Investigating the Role of Neuregulin-1 in Mitigating Haemolysis Mediated Kidney Injury in Humanized Sickle Cell Mice.

Abstract

Background: Chronic kidney disease (CKD), defined as the persistent and gradual decline in kidney function, is a common and severe complication of sickle cell disease (SCD). CKD in SCD is reported in childhood and worsens with age. More than half of adults with SCD over the age of forty (40) develop CKD, leading to end-stage renal disease. This contributes significantly to 16 18% of SCD global deaths each year. Acute kidney injury (AKI), the sudden loss of kidney function, is a major risk factor for developing chronic kidney disease (CKD). In sickle cell disease, the incidence of AKI is three times higher than in non-sickle cell patients, affecting over 30% of hospitalized individuals. Again, AKI is a leading cause of early death in critically ill SCD patients. Despite its significant role in kidney disease, treatment options for AKI in SCD remain limited and uncertain, posing a significant challenge in its clinical management. A major driver of kidney injury in SCD is intravascular haemolysis, which releases excess protein free heme into circulation, triggering kidney injury through vascular inflammation, endothelial dysfunction, oxidative stress, and cytotoxicity. Hydroxyurea (HU), the approved SCD‑modifying therapy, partly improves renal function, but its impact on heme‑driven kidney injury remains uncertain and underexplored. There is an urgent need for targeted interventions against heme driven kidney injury in SCD. Neuregulin‑1 (NRG‑1), an endothelium‑derived peptide with anti‑inflammatory, antioxidant, and cytoprotective properties, holds therapeutic potential in SCD. It is hypothesized that Neuregulin-1 reduces kidney injury in sickle cell mice by modulating haemolysis, inflammation and inducing anti-heme cytoprotective factors. This PhD work assessed the effects of NRG‑1 and HU on haemolysis, inflammation, kidney injury, and renal histopathologic changes in a humanized sickle cell mouse model that mimics clinical features of SCD. Methods: The HbSS-Townes mouse model, developed by Dr. Tim Townes’ laboratory (University of Alabama, Birmingham), which carries human haemoglobin knock-in genes and develops kidney injury resembling human disease, was used. Control mice (HbAA), which do not develop the disease, were also included. Cohorts of twelve‑week‑old Townes sickle cell (HbSS) and non‑sickle (HbAA) mice (n = 8 per group, sex-matched) were intraperitoneally administered with recombinant human neuregulin-1 (NRG-1) at doses of 5 µg/kg/day and 25 µg/kg/day; 50 mg/kg of hydroxyurea (HU) or sterile phosphate-buffered saline (vehicle) for two weeks. Following treatment with NRG-1, HU or vehicle, blood and urine samples were collected for analysis of complete blood count, haemolysis markers, and kidney injury biomarkers. Kidney tissue sections were examined for histopathologic renal changes, and immunohistochemistry was used to assess the effect of neuregulin-1 on the expression and tissue distribution of heme oxygenase-1, a heme degrading enzyme. Results: HbSS mice exhibited anaemia, leucocytosis, elevated haemolysis (total plasma heme, lactate dehydrogenase [LDH]), increased urinary kidney injury biomarkers (neutrophil gelatinase associated lipocalin [NGAL] and cystatin C), and reduced renal repair biomarkers (clusterin, epidermal growth factor [EGF]) prior treatment. Urinary levels of NGAL and Cystatin C positively correlated with total plasma heme (ρ = 0.98, p < 0.05) and LDH (ρ = 0.97, p < 0.05) respectively. Both NRG-1 and HU significantly reduced white blood cell counts, total plasma heme, lactate dehydrogenase, and pro-inflammatory kidney injury mediators (NRG-1: FN-γ, IL-6, CXCL10, VEGF-A, CCL19, MCP-5; HU: CXCL10, VEGF-A, IFN-γ) in HbSS mice. Notably, NRG-1 (25 µg/kg/day), like HU, significantly increased circulating fetal haemoglobin containing red blood cells (F-cells). HU reduced urinary cystatin C and NGAL levels more than NRG‑1, whereas NRG‑1 significantly increased urinary clusterin and EGF. HU significantly reduced medullary congestion, while NRG‑1 markedly reduced iron deposition in cortical tubular epithelial cells. Both treatments reduced glomerular congestion, Bowman’s membrane thickening, tubular brush border loss, and glomerulosclerosis. NRG‑1 enhanced HO‑1 expression in HbSS kidneys, whereas HU had no significant effect on HO-1 expression. ErbB4 was highly expressed in HbSS kidneys compared to HbAA, suggesting its involvement in kidney protection pathways. Conclusion: This work provides new insight supporting the exploration of NRG‑1 as a targeted therapeutic agent for kidney injury in SCD. The complementary renoprotective effects observed with NRG‑1 and hydroxyurea support further evaluation of their combined use to improve kidney outcomes in SCD.