NETosis Contributes to the Pathogenesis of Diabetic Kidney Disease: A Proposed Mechanistic Pathway

Background: Diabetic kidney disease (DKD) is one of the most debilitating complications of diabetes. Considerable research has focused on the key role of NADPH oxidases (NOXs) in DKD. Of note, our group has demonstrated the role of mTOR signaling pathway in mediating NOX-derived reactive oxygen species (ROS) production in DKD. Inflammation and an overactive immune response are known to be major risk factors for the development and progression of DKD. Recently, NETosis, a novel neutrophil-specific cell death process, was described to be associated with inflammation and diabetes. However, the effect of NETosis on DKD remains uninvestigated. Interestingly, increasing evidence highlights a pivotal role for the mTORC1 pathway and NOXs in regulating NETosis. Herein, we hypothesize that hyperglycemia activates the mTOR/NOX signaling pathway, leading to excess neutrophil extracellular traps (NETs) formation and eventual kidney injury. Methods: Control mice, mice treated with phorbol 12-myristate 13-acetate (PMA) to induce NETosis, and mice models of type 1 and type 2 diabetes treated either with Cl-amidine to inhibit NETosis or with Cl-amidine's vehicle were used. Functional, histological, and molecular parameters of the kidneys were determined. Human transcriptomics datasets from GEO were further used for validation. Results: Our data show that increased NETs formation mediates renal dysfunction and histopathological alterations associated with DKD. Of note, treatment with PMA mimicked diabetes-associated renal injury, as assessed by UAE, UACR, BUN and serum cystatin C, and induced glomerular hypertrophy, glomerulosclerosis, extracellular matrix expansion and podocyte depletion. Treatment with Cl-amidine attenuated diabetes-induced glomerular and podocyte injury. Increased NETs formation in diabetes was paralleled by an increase in NOX-dependent ROS production and mTOR signaling pathway activation. Our findings were further confirmed in transcriptomic analysis of human DKD, where a positive correlation between NETs and DKD was observed. Querying protein-protein interaction databases also revealed an association between NETs markers, mTOR signaling proteins, and NOXs. Conclusions: To our knowledge, this study is the first to describe the role of NETosis in DKD, identifying NETosis as one of the final mechanistic drivers of DKD. Funding: Private Foundation Support, Clinical Revenue Support