Login to Access Video or Poster Abstract: MP12-06
Sources of Funding: NIH-DK-RO1-54084 _x000D_ Carroll W. Fiest endowed chair funds

Introduction

Elevated urinary oxalate and calcium levels have independently been associated with sub-sets of idiopathic stone formers. However, precise mechanisms of interplay between elevated oxalate levels and renal tubular inflammation is not fully understood. In the present studies we set out to determine effects of oxalate on expression of pro-inflammatory genes.

Methods

Renal epithelial cell lines (HK2 cells) were used in culture to evaluate the effects of oxalate and COM crystals. We utilized microarray analysis using Affymetrix HG_U133_plus2 gene chip. Data analysis was performed suing Data Mining Tool (DMT 3.1, Affymetrix) and GeneSpring 7.2 (Silicon Genetics). Cell Intensity files were processed into expression values for all the 55,000 probe sets (transcripts) on each array and following the respective normalization step. Differentially expressed genes were classified according to the Gene Ontology functional category (GenMAPP v2) and functional significance of differentially expressed genes was determined using Ingenuity Pathways Analysis Software (Ingenuity Systems, http://www.ingenuity.com). Cluster and Heatmap images were generated using BRB-Array tools30. Changes in gene expression were further validated by relative quantitative RTPCR. Protein expression was monitored by Western Blot analysis, immune-histochemical and immunofluorescence methods.

Results

Gene Set Enrichment of the Transcriptome of human renal epithelial cells upon oxalate exposure revealed that oxalate exposure was associated with positive enrichment of genes associated with immune response, immune system processes and inflammatory response. Identification of lipopolysaccharide (LPS) gene set enrichment signature prompted us to evaluate activation of Toll-like receptor 4 (TLR4) pathway as one of the key. Oxalate induced nuclear translocation of the transcription factor NF-?B and activation of p38 MAP kinase in renal epithelial cells. Moreover, inhibition of TLR4 as well as p38 MAP kinase blocked NF-?B activation. At the protein level, effects of oxalate on expression of proinflammatory cytokines and chemokine IL-6 were similar to that of LPS treatment in renal epithelial cells.

Conclusions

These results show for the first time that oxalate and COM crystals engage TLR4 a member of the pattern recognition receptor system. Given the roles played by TLR4, we hypothesize that elevated levels of oxalate promote renal tubular inflammation by activating TLR4

Funding

NIH-DK-RO1-54084 _x000D_ Carroll W. Fiest endowed chair funds