Rac1 GTPase Inhibition Blocked Podocyte Injury and Glomerular Sclerosis during Hyperhomocysteinemia via Suppression of Nucleotide-Binding Oligomerization Domain-Like Receptor Containing Pyrin Domain 3 Inflammasome Activation

Abstract

Elevated homocysteine (Hcy) levels have been shown to activate nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome leading to podocyte dysfunction and glomerular injury. However, it remains unclear how this inflammasome activation in podocytes is a therapeutic target for reversal of glomerular injury and ultimate sclerosis. The present study tested whether inhibition of Rac1 GTPase activity suppresses NLRP3 inflammation activation and thereby blocks podocyte injury induced by elevated Hcy. In cultured podocytes, we found that L-Hcy (the active Hcy form) stimulated the NLRP3 inflammasome formation, as shown by increased colocalization of NLRP3 with apoptosis-associated speck-like protein (ASC) or caspase-1, which was accompanied by increased interleukin-1β production and caspase-1 activity, indicating NLRP3 inflammasome activation. Rac1 activator, uridine triphosphate (UTP), mimicked L-Hcy-induced NLRP3 inflammasome activation, while Rac1 inhibitor NSC23766 blocked it. This Rac1 inhibition also prevented L-Hcy-induced podocyte dysfunction. All these effects were shown to be mediated via lipid raft redox signaling platforms with nicotinamide adenine dinucleotide phosphate oxidase subunits and consequent O2– production. In animal studies, hyperhomocysteinemia (hHcy) induced by folate-free diet was shown to induce NLRP3 inflammasome formation and activation in glomeruli, which was also mimicked by UTP and inhibited by NSC23766 to a comparable level seen in Nlrp3 gene knockout mice. These results together suggest that Rac1 inhibition protects the kidney from hHcy-induced podocyte injury and glomerular sclerosis due to its action to suppress NLRP3 inflammasome activation in podocytes.

Keywords

• Rac1 GTPase
• Membrane rafts
• Inflammatory machinery
• Glomerulosclerosis
• Reactive oxygen species