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.

PurposeWe have previously shown that production of reactive oxygen species (ROS) is an important contributor to renal injury and inflammation following exposure to oxalate (Ox) or calcium-oxalate (CaOx) crystals. The present study was conducted, utilizing global transcriptome analyses, to determine the effect of Apocynin on changes in the NADPH oxidase system activated in kidneys of rats fed a diet leading to hyperoxaluria and CaOx crystal deposition.ApproachAge-, sex- and weight-matched rats were either fed regular rat chow or regular rat chow supplemented with 5% w/w hydroxy-L-proline (HLP). Half of the rats on the HLP diet were also placed on Apocynin-supplemented H2O. After 28 days, each rat was euthanized, their kidneys freshly explanted and dissected to obtain both cortex and medulla tissues. Total RNA was extracted from each tissue and subjected to genomic microarrays to obtain global transcriptome data. KEGG was used to identify gene clusters with differentially expressed genes. Immunohistochemistry was used to confirm protein expressions of selected genes.ResultsGenes encoding both membrane- and cytosolic-NADPH oxidase complex-associated proteins, together with p21rac and Rap1a, were coordinately up-regulated significantly in both renal medulla and cortex tissues in the HLP-fed rats compared to normal healthy untreated controls. Activation of NADPH oxidase appears to occur via the angiotensin-II/angiotensin-II receptor-2 pathway, although the DAG-PKC pathway of neutrophils may also contribute. Immuno histochemical staining confirmed up-regulated gene expressions. Simultaneously, genes encoding ROS scavenger proteins were down-regulated. HLP-fed rats receiving Apocynin had a complete reversal in the differential-expression of the NADPH oxidase system genes, despite showing similar levels of hyperoxaluria.ConclusionsA strong up-regulation of an oxidative/respiratory burst involving the NADPH oxidase system, activated via the angiotensin-II and most likely the DAG-PKC pathways, occurs in kidneys of hyperoxaluric rats. Apocynin treatment reversed this activation without affecting the levels of hyperoxaluria.

We used an unbiased approach of gene expression profiling to determine differential gene expression of all the macromolecular modulators (MMs) considered to be involved in stone formation, in hyperoxaluric rats, with and without treatment with the NADPH oxidase inhibitor apocynin. Male rats were fed rat chow or chow supplemented with 5% wt/wt hydroxy-l-proline (HLP) with or without apocynin-supplemented water. After 28 days, rats were euthanized and their kidneys explanted. Total RNA was isolated and microarray analysis was conducted using the Illumina bead array reader. Gene ontology analysis and the pathway analyses of the genes were done using Database for Annotation, Visualization of Integrated Discovery enrichment analysis tool. Quantitative RT-PCR of selected genes was carried out to verify the microarray results. Expression of selected gene products was confirmed using immunohistochemistry. Administration of HLP led to crystal deposition. Genes encoding for fibronectin, CD 44, fetuin B, osteopontin, and matrix-gla protein were upregulated while those encoding for heavy chains of inter-alpha-inhibitor 1, 3, and 4, calgranulin B, prothrombin, and Tamm-Horsfall protein were downregulated. HLP-fed rats receiving apocynin had a significant reversal in gene expression profiles: those that were upregulated came down while those that were downregulated stepped up. Apocynin treatment resulted in near complete absence of crystals. Clearly, there are two types of MMs; one is downregulated while the other is upregulated during hyperoxaluria and crystal deposition. Apparently gene and protein expressions of known macromolecular modulators of CaOx crystallization are likely regulated by ROS produced in part through the activation of NADPH oxidase.

Hyperoxaluria associated with renal deposition of calcium oxalate (CaOx) crystals causes renal injury and inflammation leading to number of diseases including chronic kidney disease (CKD). It is however, not been possible to separate the renal consequences of hyperoxaluria from that of CaOx crystal deposition. We decided to utilize ethylene glycol (EG) model where hyperoxaluria and CaOx crystal deposition can be separated in time. To test our hypothesis, male rats were made hyperoxaluric by administering EG, rats were euthanized and kidneys were extracted on day 14, when occasional crystal is seen in the kidneys and day 28, when all animals have developed renal CaOx crystal deposits. Total RNA was extracted for microarray analysis and genome wide analysis of differentially expressed genes was performed to investigate differences between hyperoxaluria and crystal induced alterations in the kidneys. Immunohistochemical and Hematoxylin and Eosin (H&E) staining was also done for macromolecules with significant role in stone formation. All EG fed rats became hyperoxaluric by day 7, showed a few crystal deposits on day 14, and had heavy crystal deposition by day 28. There were significant changes in the expression of genes encoding for NADPH Oxidases; macromolecular crystallization modulators; genes involved in inflammasome activation; and osteogenic marker genes. Results demonstrate major differences between hyperoxaluria and CaOx crystal induced changes in the kidneys. Injury and inflammation are mainly associated with crystal deposition indicating significant role played by crystal retention.

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Results indicate that hyperoxaluria-induced production of ROS, injury and inflammation are in part associated with the activation of Nox through renin-angiotensin-aldosterone pathway.