Increased Oxidative Stress In Sickle Cell Disease Activates The Renin-Angiotensin-TGF-β Pathway To Mediate Sickle Nephropathy

Sickle nephropathy (SN) is a common cause of morbidity and mortality in sickle cell disease (SCD), begins with hyposthenuria in childhood, and progresses to albuminuria, focal sclerosing glomerulo-sclerosis (FSGS), glomerular hypofiltration, and end stage renal disease in 30-50% adults The precise molecular mechanisms underlying SN are largely unexplored, as SN has been presumed to result from sickling associated ischemia/necrosis. Herein, we explored mechanisms of sickle renal pathologies utilizing the Berkeley sickle mouse model (HbS mice). We show that HbS mice develop renal pathologies similar to human SN, with hyposthenuria, progressive albuminuria, FSGS and nephron loss. HbS mice 4-8 weeks of age have high GFR compared to WT mice, that rapidly declines to subnormal levels by 16-24 weeks of age. We next explored the role of increased oxidant stress in mediating SN. We recently showed that sickle RBC are likely major contributors of reactive oxygen species (ROS) in SCD, and these high levels of ROS in RBC are also generated enzymatically by NADPH oxidase (George, et al Blood 2013). We now show that SCD-associated ROS initiate pathologically significant processes, including increased conversion of oxidized angiotensinogen (ANG) to angiotensin II (AT), and secondary AT receptor 1 (AT1R)-mediated generation of TGFβ1 in the HbS kidneys, which then phosphorylates Smad 2/3. We tested if activated Renin-Angiotensin-system (RAS) -AT1R-mediated TGFβ1 signaling causes albuminuria and FSGS in HbS mice. We blocked the AT1R with losartan, or its ligand AT by an angiotensin converting enzyme inhibitor, Captopril, starting at an early age (4wk) for 6-12 months. This prevented albuminuria and FSGN development in HbS mice. However, sickle hyposthenuria was worsened with losartan, and was even more severe with captopril. These data suggest that excessive AT1R signaling causes sickle glomerulopathy, and AT1R promotes urine concentrating ability; however, the captopril effect suggests that AT binds another receptor to further mediate urine concentrating ability. Increased RAS signaling is known to mediate glomerulopathy in other diseases, but its role in urine concentration has not been described. AT can also bind AT2 receptor that has been identified as a renoprotective receptor. We therefore investigated the role of AT1R and AT2R in sickle glomerulopathy and hyposthenuria by transplanting bone marrow from HbS mice into WT mice, AT1R-/- mice (HbS/AT1R-/-) and AT2R-/- mice (HbS/AT2R-/-) and followed them for 6-12 months. Bone marrow from WT mice was concurrently transplanted into WT, AT1R and AT2R deficient mice as controls. HbS/WT mice developed similar SN as in HbS mice with progressive albuminuria and hyposthenuria, the former reversible with losartan and captopril, and the latter worsened by these drugs as described above. However, HbS/AT1R mice were protected from development of albuminuria and FSGS, had reduced active TGFβ1 and ^PSmad-2/3, unlike HbS/WT mice, but developed significant hyposthenuria, which was worse than HbS/WT mice, and reminiscent of the effect of losartan. The HbS/AT2R mice also developed significantly worse hyposthenuria than HbS/WT mice, and were additionally not protected from albuminuria. These data suggest both AT1R and AT2R mediate urine concentrating ability, an effect blocked more effectively by captopril than losartan. AT1R signaling is known to activate NADPH oxidase to generate ROS. Indeed, mice placed on Captopril and Losartan had reduced ROS in RBC and platelets (cell types known to express AT1R) and kidneys, and consequently reduced RAS activation (significantly less oxidized ANG and AT), breaking the ROS-RAS-AT1R feedback loop. Significantly higher RBC and platelet ROS, oxidized ANG, and AT levels were also confirmed in patients with SCD as compared to their unaffected sibling controls. In summary, our data show that SN occurs from two distinct mechanisms – a) glomerulopathy that results in albuminuria, glomerulosclerosis and renal failure, which occurs primarily from increased AT1R signaling, and b) a tubulopathy, that results in inability to concentrate urine, and is worsened by AT1R signaling blockade, and AT2R signaling protects tubules against worsening hyposthenuria. Targeted therapies that block AT1R signaling but increase AT2R signaling may improve both glomerular and tubular pathologies in SCD and can now be explored.