ABSTRACT
TonEBP/NFAT5 is a major regulator of the urinary concentrating process and is essential for the osmoadaptation of renal medullary cells. Focal adhesion kinase (FAK) is a mechanosensitive non-receptor protein tyrosine kinase expressed abundantly in the renal medulla. Since osmotic stress causes cell shrinkage, the present study investigated the contribution of FAK on TonEBP/NFAT5 activation. Osmotic stress induced time-dependent activation of FAK as evidenced by phosphorylation at Tyr-397, and furosemide reduces FAK Tyr-397 phosphorylation in the rat renal medulla. Both pharmacological inhibition of FAK and siRNA-mediated knockdown of FAK drastically reduced TonEBP/NFAT5 transcriptional activity and target gene expression in HEK293 cells. This effect was not mediated by impaired nuclear translocation or by reduced transactivating activity of TonEBP/NFAT5. However, TonEBP/NFAT5 abundance under hypertonic conditions was diminished by 50% by FAK inhibition or siRNA knockdown of FAK. FAK inhibition only marginally reduced transcription of the TonEBP/NFAT5 gene. Rather, TonEBP/NFAT5 mRNA stability was diminished significantly by FAK inhibition, which correlated with reduced reporter activity of the TonEBP/NFAT5 mRNA 3' untranslated region (3'-UTR). In conclusion, FAK is a major regulator of TonEBP/NFAT5 activity by increasing its abundance via stabilization of the mRNA. This in turn, depends on the presence of the TonEBP/NFAT5 3'-UTR.
ABSTRACT
Nephrocalcinosis, acute calcium oxalate (CaOx) nephropathy, and renal stone disease can lead to inflammation and subsequent renal failure, but the underlying pathological mechanisms remain elusive. Other crystallopathies, such as gout, atherosclerosis, and asbestosis, trigger inflammation and tissue remodeling by inducing IL-1ß secretion, leading us to hypothesize that CaOx crystals may induce inflammation in a similar manner. In mice, intrarenal CaOx deposition induced tubular damage, cytokine expression, neutrophil recruitment, and renal failure. We found that CaOx crystals activated murine renal DCs to secrete IL-1ß through a pathway that included NLRP3, ASC, and caspase-1. Despite a similar amount of crystal deposits, intrarenal inflammation, tubular damage, and renal dysfunction were abrogated in mice deficient in MyD88; NLRP3, ASC, and caspase-1; IL-1R; or IL-18. Nephropathy was attenuated by DC depletion, ATP depletion, or therapeutic IL-1 antagonism. These data demonstrated that CaOx crystals trigger IL-1ß-dependent innate immunity via the NLRP3/ASC/caspase-1 axis in intrarenal mononuclear phagocytes and directly damage tubular cells, leading to the release of the NLRP3 agonist ATP. Furthermore, these results suggest that IL-1ß blockade may prevent renal damage in nephrocalcinosis.