Glomerular Hypertrophy and Alterations in Renin-Angiotensin System Activation Are Associated with Diminished Systemic Blood Pressure in Aging Mice with Sickle Cell Anemia
Authors: Brito Pamela L. , Gotardo Erica M.F. , Chweih Hanan , Ferreira Wilson A. , Leonardo Flavia C. , Franco-Penteado Carla F. , Oliveira Mariana Gonçalves , Antunes Edson , Costa Fernando F. , Conran Nicola 
Published: October 2019

Abstract
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.

Disclosures
No relevant conflicts of interest to declare.