Activation of the NLRP3 Inflammasome in Association with Calcium Oxalate Crystal Induced Reactive Oxygen Species in Kidneys

Purpose: We investigated the association of hyperoxaluria and calcium oxalate crystal induced production of reactive oxygen species with activation of the NLRP3 inflammasome.

Materials And Methods: Eight-week-old male rats were given hydroxy-L-proline to induce hyperoxaluria. A group of rats on the hydroxy-L-proline diet also received apocynin, an antioxidant and nonspecific inhibitor of NADPH oxidase. At 28 days the rats were sacrificed and the kidneys were extracted. Microarray analysis was done with the BeadArray™ Reader. Gene ontology and gene pathway analyses were done with the DAVID (Database for Annotation, Visualization of Integrated Discovery) enrichment analysis tool. Quantitative real-time polymerase chain reaction and immunohistochemical staining were performed to confirm microarray results.

Results: Analysis of 22,226 genes revealed that 20 and 24 pathways were highly significant in the cortex and medulla, respectively. In the cortex extracellular matrix-receptor interaction, complement and coagulation cascades, focal adhesion and hypertrophic cardiomyopathy were the most significant pathways. In the medulla complement and coagulation cascades, extracellular matrix-receptor interaction and dilated cardiomyopathy were the major pathways. Genes encoding for PYCARD (ASC), TXNIP, NLRP3, caspase-1, and IL-1β and 18 were significantly up-regulated in hydroxy-L-proline fed rats but in the group that received apocynin these genes were down-regulated in the cortex and medulla. Results were verified by quantitative real-time polymerase chain reaction with SYBR® Green assay and immunohistochemical staining.

Conclusions: Results indicate a role for reactive oxygen species in activation of the NLRP3 inflammasome via TXNIP. This led to a robust inflammatory response in the kidneys of rats with hyperoxaluria and calcium oxalate nephrolithiasis.