1 2 3 4 5 6 Oxalate up - regulates expression of IL - 2 R β and activates IL - 2 R signaling in HK - 2 cells , a 7 line of human renal epithelial cells

45 46 Purpose: The role of inflammation in oxalate induced nephrolithiasis is debated. Our 47 gene expression study indicated an increase in Interleukin-2 Receptor beta (IL-2Rβ) 48 mRNA in response to oxalate. Herein, we evaluated IL-2Rβ expression and its down49 stream signaling pathway in HK2 cells in an effort to understand the mechanisms of 50 oxalate nephrotoxicity. 51 Materials and Methods: HK-2 cells were exposed to oxalate for various time points in 52 the presence or absence of SB203580, a specific p38 MAP kinase inhibitor. Gene 53 expression data were analyzed by Ingenuity Pathway Analysis software. mRNA 54 expression was quantitated via Real Time-PCR and changes in protein expression/kinase 55 activation were analyzed by western blot. 56 Results: Exposure of HK-2 cells to oxalate resulted in increased transcription of IL-2Rβ 57 mRNA and increased protein levels. Oxalate treatment also activated IL-2Rβ signaling 58 pathway (JAK1/STAT5 phosphoryylation). Moreover, the increase in IL-2Rβ protein was 59 dependent upon p38-MAPK activity. 60 Conclusions: These results suggest that oxalate-induced activation the IL-2Rβ pathway 61 may lead to a plethora of cellular changes, the most common of which is the induction of 62 inflammation. These results suggest a central role for p38-MAPK pathway in mediating 63 the effects of oxalate in renal cells, and additional studies may provide the key to 64 unlocking novel biochemical targets in stone disease. 65 Introduction: 66 Kidney stones are a common source of urologic morbidity. In fact, the treatment 67 of kidney stone disease represents one of the most costly urological conditions in the 68 United States (25). The formation of calcium oxalate (CaOx) stones in the kidney is the 69 most common pathological condition involving oxalate. Oxalate is a metabolic end 70 product, which is filtered at the glomerulus and undergoes bidirectional transport in the 71 renal tubules before being excreted by the kidney (13, 14, 24). In addition to kidney 72 stones, oxalate deposits are associated with renal cysts in acquired renal cystic disease, 73 benign neoplasms of the breast, and hyperplastic thyroid glands, amongst others (7, 10, 74 28, 34). Many of these conditions are associated with, tissue inflammation, atypical cell 75 proliferation and cell death. Unsurprisingly, the mechanism and modifications of calcium 76 oxalate deposition and stone formation is more complex than the simple precipitation of 77 CaOx crystals (18). 78 79 Previous studies from our laboratory and those of others have demonstrated that 80 the oxalate-renal cell interaction induces the initiation of DNA synthesis, cell growth, 81 apoptosis, and changes in gene expression consistent with cellular stress (1,12, 15, 17, 82 30). These data suggest that oxalate toxicity may result in tissue damage and/or 83 inflammation. However, the cellular signaling pathways activated in renal cells following 84 oxalate exposure are not well delineated and continue to be a large area of interest and 85 study. 86