Mesenchymal Stem Cells Attenuate Diabetic Kidney Disease by Inhibiting the mTOR/NADPH Oxidase/NETosis Signaling Pathway: A Novel Therapeutic Target

Abstract

Background: Diabetic kidney disease (DKD) is one of the most debilitating complications of diabetes. Despite recent advances made in the field, the underlying mechanisms of DKD remain to be further elucidated. Considerable research has focused on the key role of the mTOR/NADPH oxidase signaling axis in diabetes, yet, more research is necessary to fully understand its implication in DKD. Recently, a novel neutrophil-specific cell death process termed NETosis was found to be associated with diabetes. Of note, NADPH oxidases play a pivotal role in the regulation of NETosis. Concomitantly, a large body of evidence has revealed that treatment with mesenchymal stem cells (MSCs) attenuates inflammation and induces glomerular repair. However, the renoprotective mechanistic pathways of MSCs in DKD remain largely unexplained.

Hypothesis: In this work, we explore the hypothesis that hyperglycemia activates NADPH oxidase signaling through an mTOR-dependent mechanism, leading to enhanced formation of neutrophil extracellular traps (NETs) and eventual renal injury. We also assess whether treatment with MSCs-conditioned media (MSCs-CM) or MSCs restores the homeostatic balance of the mTOR/NADPH oxidase signaling pathway, thus regulating NETosis and preventing the progression of DKD.

Design: Three sets of animal studies were performed to assess our hypothesis. Control mice, type 1 diabetic mice, and type 1 diabetic mice treated with either rapamycin (mTORC1 inhibitor), JR-AB2-011(mTORC2 inhibitor) or PP242 (mTORC1/2 inhibitor) were used. In addition, 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. In parallel experiments, control rats, type 1 diabetic rats, and type 1 diabetic rats treated with either MSCs-CM or MSCs were used. Functional, histological and molecular parameters of the kidneys were determined. In our clinical study, control subjects and patients with type 2 diabetes were recruited from Hamad General Hospital in Qatar and serum NETosis markers were measured.

Results: Treatment with rapamycin, JR-AB2-011 or PP242 significantly attenuated renal injury by inhibiting hyperglycemia-induced oxidative stress and NADPH oxidase activity. In addition, data from our experimental animal models and from our clinical study reveal that NETs formation, as assessed by increased NETosis markers, was positively correlated with renal injury. Of note, treatment with rapamycin, JR-AB2-011 or PP242 significantly decreased diabetes-associated NETs formation, suggesting a plausible crosstalk between the mTOR signaling pathway and NETosis. Treatment with MSCs-CM or MSCs attenuated diabetes-induced renal injury by inhibiting the mTOR/NADPH oxidase signaling pathway and by markedly decreasing NETs formation. Taken together, the results highlight the importance of MSCs-CM or MSCs in restoring homeostasis by correcting the alterations observed in different signaling pathways.

Conclusion: To our knowledge, this is the first study that describes the novel mechanistic link between mTOR/NADPH oxidase signaling axis and NETosis in DKD. In addition, this study highlights the importance of MSCs and more importantly of MSCs-CM as possible therapeutic modalities for DKD.