Uncoupling Sickle Hematopoiesis Induced Angiotensin Receptor Signaling in Sickle Nephropathy

Angiotensin II (Ang-II) is a major bioactive peptide of the renin-angiotensin system. We recently reported hyperangiotensinemia in mice and patients with sickle cell disease (SCD), likely mediated by SCD-associated vascular damage (Chang, et al Nat Commun 2015). Herein, we explored whether the hyperangiotensinemia was driven by sickle hematopoiesis and its role in sickle nephropathy. We transplanted bone marrow from Berkeley sickle mice (SS) into C57Bl/6 WT mice (WT) and followed mice fully chimeric for sickle hematopoiesis for 6-9 months. Control WT mice were transplanted with bone marrow from WT mice. We observed that the SS/WT sickle hematopoietic chimeras developed elevated plasma Ang-II levels while the control chimeras did not, suggesting that sickle hematopoietic cells instigate hyperangiotensinemia. We then placed SS/WT chimeric mice or untransplanted Berk-SS mice on captopril, an angiotensin converting enzyme inhibitor (ACE-inhibitor) that would lower plasma Ang-II levels, and as expected Ang-II levels returned to baseline levels found in control mice. Additionally, an improvement in albuminuria and focal segmental glomerulo-necrosis (FSGN) was also noted, implying that sickle glomerulopathy is dependent on sickle hematopoiesis driven hyperangiotensinemia, and blocking this signaling can reverse the glomerular pathology. Similar results were also seen with the use of losartan, an Ang-II receptor 1 blocker (AT₁R blocker) in SS/WT chimeras and untransplanted Berk-SS mice. This finding was confirmed by transplanting Berk-SS bone marrow in AT₁R knockout mice (SS/AT₁R^-/- chimeras). Ang-II-AT₁R signaling resulted in increased Smad1/2 phosphorylation, nitrotyrosine and TGFβ1 production in SS/WT glomeruli, resulting in FSGS that was absent in SS/AT₁R^-/- mice. While AT₁R blockade improved glomerular pathology and albuminuria, the urine concentration defect (hyposthenuria), present in SCD, worsened with AT₁R blockade with losartan. Hyposthenuria was even more severe in captropril treated Berk-SS mice, resulting in high mortality, which was averted only if they received supplemental hydration. Hence, while increased Ang-II signaling caused sickle glomerulopathy, Ang-II protected against severe hyposthenuria instigated by SCD, by signaling through AT₁R and likely through a second receptor, conceivably AT₂R. AT₂R is known to have renoprotective functions, and AT₂R^-/- mice develop worse experimentally induced acute tubular necrosis. We investigated the mechanism of induction of hyposthenuria further using mice deficient in both Ang-II receptors and generating SS/AT₁R^-/- and SS/AT2R^-/- chimeras along with the appropriate controls (WT/AT₁R^-/-, WT/AT₂R^-/-, WT/WT and SS/WT chimeric mice), by transplanting bone marrow from Berk-SS or WT mice into WT, AT₁R^-/- and AT₂R^-/- mice. Dual AT₁R and AT₂R deficiency is embryonic lethal. WT/WT mice had no hyposthenuria, while SS/WT chimeras developed the classic hyposthenuria associated with SCD. In AT₁R chimeras: Both WT/AT1R^-/- and SS/AT₁R^-/- chimeras developed hyposthenuria compared to WT/WT mice, demonstrating that Ang-II-AT₁R signaling helps concentrate urine both in normal and sickle mice. In AT₂R chimeras: WT/AT₂R^-/- chimeras had no hyposthenuria as WT/WT chimeras, suggesting that AT₂R plays little role in concentrating urine in normal situations. However, SS/AT₂R^-/- chimeras developed significantly worse hyposthenuria than SS/WT chimeras, suggesting that under the stress of sickle cell disease, Ang-II-AT₂R signaling improves hyposthenuria caused by SCD. Hence, while increased Ang-II signaling causes glomerulopathy via AT₁R, it improves urine concentration in SCD both via AT₁R and AT₂R signaling. Indeed, a combination of AT₁R inhibition with losartan, and an AT₂R agonism via C21 (kindly provided by Vicore Pharma) improved both albuminuria and hyposthenuria in sickle mice. In summary, we show the molecular basis of sickle nephropathy and demonstrates that sickle hematopoiesis induces pathologic hyperangiotensinemia; increased Ang-II-AT₁R signaling leads to glomerulopathy and end stage renal disease, despite its protective role in urine concentration; Ang-II-AT2R signaling improves hyposthenuria. Therefore, clinically, reduced AT₁R signaling can improve sickle nephropathy, and dual AT₁R blockade with AT₂R agonism can be explored as a preferred therapeutic target.