Glomerular Hypertrophy and Alterations in Renin-Angiotensin System Activation Are Associated with Diminished Systemic Blood Pressure in Aging Mice with Sickle Cell Anemia
Glomerular Hypertrophy and Alterations in Renin-Angiotensin System Activation Are Associated with Diminished Systemic Blood Pressure in Aging Mice with Sickle Cell Anemia
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肾小球肥大和肾素-血管紧张素系统激活的改变与镰状细胞性贫血老年小鼠的全身血压降低有关
作者：
P. L. Brito;É. Gotardo;H. Chweih;W. A. Ferreira;F. C. Leonardo;C. Franco;Mariana Gonçalves Oliveira;E. Antunes;F. Costa;N. Conran
镰状细胞肾病是镰状细胞性贫血（SCA）的主要且严重的临床并发症。肾髓质的缺氧微环境导致高渗透压和酸中毒，这有利于脱氧血红蛋白 S (HbS) 聚合和随后的红细胞镰状化，促进器官中的血管闭塞过程。肾缺血和肾小球血流可以调节血管紧张素 II (AngII) 的产生，血管紧张素 II 是肾素-血管紧张素系统 (RAS) 的血管收缩激素衍生物，可调节血压 (BP) 等功能。考虑到 SCA 患者的血容量和心输出量升高，血压可能会升高；然而，SCA 患者通常表现出较低的 PB（Pikilidou 等人，J Clin Hypertens.，2015），并且随着年龄的增长而改变。先前已有报道称患有 SCA 的雄性小鼠血浆 AngII 降低（Dos Santos 等人，Life Sci.，2014），因此我们旨在研究 SCA 动物模型中 PB 和 RAS 的变化与衰老和肾脏改变之间的关系。无SCA的雄性嵌合小鼠(QCON)和有SCA的雄性嵌合小鼠(QSCA)是通过对8周龄的C57BL6小鼠进行照射，然后分别从C57BL6或转基因Berkeley SCA小鼠进行骨髓细胞移植而产生的。由于循环和组织 RAS 明显受到雌激素的影响，因此我们在所有方案中仅使用雄性小鼠。动物被细分为青壮年（5个月大）和成年（8-9个月大）。确认移植的SCA小鼠的表型后，通过无创方法（NBP-AdInstruments）测量血压，并在5/9个月时处死动物以收集生物材料。通过 ELISA 对血浆 AngII 和血管紧张素转化酶 (ACE) 进行定量。为了对肾脏进行组织学分析，将器官标本固定并包埋在组织树脂中，切片并用苏木精和伊红染色。通过蛋白质印迹法对速冻肾脏的肾 AngI/II 进行定量。 5月龄和8月龄时QSCA的平均血压（分别为87.9±1.9mmHg，n=12；和94.31±4.4mmHg，n=2；）显着低于同龄QCON的平均血压（分别为108.9±5mmHg，n=10；和110.6±2.2mmHg，n=9），表明该模型模拟了SCA 患者发生的血流病理变化。鉴于 AngII 和 PB 调节之间的密切关系，我们研究了这些年龄段的 QSCA 小鼠中 RAS 是否发生改变。与 QCON 动物（青年，38.77 ± 9，n = 10；成年，35.52 ± 5.3 pg/ml，n = 10）相比，青年和成年雄性 QSCA 小鼠均表现出较低的血浆 AngII 浓度（青年，24.18 ± 3 pg/ml，n = 11；成年，16.44 ± 2 pg/ml，n = 4）。与 QSCA 相比，P<0.05）；然而，没有观察到血压降低和血浆 AngII 降低之间存在显着相关性。 AngII是由ACE裂解AngI产生的；与年轻成年 QCON 小鼠（21.9 ± 4.9 pg/ml，n=9）相比，年轻成年 QSCA 小鼠的血浆 ACE 浓度也降低（12.5 ± 1.8 pg/ml，n=11）。此外，年轻成人/成人 QSCA 中 ACE 浓度降低与 AngII 血浆水平相关（rs=0.785，P<0.001，n=15）。研究结果表明，SCA 小鼠器官中的 AngII 生成可能受到损害；事实上，肾内 AngI/II 蛋白表达分析表明，与成年 QCON 小鼠 (1.6 ± 0.2 AU) 相比，成年 QSCA 小鼠肾 AngI/II 表达显着降低 (0.71 ± 0.2 AU，标准化为 β-肌动蛋白)，这表明在高龄 SCA 中这种激素的产生量较低。心血管和肾脏疾病模型（包括高血压和 SCA）中肾小球肥大增加。定量形态分析显示，与年轻成年和成年 QCON 小鼠 (324.1 ± 10.8 µm2；332.8 ± 12.4) 相比，QSCA 小鼠的肾小球毛细血管凝灰岩面积扩大，并随着年龄的增长而增加 (年轻成年小鼠：372.2 ± 12.8 µm2；成年：412.9 ±12.1 µm2，n=3) µm2，n=3，p<0.001）。因此，肾小球肥大可能反映了 SCA 小鼠的过度滤过和肾损伤，这种情况显然随着年龄的增长而加剧。随后肾小球压力的变化可能会引起 RAS 的适应性反应，进而减少 ACE 和 AngII 的产生，并与全身血压的变化相关。了解 SCA 中的 RAS 可能会提高我们对 SCA 中肾损伤及其系统性后果的理解，并且对于确定如何在这些患者中最好地利用 ACE 抑制剂和 AngII 受体阻滞剂药物非常重要。
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Sickle cell nephropathy is a major and severe clinical complication of sickle cell anemia (SCA). The hypoxic microenvironment of the renal medulla leads to hyperosmolarity and acidosis, which favors deoxygenated hemoglobin S (HbS) polymerization and subsequent sickling of erythrocytes, promoting vaso-occlusive processes in the organ. Renal ischemia and glomerular blood flow can modulate angiotensin II (AngII) production, a vasoconstrictor hormone derivative of the Renin-Angiotensin System (RAS), which regulates blood pressure (BP), among other functions. Considering the elevated blood volume and cardiac output in individuals with SCA, an increase in BP might be expected; however SCA patients often present lower PB (Pikilidou et al., J Clin Hypertens., 2015), which modifies with aging. Reduced plasma AngII in male mice with SCA has been reported previously (Dos Santos et al., Life Sci., 2014), thus we aimed to investigate changes in PB and RAS, in association with aging and renal alterations in an animal model of SCA. Male chimeric mice without SCA (QCON) and male chimeric mice with SCA (QSCA) were generated by the irradiation of 8-week old C57BL6 mice followed by bone marrow cell transplantation from C57BL6 or transgenic Berkeley SCA mice, respectively. As circulating and tissue RAS are markedly affected by estrogens, we used only male mice for all protocols. Animals were subdivided into young adults (5 months of age) and adults (8 - 9 months of age). After confirming the phenotype of the transplanted SCA mice, BP was measured by a noninvasive method (NBP - AdInstruments) and animals were sacrificed at the age of 5/9 months for biological material collection. Plasma AngII and Angiotensin Convertor Enzyme (ACE) were quantified by ELISA. For histological analysis of the kidneys, organ specimens were fixed and embedded in historesin, sectioned and stained with hematoxylin and eosin. Renal AngI/II was quantitated by Western Blot from snap-frozen kidneys. The mean BPs of QSCA at 5 and 8 months old were significantly lower (87.9 ± 1.9 mmHg, n=12; and 94.31 ± 4.4 mmHg, n=2; respectively) than those of QCON of the same age (108.9 ± 5 mmHg, n=10; and 110.6 ± 2.2 mmHg, n=9; respectively), indicating that this model mimics the pathological changes in the blood flow that occur in SCA patients. Given the close relationship between AngII and PB regulation, we investigated whether the RAS is altered in QSCA mice at these ages. Both young adult and adult male QSCA mice presented lower plasma AngII concentrations (young adult, 24.18 ± 3 pg/ml, n= 11; adult, 16.44 ± 2 pg/ml, n=4), compared to QCON animals (young adult, 38.77 ±9, n= 10; adult, 35.52 ± 5.3 pg/ml, n=10, P<0.05, compared to QSCA); however no significant correlation between reduced BP and reduced plasma AngII was observed. AngII is produced by the cleavage of AngI by ACE; plasma ACE concentrations were also reduced in young adult QSCA mice (12.5 ± 1.8 pg/ml, n=11), compared to young adult QCON mice (21.9 ± 4.9 pg/ml, n=9). Moreover, reduced ACE concentrations in young adult/adult QSCA correlated with plasma levels of AngII (rs=0.785, P<0.001, n=15). Findings imply that AngII generation could be compromised in the organs of SCA mice; indeed, intra-renal AngI/II protein expression analysis showed that adult QSCA mice presented a significantly reduced expression of renal AngI/II (0.71 ± 0.2 AU, normalized to β-actin) compared to adult QCON mice (1.6 ± 0.2 AU), suggesting a low production of this hormone at advanced age in SCA. Glomerular hypertrophy increases in cardiovascular and renal disease models, including in hypertension and SCA. Quantitative morphometric analyses showed expansion of the glomerular-capillary tuff area of QSCA mice, which increased with age (young-adult: 372.2 ± 12.8 µm2; adult: 412.9 ±12.1 µm2, n=3), compared to young-adult and adult QCON mice (324.1 ± 10.8 µm2; 332.8 ± 12.4 µm2, n=3, p<0.001). As such, glomerular hypertrophy may reflect hyperfiltration and renal damage in the SCA mice, which apparently augments with age. Subsequent alterations in glomerular pressure may incur adaptive responses in the RAS, in turn diminishing ACE and AngII production, in association with alterations in systemic blood pressure. An understanding of the RAS in SCA may improve our understanding of renal damage and its systemic consequences in SCA and will be important for determining how ACE inhibitor and AngII receptor blockers drugs may be best utilized in these patients.
No relevant conflicts of interest to declare.