Time Window Of Accelerated Target Organ Damage In Salt Loaded Spontaneously Hypertensive Rats

Objective:Salt intake is one of the key environmental factors that contribute to elevated blood pressure level. In addition to its direct effect on blood pressure level, salt could induce cardiac, brain renal and large artery injury independent of blood pressure level. Spontaneously hypertensive rats (SHR) is the most frequently used hypertensive animal model. However, it is time consuming to induce obvious end organ injury in this model. For example, during the development of elevated blood pressure level, left ventricular (LV) hypertrophy will occur at age of～15 to 20 weeks, and overt cardiac dysfunction will develop till age of～52 to 90 weeks. Therefore, application of salt loading in SHR has been reported in literature to accelerate the process of target organ damage. Meanwhile, the time window of end organ injury using excess salt intake in SHR is not consistent. Thus, the purpose of the current work is to investigate the exact time window of salt loading induced cardiac, renal and aortic injury in SHR, and compared the changes with those of normotensive WKY rats, which would be a premise to for further interventional studies.Methods:Sixty male SHR with age of 8 weeks, were randomly divvied into three groups:0.5%NaCl in chow (normal salt group, NS group),4.0%NaCl (4.0%high salt group, HS4 group) and 8.0%NaCl (8%high salt group, HS8 group).36 male weight and age matched WKY rats consuming above NaCl gradient in chow served as controls. Blood pressure (systolic blood pressure) was measured by tail cuff method every two or four weeks.24 h urine was harvested every two weeks using metabolic cage, to determine total microalbumin by ELISA. Non-invasive LV function was determined by transthoracic echocardiography every four weeks. At age of 20 weeks, invasive hemodynamic analysis by using two pressure catheters (one placed above aortic valve via right carotid artery, another was placed below renal artery in abdominal aorta) was carried out to determine aortic pulse wave velocity (PWV). Then, rats were sacrificed, and cardiac, renal and aortic tissue were harvested. Part of cardiac tissue was prepared for paraffin-embedding and collagen-specific picrosirius red staining. Tissue images were captured by charged couple device, and stored into JPEG format for measurement of collagen volume fraction and perivascular collagen area to luminal area ratio using a professional image analysis software (Image Pro Plus version 4.5, Media Cybernatics, US), to evaluate the extent of fibrosis in cardiac interstitial and perivascular area.Results:(1) Blood pressure changes:all rats exhibited an increasing trend for blood pressure level across time, except for WKY NS group (0.5%NaCl). At the end of follow-up, blood pressure level in SHR were significantly elevated compared those of WKY rats. However, there were no statistical significance between HS4 and HS8 group of SHR, and HS4 and HS8 of WKY rats.(2) All rats of HS4 and HS8 group of SHR, and HS4 and HS8 of WKY rats showed an overt damage in left ventricular structure and function, and there were no statistical significance between HS4 and HS8 group of SHR, and HS4 and HS8 of WKY rats. Moreover, these results showed that the period between 8 weeks and 12 weeks after high salt loading is the key duration when compensated LV remodeling develops to decompensated remodeling. In addition, there was an significant augmentation in cardiac CVF, PVCA/LA and CSA by histopathology analysis, showing increased cardiac fibrosis and myocardium hypertrophy in HS4 and HS8 group of SHR, and HS4 and HS8 of WKY rats.However, there were no statistical significance between HS4 and HS8 group of SHR, and HS4 and HS8 of WKY rats.(3) Urinary protein excretion:all rats showed an increasing trend for protein excretion except for normal salt WKY rats. At age of 20 weeks (12 weeks after intervention), the order of urinary microalbumin from high to low was as the following:SHR HS8>WKY HS8>WKY HS4>SHR HS4>SHR NS>WKY NS, indicating a positive correlation between salt loading and urinary microalbumin value. In addition, baseline value of 24 h urinary microalbumin of SHR was higher than those of WKY rats, indicating subtle renal damage in SHR at age of 8 weeks.(4) PWV:Compared with WKY NS group, PWV of HS4 and HS8 in SHR were significantly elevated [2.00±0.60m/s (SHR HS4 group),1.98±0.51m/s (SHR HS8 group), vs.1.45±0.30m/s (WKY NS group), all P<0.05]. However, there were no statistical significance between HS4 and HS8 group of SHR, and HS4 and HS8 of WKY rats.Conclusion:1. there were no statistical significance in blood pressure, cardiac structure and function and aortic compliance between HS4 and HS8 group of SHR, and HS4 and HS8 of WKY rats.2. The period between 8 weeks and 12 weeks after high salt loading is the key duration when compensated LV remodeling develops to decompensated remodeling.3. There was an significant augmentation in cardiac collagen concentration and aortic stiffness in HS4 and HS8 group of SHR, but not in HS4 and HS8 group of WKY rats.4. There is a positive correlation between salt loading and urinary microalbumin value independent blood pressure level.