Uromodulin p.Cys147Trp mutation drives kidney disease by activating ER stress and apoptosis
Bryce G. Johnson, Lan T. Dang, Graham Marsh, Allie M. Roach, Zebulon G. Levine, Anthony Monti, Deepak Reyon, Lionel Feigenbaum, Jeremy S. Duffield
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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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 7

Silencing of TRIB3 in primary human renal epithelial cells rescues them from ER stress–induced sensitivity to TNF-α– and TRAIL–mediated apoptosis.

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Silencing of TRIB3 in primary human renal epithelial cells rescues them ...
(A) Quantitative PCR of human UMOD-producing cell lines for the expression of genes related to ER stress–mediated apoptosis. (B and C) In vitro protocol for inducing mutant UMOD protein–mediated apoptosis. (B) Timeline diagram indicating the experimental setup for transient treatment of UMOD-producing cell lines with the ER stress inducer brefeldin A (5 and 10 μg/ml). (C) Quantitative PCR of human UMOD-producing cell lines treated as indicated in panel B for key genes related to ER stress. Note the dose responsiveness to increasing concentrations of brefeldin A. (D–F) In vitro platform for assessing the role of TRIB3 in ER stress–mediated apoptosis in primary human UMOD-producing cell lines. (D) Schema of the experimental protocol for TRIB3 silencing, transient induction of ER stress with brefeldin A, recovery, stimulation with cytokines to induce apoptosis, and quantification of caspase-3 and caspase-7 activity. (E) Images of caspase-3, -7 activity (in purple) of human UMOD-producing cell lines after 24 hours of treatment with vehicle, TNF-α (50 ng/ml), or TRAIL (50 ng/ml). Scale bars: 300 μm. (F) Quantification of caspase-3, -7 activity normalized to the cell area. Note the marked reduction in response to TNF-α and TRAIL treatment in the mutant cell line with TRIB3 silencing. Representative data from 1 of 3 experiments are shown (A, C, E, and F). Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001, by 2-tailed Student’s t test or 2-way ANOVA with post-hoc testing. n = 3–6 per group.