Calcium oxalate crystals induce renal inflammation by NLRP3-mediated IL-1β secretion
Shrikant R. Mulay, … , Helen Liapis, Hans-Joachim Anders
Shrikant R. Mulay, … , Helen Liapis, Hans-Joachim Anders
Published December 10, 2012
Citation Information: J Clin Invest. 2013;123(1):236-246. https://doi.org/10.1172/JCI63679.
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Research Article Nephrology

Calcium oxalate crystals induce renal inflammation by NLRP3-mediated IL-1β secretion

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-1β 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-1β 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-1β–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-1β blockade may prevent renal damage in nephrocalcinosis.

Authors

Shrikant R. Mulay, Onkar P. Kulkarni, Khader V. Rupanagudi, Adriana Migliorini, Murthy N. Darisipudi, Akosua Vilaysane, Daniel Muruve, Yan Shi, Fay Munro, Helen Liapis, Hans-Joachim Anders

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Figure 2

Mechanisms of CaOx crystal–induced IL-1β secretion.

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Mechanisms of CaOx crystal–induced IL-1β secretion. (A) BMDCs were prime...
(A) BMDCs were primed with LPS (1 μg/ml) and stimulated with CaOx crystals in the presence or absence of cytochalasin D. TEM showed that CaOx crystals appeared outside the cell (asterisks) and inside intracellular endosomes (arrowheads), whereas cytochalasin D treatment inhibited their uptake in DCs. (B) Incubation with cytochalasin D, CA074Me, and N-acetyl cysteine (NAC) affected IL-1β secretion, measured by ELISA in cell culture supernatants after 6 hours. (C) Similar experiments were performed using 75 mM KCl in cell culture medium to block potassium efflux from the DCs. NaCl of an identical molarity was used as a molarity control. High extracellular potassium prevented CaOx-induced IL-1β secretion. (D) DCs were isolated from WT and P2x7–/– mice. ATP-induced, but not CaOx-induced, IL-1β secretion depended on the presence of P2X7. Data are means ± SD from 3 independent experiments. (E) Renal ECs, MCs, TECs, and renal DCs were primed with LPS and exposed to CaOx crystals, and IL-1β secretion was measured. ATP was used as a positive control for NLRP3 activation. (F) Western blotting. LPS priming induced pro–IL-1β expression only in DCs. (G) Primary TECs were isolated from WT mice and cultured with increasing concentrations of CaOx. ATP release was quantified in cell culture supernatant upon 18 hours of CaOx crystal exposure with and without increasing concentrations of apyrase. (H) Coculture of LPS-primed DCs with CaOx (100 μg/ml) damaged TECs with or without apyrase (1 U/ml). IL-1β secretion was measured by ELISA after 24 hours. Data are means ± SD from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. medium (B, C, and E); vs. WT (D); vs. 1,000 μg/ml CaOx alone (G); or as indicated by brackets.