Investigating the Interplay between Growth Differentiation Factor 15 (GDF-15) and Neutrophil Extracellular Traps (NETs) in the Pathophysiology of Diabetic Nephropathy

Abstract

Background: Diabetes mellitus is a heterogeneous group of metabolic disorders characterized by hyperglycemia due to an absolute or relative deficit in insulin secretion, action, or both. The increased prevalence of diabetes globally has led to an increase in the number of both microvascular and macrovascular complications. Among the microvascular complications is diabetic nephropathy, also known as Diabetic Kidney Disease (DKD), which is the single strongest predictor of mortality in patients with diabetes. The main hallmarks of DKD are hyperglycemia-induced progressive renal dysfunction, inflammation, and fibrosis; all of which are mediated by the overproduction of reactive oxygen species (ROS). Growth Differentiation Factor 15 (GDF-15), a member of the TGF-β superfamily, that has been previously recognized for its role in cellular stress response and apoptosis, has recently emerged as a potential mediator in the intricate network of inflammatory pathways associated with DKD. Yet, it is important to note that in the context of DKD, the dual nature of GDF-15—both protective and deleterious—is a subject of debate. On the other hand, neutrophils, via the process of NETosis, play a crucial role in the pathogenesis of DKD through the release of Neutrophil Extracellular Traps (NETs), a novel form of neutrophil-specific cell death. Herein, we investigate the role of GDF-15 in the pathogenesis of DKD and its crosstalk with Neutrophil Extracellular Traps (NETs) in type 2 diabetes. Hypothesis: In this thesis work, we hypothesize that the inhibition of GDF-15 attenuates NETosis, thereby preventing the progression of diabetic kidney disease. Design: T2DM was induced using the high fat diet/STZ model in two sets of experiments over a short- (8 weeks) and long- (15 weeks) term periods respectively. In the first study, C57BL/6J mice were divided into six groups: control, control receiving GDF-15 monoclonal antibody AV-380 (7.5mg/kg or 20mg/kg), T2DM, and T2DM receiving AV-380 (7.5mg/kg or 20mg/kg) for 8 weeks. In the second study, C57BL/6J mice were divided into four groups: control, T2DM, and T2DM receiving AV-380 (7.5mg/kg or 20mg/kg) for 15 weeks. Functional, histopathological, and molecular studies were performed on kidney tissues from all groups. Results: Our findings show that elevated GDF-15 levels in T2DM mouse models of both studies lead to increased NADPH-associated ROS overproduction, contributing to DKD. Treatment of T2DM mice with a neutralizing anti-GDF-15 antibody (AV-380) attenuated renal injury as assessed by reductions in urinary albumin excretion, urinary albumin to creatinine ratio, proteinuria, blood urea nitrogen levels, glomerular hypertrophy, glomerulosclerosis, and fibrosis. Additionally, inhibition of GDF-15 signaling significantly decreased T2DM-associated NETs formation, oxidative stress, NADPH oxidase activity and NOX4 expression. Our results further indicate that GDF-15 acts upstream of NETs and NOX4 in mediating renal injury. Furthermore, short-term (8 weeks) and long-term (15 weeks) studies confirm that higher doses of AV-380 (20 mg/kg) are more effective in reducing GDF-15 levels and its pathological effects in the context of DKD. Conclusion: Our findings suggest an interplay between GDF-15 and NETosis in the context of DKD, resulting in NADPH oxidase-dependent ROS overproduction. The inhibition of GDF-15 signaling with the AV-380 monoclonal antibody restores renal function, reduces oxidative stress, and regulates NETosis, thereby slowing DKD progression. Taken together, targeting GDF-15 signaling pathway and activity through pharmacological interventions could present a promising strategy for treating DKD.