Targeting hemolysis and raas: Osivelotor and losartan ameliorate sickle cell renal pathology

Abstract In sickle cell disease (SCD), progressive renal dysfunction is common and driven by mechanisms such as chronic hemolysis, vascular damage, and ischemia. Studies suggest that Losartan, an FDA-approved antihypertensive targeting the renin-angiotensin-aldosterone system (RAAS), may offer renoprotective benefits in SCD. Osivelotor (GBT021601), a second-generation inhibitor of sickle hemoglobin (Hb) polymerization, shares a mechanism with voxelotor, but has improved pharmacokinetics. By reducing hemolysis and circulating cell-free Hb, Osivelotor may decrease Hb-induced renal toxicity.We hypothesized that combined treatment with Osivelotor and Losartan, would improve kidney function and histopathology in SCD mice. Transgenic sickle mice (Hb SS; Townes model, Jackson Laboratory) were treated starting at 12 weeks of age for 14 weeks with either control chow (C), Osivelotor (O), Losartan (L), or Osivelotor + Losartan (O+L). Osivelotor (300mg/kg/day) was milled in standard rodent diet and Losartan (0.2mg/mL) was dissolved in drinking water. Each group included 12 mice (6 males, 6 females). Blood, urine and kidney tissues were collected at 26 weeks. We assessed markers of hemolysis (Hb, reticulocyte %, hemoglobinuria), reactive oxygen species (thiobarbituric acid reactive substances [TBARS]), kidney injury (kidney injury molecule-1 [KIM-1], nephrin), and kidney function (albuminuria, serum cystatin C and creatinine). Serum erythropoietin was normalized to hematocrit (HCT). Histopathology was assessed using picrosirius red staining. Group comparisons were analyzed by ANOVA adjusted for sex. Data are shown as mean ± standard error.SCD mice treated with Osivelotor had significantly higher Hb concentrations (O: 12.2±0.4 g/dL, O+L: 8.5±0.5g/dL) compared to control (C: 6.8±0.3g/dL) or Losartan-only (L: 4.8±0.3g/dL) treated SCD mice (P<0.001). Hemolytic biomarkers, including absolute reticulocyte count (C: 3599±110, O: 2372±147, L: 3139±158, O+L: 2377±171 x103/µL; P<0.001) and urine cell-free Hb concentrations (C: 9.1±1.1, O: 3.2±0.8, L: 6.8±1.5, O+L: 5.4±1.7 ng/24h; P=0.02), improved with Osivelotor treatment. Serum erythropoietin-to-HCT concentration, a surrogate biomarker of hypoxia sensed by peritubular cells in the kidney cortex, improved with Osivelotor treatment (C: 3204±485, O: 1700±172, L: 9408±3250, O+L: 2804±462 pg/mL/HCT; P=0.001). Hemolytic biomarkers were not improved with Losartan alone.Biomarkers of oxidative stress (TBARS; C: 86.4±6.6, O: 69.8±5.5, L: 43.2±3.2, O+L: 33.9±3.6 nmol/24h; P<0.001), glomerular injury (nephrin; C: 9.4±0.3, O: 8.2±0.3, L: 1.1±0.1, O+L: 0.9±0.07 ng/24h; P<0.001 ), and tubular injury (KIM-1; C: 606±125, O: 564±107, L: 424±108, O+L 271±58 pg/24h; P=0.0033) progressively improved with Osivelotor, Losartan, and the combination of Osivelotor+Losartan treatment, respectively.Kidney function demonstrated a progressive reduction in albuminuria with treatment (C: 120±16, O: 101±8, L: 31±5, O+L: 20±4 µg/24h; P<0.001). We did not observe substantial changes in serum concentrations of cystatin C (C: 0.49±0.03, O: 0.46±0.02, L: 0.62±0.04, O+L: 0.54±0.04 µg/ml) or serum creatinine (C: 0.09±0.005, O: 0.09±0.006, L: 0.12±0.007, O+L: 0.13±0.013 mg/dL) with treatment, although both arms of treatment that included Losartan had mild elevations in these values.Histopathology demonstrated global and diffuse glomerular mesangial expansion and mild interstitial fibrosis in the control group. The Osivelotor+Losartan group exhibited reduced severity, with only focal and segmental mesangial expansion and minimal interstitial fibrosis while Osivelotor and Losartan-alone groups displayed intermediate features.In conclusion, this study demonstrates that targeting both hemolysis and RAAS provides additive renoprotective effects in SCD. Osivelotor, a potent inhibitor of Hb S polymerization, moderated hemolysis leading to reduced circulating cell-free Hb and oxidative stress, two key drivers of tubular injury in SCD. Losartan, an angiotensin II receptor blocker, mitigates RAAS-mediated pathways involved in glomerular hypertension, mesangial expansion, and interstitial fibrosis. The combination treatment led to the greatest improvement across functional, molecular and structural indicators of kidney damage. These findings suggest that co-targeting hemolytic and RAAS pathways addresses the multifactorial nature of SCD nephropathy, offering a potentially synergistic therapeutic strategy.