Cytochrome b₅ reductase-3 overexpression reduces renal damage associated to intravascular hemolysis

Intravascular hemolysis can cause both acute kidney injury (AKI) and chronic kidney disease (CKD). When erythrocytes break down in the bloodstream, they release hemoglobin and heme derivatives, which accumulate in renal cells and lead to increased oxidative stress, inflammation, cell death, and mitochondrial dysfunction. The enzyme NADH-cytochrome b₅ reductase 3 (CYB5R3) plays a role in NAD⁺ metabolism and helps reduce oxidative stress and inflammation while enhancing mitochondrial function. In this study, we examined whether overexpressing CYB5R3 offers kidney protection against AKI caused by intravascular hemolysis and prevents the progression from AKI to CKD, including analysis of sex-based differences. We created an experimental model to assess acute and chronic kidney damage related to intravascular hemolysis in wild-type (WT) and CYB5R3-overexpressing transgenic (TG) mice. Additionally, we performed in vitro experiments to determine if pharmacological induction of CYB5R3 with tetrahydroindenoindole (THII) reduced heme-mediated nephrotoxicity. AKI resulting from intravascular hemolysis impaired kidney function, increased damage to tubular epithelium and podocytes, promoted oxidative stress and inflammation, caused mitochondrial dysfunction, altered autophagy-related markers, and cell death-effects that were more severe in males than females. CYB5R3 overexpression notably diminished these pathological changes and slowed CKD progression by decreasing inflammation and fibrosis, regardless of sex. In cellular systems, induction of CYB5R3 with THII lowered heme-induced reactive oxygen species (ROS) production, inflammation, and cell death, and restored NAD⁺ levels. These protective effects were eliminated by pharmacological inhibition of CYB5R3 activity. Our findings suggest that CYB5R3 could be a promising therapeutic target for preventing AKI related to intravascular hemolysis and its progression to CKD.