Abstract
Intravascular hemolysis releases cell-free hemoglobin and heme into the circulation. When haptoglobin and hemopexin are depleted, as observed in sickle cell anemia, cell-free hemoglobin and heme can cause direct oxidative injury, upregulation of inflammatory and immune response pathways, and depletion of nitric oxide. The kidneys are the primary route for clearance of non-scavenged cell-free hemoglobin and heme and are particularly susceptible to these deleterious pathways (PMID 31455889).
In patients with sickle cell anemia, increased markers of hemolysis and hemoglobinuria are risk factors for kidney disease and kidney disease progression (PMID 24329963). Voxelotor is a small molecule allosteric modulator that binds and maintains sickle hemoglobin in the oxygenated state, thereby preventing hemoglobin S polymerization and red blood cell sickling. In a phase 3 study, voxelotor improved the degree of hemolysis, as reflected by a rise in hemoglobin concentration and a reduction in indirect bilirubin and reticulocyte percentage (PMID 31199090). The benefits of reducing hemolytic anemia with voxelotor on kidney function are unknown.
We investigated whether GBT1118, an analogue of voxelotor with improved pharmacokinetic properties in transgenic sickle mice, would improve biomarkers of kidney damage and kidney function in sickle cell anemia. Transgenic sickle mice (Hb SS; Townes model, Jackson Laboratory) were treated with either GBT1118 (5 male, 5 female) or control (5 male, 5 female) chow from 12 to 24 weeks of age. Urine was collected using metabolic cages for 24 hours at 3 week intervals and blood was collected at baseline, 6 weeks, and 12 weeks of therapy. We compared markers of hemolysis (hemoglobin, reticulocyte percentage), reactive oxygen species (thibarbituric acid reactive substances [TBARS], kidney injury (kidney injury molecule-1 [KIM-1], nephrin), and kidney function (urine albumin and protein, serum blood urea nitrogen [BUN] and creatinine) at the respective time points using ANOVA and adjusting for gender. Mean and standard error values are provided.
Consistent with the clinical results observed in the voxelotor phase 3 study, we observed significant improvements in the hemoglobin concentrations and reticulocyte percentages of Hb SS mice treated with GBT1118 versus control (Figure 1A and 1B). The hemoglobin occupancy of GBT1118 after 6 weeks (33 ± 2%) and 12 weeks (28 ± 2%) of therapy were also similar to what was observed in the phase 3 clinical study. An improvement in hemolysis led to reduced hemoglobinuria (6 weeks: treated = 0.07 ± 0.03 vs. control = 0.72 ± 0.2 ng/day, P = 0.004; 12 weeks: treated = 0.04 ± 0.03 vs. control = 1.06 ± 0.32 ng/day, P = 0.006) and less oxidant damage in the kidneys, assessed by urine TBARS concentration, in the treated versus control mice (6 weeks: treated = 31 ± 6 vs. control = 60 ± 10 nmol/day, P = 0.001; 12 weeks: treated = 29 ± 4 vs. control = 71 ± 9 nmol/day, P < 0.001).
At 24 weeks of age, markers of glomerular (nephrin: treated = 3.5 ± 0.7 vs. control = 8.5 ± 1.6 ng/day) and tubular injury (KIM-1: treated = 169.6 ± 19.4 vs. control = 348.9 ± 48.2 pg/day) were significantly improved in the treated versus control Hb SS mice (P< 0.001) (Figure 1C). Albuminuria remained stable in the treated Hb SS mice and was relatively close in value to the degree of albuminuria observed in Hb AA mice (Figure 1D). In contrast, albuminuria progressively increased with older age in the control Hb SS mice. A similar pattern was observed for proteinuria (24 week: Hb SS treated = 2.02 ± 0.17 mg/day, Hb SS control = 3.65 ± 0.44 mg/day, Hb AA = 1.65 ± 0.27 mg/day). At 24 weeks of age, the treated Hb SS mice had serum BUN and creatinine values that were similar to what was observed at 12 weeks of age in the Hb SS mice and to the 24-week old Hb AA mice (Figure 1E, 1F). Furthermore, the values for serum BUN and creatinine were significantly lower in the treated versus control Hb SS mice at 24 weeks of age (P ≤ 0.04).
In conclusion, we demonstrate that an improvement in hemolysis results in preserved kidney function in transgenic sickle mice. Our findings highlight the clinical importance of hemolytic anemia in the pathophysiology of sickle cell nephropathy. Our results also provide support for developing strategies to mitigate hemolysis in sickle cell anemia in order to provide a targeted approach to improve kidney disease, a devastating complication associated with high morbidity and mortality, in sickle cell anemia.
Gordeuk: Modus Therapeutics: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Emmaus: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy. Saraf: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding.
