects the water-sodium retention which was regulated by several factors:the activation of cardio Corin-ANP pathway as well as CD73-A1AR pathway lead to inhibition of reabsorption of water and sodium by ANP; significant increase in 24h urine volume and 24h urinary sodium excretion; renal CD73-A1AR activation (observed in previous studies) implies the upregulation of tubuloglomerular feedbacks. In A1AR-/- DOCA-Salt mice, there is no tubuloglomerular feedbacks and the increase in 24h urine volume was more prominent. The blood pressure was not elevated. A1AR may exacerbate water-sodium retention in DOCA-Salt mice.C. In A1AR-/- DOCA-Salt mice, there was interstitial fibrosis observed in 1 out of 6 specimens but no early changes in heart weight index and microvessel density. There was no protective role in cardiac hypertrophy, inflammation, and fibrosis in DOCA-Salt mice. |
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