Purpose To investigate association between hyperoxaluria and calcium oxalate (CaOx) crystal induced production of reactive oxygen species (ROS) and activation of NLRP3 inflammasome. Materials and Methods Eight week old male rats were given hydroxy-l-proline (HLP) to induce hyperoxaluria. A group of rats on HLP diet also received apocynin, antioxidant and non-specific inhibitor of NADPH oxidase. After 28 days rats were euthanized and kidneys were extracted. Microarray analysis was conducted using Illumina bead array reader™. Gene ontology (GO) analysis and the pathway analysis of the genes was done using DAVID (Database for Annotation, Visualization of Integrated Discovery) enrichment analysis tool. Quantitative RT PCR and immunohistochemical staining were performed to confirm microarray results. Results Analysis of 22,226 genes revealed 20 and 24 pathways were highly significant in cortex and medulla respectively. In cortex, ECM-receptor interaction, complement and coagulation cascades, focal adhesion and hypertrophic cardiomyopathy were most significant pathways, whereas in medulla, complement and coagulation cascades, ECM-receptor interaction and dilated cardiomyopathy were major pathways. Genes encoding for PYCARD (ASC: apoptosis –associated spec-like protein containing a CARD), TXNIP (Thioredoxin-interacting protein), NLRP3, Caspase-1, IL-1β and IL-18 were significantly up regulated in HLP-fed rats, but the group receiving apocynin had these genes down regulated in the cortex and medulla respectively. These results were verified using quantitative RT PCR with Syber Green assays and Immunohistochemical staining. Conclusions Results indicate a role for ROS in activation of NLRP3 inflammasome via TXNIP leading to a robust inflammatory response in kidneys of rats with hyperoxaluria and CaOx nephrolithiasis.

A major role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes is to catalyze the production of superoxides and other reactive oxygen species (ROS). These ROS, in turn, play a key role as messengers in cell signal transduction and cell cycling, but when they are produced in excess they can lead to oxidative stress (OS). Oxidative stress in the kidneys is now considered a major cause of renal injury and inflammation, giving rise to a variety of pathological disorders. In this review, we discuss the putative role of oxalate in producing oxidative stress via the production of reactive oxygen species by isoforms of NADPH oxidases expressed in different cellular locations of the kidneys. Most renal cells produce ROS, and recent data indicate a direct correlation between upregulated gene expressions of NADPH oxidase, ROS, and inflammation. Renal tissue expression of multiple NADPH oxidase isoforms most likely will impact the future use of different antioxidants and NADPH oxidase inhibitors to minimize OS and renal tissue injury in hyperoxaluria-induced kidney stone disease.

Many calcium oxalate (CaOx) kidney stones develop attached to renal papillary subepithelial deposits of calcium phosphate (CaP), called Randall’s plaque (RP). Pathogenesis of the plaques is not fully understood. We hypothesize that abnormal urinary environment in stone forming kidneys leads to epithelial cells losing their identity and becoming osteogenic. To test our hypothesis male rats were made hyperoxaluric by administration of hydroxy-l-proline (HLP). After 28 days, rat kidneys were extracted. We performed genome wide analyses of differentially expressed genes and determined changes consistent with dedifferentiation of epithelial cells into osteogenic phenotype. Selected molecules were further analyzed using quantitative-PCR and immunohistochemistry. Genes for runt related transcription factors (RUNX1 and 2), zinc finger protein Osterix, bone morphogenetic proteins (BMP2 and 7), bone morphogenetic protein receptor(BMPR2), collagen, osteocalcin, osteonectin, osteopontin (OPN), matrix-gla-protein (MGP), osteoprotegrin (OPG), cadherins, fibronectin (FN) and vimentin (VIM) were up regulated while those for alkaline phosphatase (ALP) and cytokeratins 10 and 18 were down regulated. In conclusion, epithelial cells of hyperoxaluric kidneys acquire a number of osteoblastic features but without CaP deposition, perhaps a result of down regulation of ALP and up regulation of OPN and MGP. Plaque formation may additionally require localized increases in calcium and phosphate and decrease in mineralization inhibitory potential.