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Research Article
* These authors contributed equally
This study established a novel in vivo beagle model of chronic moderate renal insufficiency combined with atrial fibrillation via transcatheter renal artery embolization and rapid atrial pacing, and demonstrated that this combination severely impaired left atrial endothelial function, enhanced procoagulant activity, and elevated thrombosis risk through inflammatory and RAAS-mediated pathways.
The co-occurrence of atrial fibrillation (AF) and renal insufficiency (RI) represents a clinically complex phenomenon with high prevalence. This study investigated the impact of chronic moderate RI combined with AF on left atrial endothelial function in beagles. Chronic RI was induced by transcatheter embolization of the main branch of the left renal artery using gelatin sponge granules, with animals maintained for 3 months to allow chronic renal parenchymal damage to develop. AF was subsequently induced by continuous rapid atrial pacing (basic cycle length, 60 ms; voltage, 4 times the diastolic pacing threshold; 180 min). Histological changes were assessed using hematoxylin and eosin staining. Parameters associated with endothelial function, coagulation, fibrinolysis, inflammation, and renin–angiotensin–aldosterone system (RAAS) activity in plasma and heart tissues were examined using enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, and western blotting. The results demonstrated successful establishment of a chronic kidney damage model with moderate RI, confirmed by significantly elevated plasma creatinine, urea nitrogen, and reduced creatinine clearance. Left atrial endothelial function was significantly impaired in beagles with combined RI and AF, accompanied by enhanced procoagulant effects and decreased fibrinolysis. Inflammatory markers were elevated following the RI procedure or AF induction and were further enhanced in the combined RI+AF group. Additionally, RAAS activity, evidenced by increased renin and aldosterone levels, was elevated following RI and further augmented upon combination with AF. In summary, this study successfully established an in vivo animal model of chronic moderate RI combined with AF in beagles, demonstrating that this combination significantly disturbed left atrial endothelial function and increased thrombotic risk.
Atrial fibrillation (AF), the most prevalent form of sustained cardiac arrhythmia, is closely linked to unfavorable clinical outcomes. Owing to the systemic pathophysiological characteristics of AF, this arrhythmia frequently engages in intricate pathophysiological crosstalk with a range of concomitant comorbidities, including cognitive decline, depression, and systemic embolism1. Chronic kidney disease (CKD) has been identified as an independent risk factor for both the development of AF and the occurrence of thromboembolic events. Approximately 15% to 20% of patients diagnosed with CKD are affected by AF, while the proportion of patien....
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This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health (Publication No. 85-23, revised 1996). The study protocol was approved by the Institutional Animal Care and Use Committee of Beijing Anzhen Hospital (Approval No. 81700293). The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee of our institution. All efforts were made to minimize animal suffering and reduce the number of animals used in the study. The reagents and the equipment used are listed in the Table of Materials.
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Animal model of chronic moderate RI and AF
A schematic overview of the experimental design and timeline is presented in Figure 1. The representative images of left renal artery angiography before and after transcatheter embolization, shown in Figure 2A,B revealed that the small renal artery branches were successfully occluded following transcatheter embolization, as evidenced by the absence of distal .......
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The present study established an in vivo animal model of moderate RI combined with AF and explored the pathogenesis of left atrial endothelial dysfunction under these pathological conditions. The main findings of this study are as follows: firstly, embolization of the left main renal artery for 3 months in beagles successfully induced moderate RI. Secondly, an AF model was effectively established in beagles. Thirdly, left atrial endothelial function was significantly impaired in animals with both RI and AF, acco.......
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The authors declare no competing interests.
This work was supported by the Noncommunicable Chronic Disease-National Science and Technology Major Project (No. 2024ZD052500, No. 2024ZD0521504), the National Natural Science Foundation of China (Grant No. 81700293 and No. 81970272) and the Beijing Anzhen Hospital High Level Research Funding (Funding Project No. 2025AZB5005).
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 6-0 Polypropylene suture | Ethicon (Johnson & Johnson) | 8706H | For femoral artery repair |
| 5F Multipurpose catheter | Cordis (Cardinal Health) | 521-550S | For renal artery cannulation and embolization |
| 6F Quadripolar electrode catheter | St. Jude Medical | 401443 | For right atrial pacing |
| 6F Vascular introducer sheath | Terumo | RS*A60K10SQ | For femoral artery access |
| ABI 7500 Real-Time PCR System | Applied Biosystems (Thermo Fisher Scientific) | 4351105 | For qRT-PCR thermal cycling and fluorescence detection |
| Absorbable gelatin sponge granules (50 mg diameter) | Jinling Pharmaceutical | — | For transcatheter renal artery embolization; prepared as slurry with contrast medium and saline (1:1) |
| ADMA ELISA Kit (canine) | Cusabio | CSB-E09297c | For plasma asymmetric dimethylarginine measurement |
| Aldosterone ELISA Kit (canine) | Cusabio | CSB-E08014c | For plasma aldosterone measurement |
| BCA Protein Assay Kit | Thermo Fisher Scientific | 23225 | For protein concentration quantification |
| Beagles (male, 4–5 years, 10–12 kg) | Shanghai Jiao Tong University Agricultural Experimental Animal Farm Co., Ltd. | — | Healthy male beagles; housed individually under standard conditions |
| C-arm Fluoroscopy System | GE Healthcare | OEC 9900 Elite | For fluoroscopic guidance during catheterization and angiography |
| Chemiluminescence Imaging System | Bio-Rad | ChemiDoc XRS+ | For Western blot band visualization |
| Creatinine ELISA Kit (canine) | Nanjing Jiancheng Bioengineering Institute | C011-2-1 | For plasma creatinine measurement |
| Cuffed endotracheal tube (ID 7.0–8.0 mm) | Covidien (Medtronic) | 86449 | For endotracheal intubation and mechanical ventilation |
| DF-5A Cardiac Stimulator | Suzhou Dongfang Electronic Technology Research Institute | DF-5A | For rapid atrial pacing; settings: BCL 60 ms, pulse width 2 ms, voltage 4× threshold |
| ECL Western Blotting Substrate | Thermo Fisher Scientific | 32106 | Enhanced chemiluminescence kit for blot visualization |
| EDTA Anticoagulant Tubes | BD Vacutainer | 367835 | For blood collection (plasma separation) |
| eNOS Primary Antibody (rabbit) | Cell Signaling Technology | 32027 | Dilution 1:1000; for Western blotting (~130 kDa) |
| Eosin Y Solution (aqueous) | Solarbio | G1100 | For H&E counterstaining (2 min) |
| Foley Urinary Catheter (8 Fr) | C.R. Bard | 0165SI08 | For timed urine collection during CCr determination |
| GAPDH Primary Antibody (rabbit) | Cell Signaling Technology | 5174 | Dilution 1:1000; internal control for Western blotting (~36 kDa) |
| GraphPad Prism (version 8.0) | GraphPad Software | — | https://www.graphpad.com; for statistical analysis (one-way ANOVA, Dunnett's test) |
| Hematoxylin Solution (Harris) | Solarbio | G1140 | For H&E nuclear staining (5 min) |
| Heparin Sodium Injection (unfractionated) | Changzhou Qianhong Biopharma | H32022088 | 4000 IU diluted in 10 mL normal saline; for anticoagulation during catheterization |
| HRP-conjugated Goat Anti-Mouse IgG Secondary Antibody | Cell Signaling Technology | 7076 | Dilution 1:5000; for Western blotting |
| HRP-conjugated Goat Anti-Rabbit IgG Secondary Antibody | Cell Signaling Technology | 7074 | Dilution 1:5000; for Western blotting |
| hs-CRP ELISA Kit (canine) | Cusabio | CSB-E08557c | For plasma high sensitivity C-reactive protein measurement |
| IL-6 ELISA Kit (canine) | R&D Systems | CA6000 | For plasma interleukin-6 measurement |
| ImageJ Software (version 1.53) | National Institutes of Health | — | https://imagej.nih.gov/ij/; for Western blot densitometric analysis |
| iNOS Primary Antibody (rabbit) | Abcam | ab178945 | Dilution 1:1000; for Western blotting (~130 kDa) |
| Lactated Ringer's Solution | Baxter | 2B2324 | Intravenous fluid; infused at 5–10 mL/kg/h during procedures |
| Light Microscope | Olympus | BX53 | For histological examination of HE-stained kidney sections |
| Mechanical Ventilator (veterinary) | Midmark | Matrx Model 3000 | Tidal volume 15 mL/kg; respiratory rate 12–15 breaths/min |
| Microplate Reader | BioTek | Synergy HTX | For ELISA absorbance measurement at 450 nm |
| Neutral Buffered Paraformaldehyde (10%) | Solarbio | P1110 | For kidney tissue fixation (24 h) |
| NO ELISA Kit (canine) | Nanjing Jiancheng Bioengineering Institute | A012-1-2 | For plasma nitric oxide measurement |
| Non-ionic Iodinated Contrast Medium (Iohexol) | GE Healthcare | Omnipaque 350 | 5–8 mL per injection at 3 mL/s for renal artery angiography |
| PAI-1 ELISA Kit (canine) | Cusabio | CSB-E13043c | For plasma plasminogen activator inhibitor-1 measurement |
| PAI-1 Primary Antibody (rabbit) | Abcam | ab66705 | Dilution 1:1000; for Western blotting (~45 kDa) |
| Paraffin Wax | Leica Biosystems | 39601006 | For tissue embedding |
| PrimeScript RT Reagent Kit | Takara Bio | RR037A | For reverse transcription of RNA to cDNA |
| Protease and Phosphatase Inhibitor Cocktail | Thermo Fisher Scientific | 78440 | Added to RIPA buffer for protein extraction |
| Pulse Oximeter (veterinary) | Nonin Medical | 9847V | For continuous SpO2 monitoring during procedures |
| PVDF Membranes (0.45 μm) | Millipore | IPVH00010 | For protein transfer in Western blotting |
| Renin ELISA Kit (canine) | Cusabio | CSB-E08700c | For plasma renin measurement |
| RIPA Lysis Buffer | Beyotime | P0013B | 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS |
| Rotary Microtome | Leica Biosystems | RM2235 | For cutting 5 μm paraffin sections |
| SDS-PAGE Gel Preparation Kit (12%) | Beyotime | P0012A | For protein electrophoresis separation |
| Serum Separator Tubes | BD Vacutainer | 367986 | For blood collection (serum separation) |
| Sodium Pentobarbital | Sigma-Aldrich | P3761 | Anesthesia: 20 mg/kg IV induction; 150 mg/kg IV for euthanasia |
| Spectrophotometer (NanoDrop) | Thermo Fisher Scientific | ND-2000 | For RNA purity and concentration assessment (A260/A280) |
| SYBR Green PCR Master Mix | Applied Biosystems (Thermo Fisher Scientific) | 4309155 | For quantitative real-time PCR |
| Three-lead ECG Monitor | Philips | IntelliVue MX40 | For continuous heart rate and rhythm monitoring |
| TM (Thrombomodulin) ELISA Kit (canine) | Cusabio | CSB-E12913c | For plasma thrombomodulin measurement |
| TM Primary Antibody (rabbit) | Abcam | ab230010 | Dilution 1:1000; for Western blotting (~60 kDa) |
| TNF-α ELISA Kit (canine) | R&D Systems | CATA00 | For plasma tumor necrosis factor-α measurement |
| t-PA ELISA Kit (canine) | Cusabio | CSB-E08438c | For plasma tissue plasminogen activator measurement |
| t-PA Primary Antibody (rabbit) | Abcam | ab157469 | Dilution 1:1000; for Western blotting (~70 kDa) |
| TRIzol Reagent | Invitrogen (Thermo Fisher Scientific) | 15596026 | For total RNA extraction from LAA tissues |
| Ultrasound System with 3.5 MHz Phased-Array Transducer | Philips | EPIQ 7C | For pre-operative transthoracic echocardiography |
| Urea Nitrogen ELISA Kit (canine) | Nanjing Jiancheng Bioengineering Institute | C013-2-1 | For plasma urea nitrogen measurement |
| vWF ELISA Kit (canine) | Cusabio | CSB-E08437c | For plasma Von Willebrand factor measurement |
| vWF Primary Antibody (rabbit) | Abcam | ab6994 | Dilution 1:1000; for Western blotting (~260 kDa) |