Erschienen in:
01.08.2015 | Original Article
Apoptosis induced by an uromodulin mutant C112Y and its suppression by topiroxostat
verfasst von:
Sulistiyati Bayu Utami, Endang Mahati, Peili Li, Nani Maharani, Nobuhito Ikeda, Udin Bahrudin, Chishio Munemura, Makoto Hosoyamada, Yasutaka Yamamoto, Akio Yoshida, Yuji Nakayama, Katsumi Higaki, Eiji Nanba, Haruaki Ninomiya, Yasuaki Shirayoshi, Kimiyoshi Ichida, Kazuhiro Yamamoto, Tatsuo Hosoya, Ichiro Hisatome
Erschienen in:
Clinical and Experimental Nephrology
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Ausgabe 4/2015
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Abstract
Background
Familial juvenile hyperuricemic nephropathy (FJHN) is an autosomal dominant disorder caused by mutations in UMOD that encodes uromodulin. Topiroxostat, a novel non-purine analog, selectively inhibits xanthine oxidase and reduces the serum uric acid levels and the urinary albuminuria.
Methods
Genomic DNA of a patient was extracted from peripheral white blood. Exons and flanking sequences of UMOD were amplified by PCR with primers. Mutation analysis was performed by direct sequencing of the PCR products. The wild-type and mutant uromodulin were expressed in HEK293 cells and analyzed by western blotting, immunoprecipitation, immunofluorescence, and flow cytometry.
Results
We identified an FJHN patient who carried a novel UMOD mutation G335A (C112Y). The levels of both cytosolic and secreted C112Y protein were significantly decreased compared with the wild-type, whereas the level of ubiquitination was higher in C112Y than that in the wild type. The half-life of C112Y was shortened and it was restored by a proteasome inhibitor MG132. Immunofluorescence revealed decreased levels of C112Y in the Golgi apparatus and on the plasma membrane. Expression of C112Y induced cellular apoptosis as revealed by flow cytometry. Apoptosis induced by C112Y was suppressed by topiroxostat.
Conclusion
C112Y causes its protein instability resulting cellular apoptosis which could be suppressed with topiroxostat.