Uromodulin p.Cys147Trp mutation drives kidney disease by activating ER stress and apoptosis
Bryce G. Johnson, … , Lionel Feigenbaum, Jeremy S. Duffield
Bryce G. Johnson, … , Lionel Feigenbaum, Jeremy S. Duffield
Published October 9, 2017
Citation Information: J Clin Invest. 2017;127(11):3954-3969. https://doi.org/10.1172/JCI93817.
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Research Article Cell biology Nephrology

Uromodulin p.Cys147Trp mutation drives kidney disease by activating ER stress and apoptosis

Abstract

Uromodulin-associated kidney disease (UAKD) is caused by mutations in the uromodulin (UMOD) gene that result in a misfolded form of UMOD protein, which is normally secreted by nephrons. In UAKD patients, mutant UMOD is poorly secreted and accumulates in the ER of distal kidney epithelium, but its role in disease progression is largely unknown. Here, we modeled UMOD accumulation in mice by expressing the murine equivalent of the human UMOD p.Cys148Trp point mutation (UmodC147W/+ mice). Like affected humans, these UmodC147W/+ mice developed spontaneous and progressive kidney disease with organ failure over 24 weeks. Analysis of diseased kidneys and purified UMOD-producing cells revealed early activation of the PKR-like ER kinase/activating transcription factor 4 (PERK/ATF4) ER stress pathway, innate immune mediators, and increased apoptotic signaling, including caspase-3 activation. Unexpectedly, we also detected autophagy deficiency. Human cells expressing UMOD p.Cys147Trp recapitulated the findings in UmodC147W/+ mice, and autophagy activation with mTOR inhibitors stimulated the intracellular removal of aggregated mutant UMOD. Human cells producing mutant UMOD were susceptible to TNF-α– and TRAIL-mediated apoptosis due to increased expression of the ER stress mediator tribbles-3. Blocking TNF-α in vivo with the soluble recombinant fusion protein TNFR:Fc slowed disease progression in UmodC147W/+ mice by reducing active caspase-3, thereby preventing tubule cell death and loss of epithelial function. These findings reveal a targetable mechanism for disease processes involved in UAKD.

Authors

Bryce G. Johnson, Lan T. Dang, Graham Marsh, Allie M. Roach, Zebulon G. Levine, Anthony Monti, Deepak Reyon, Lionel Feigenbaum, Jeremy S. Duffield

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Figure 6

Autophagy enhancement degrades accumulated mutant UMOD in primary human kidney epithelial cells.

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Autophagy enhancement degrades accumulated mutant UMOD in primary human ...
(A) Quantitative PCR of human UMOD-producing cell lines (hUMOD+) for key autophagy-related genes. (B) Western blot analysis to detect LC3B (LC3B I inactive, LC3B II active). (C) Ratio of active to inactive LC3B by densitometric analysis. (D) Images of LC3B-GFP-RFP puncta in hUMOD+ C147W-mutant cell line with either DMSO (0.1%), rapamycin (50 nM), or torkinib (10 μM) at 0, 6, and 12 hours. Scale bars: 300 μm (original magnification, ×10). Note the bright-field representation at the top with yellow puncta. Blue lines in the images indicate the cell outline. (E and F) Quantification of the percentage of reduction in the number of LC3B-GFP-RFP puncta from 0 to 6 hours and 0 to 12 hours. (G) Western blot analysis of p-mTOR, UMOD, and LC3B (LC3B I inactive, LC3B II active) in the hUMOD+ C147W-mutant cell line. Note the clearance of the lower, aggregate-sized band of UMOD with treatment of the autophagy enhancers rapamycin and torkinib. (H) Densitometric analysis of the ratio of aggregated to membrane-bound forms of UMOD. Representative data from 1 of 3 (A–F) or 1 of 5 (G and H) experiments are shown. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-tailed Student’s t test or 2-way ANOVA with post-hoc testing. n = 3–6 per group.