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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

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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 1

UmodC147W/+ mice exhibit kidney failure at 24 weeks.

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UmodC147W/+ mice exhibit kidney failure at 24 weeks.
(A) Map of murine ...
(A) Map of murine UMOD locus with annotated WT and mutant p.Cys147Trp sequences (relevant codons are indicated in blue and the point mutation in red). (B) Sequencing results of a representative F0 founder male with a correctly targeted point mutation (in red) and silent mutations (thick black line). (C) BUN results from blood of 24-week-old mice. (D) sCr results from blood of 24-week-old mice. (E) Body weight of 24-week-old male and female mice. (F) Western blot to detect UMOD protein from whole-kidney tissue; the glycosylated band runs larger than the mutant, nonglycosylated band. (G) Western blot to detect UMOD proteins precipitated from urine; levels were undetectable in the mutant urine samples. (H) Immunofluorescence images of kidney sections (7-μm-thick) labeled for the ER marker calnexin (red) and UMOD (green). Arrows indicate an overlap of calnexin and UMOD. Scale bars: 50 μm (original magnification, ×40). (I) Quantitative PCR results for fibrosis-related genes expressed in whole kidneys from 24-week-old mice. (J) Western blot analysis of proteins from whole-kidney tissue to detect markers of fibrosis at the 24-week time point. Note: The blot shown in F was stripped and reprobed for fibrosis markers. (K) Histological images of whole-kidney sections (4-μm-thick) stained with Masson’s trichrome preparation (connective tissue/collagens in blue). Scale bars: 50 μm (original magnification, ×10 [cortex and medulla] and ×4 [kidney]). (L) Quantification of Masson’s trichrome–stained images of collagens. 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. n = 6–8 per group. +/+, Umod+/+; C147W/+, UmodC147W/+.
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