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Apoptosis induced by an uromodulin mutant C112Y and its suppression by topiroxostat

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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.

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Conflict of interest

This study was partially supported by Sanwa Kagaku Kenkyusho Co., Ltd. and Fuji Yakuhin Co., Ltd.

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Authors and Affiliations

  1. Division of Regenerative Medicine and Therapeutics, Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University Graduate School of Medical Science, 86 Nishichou, Yonago, 683-8503, Japan

    Sulistiyati Bayu Utami, Endang Mahati, Peili Li, Nani Maharani, Nobuhito Ikeda, Yasutaka Yamamoto, Akio Yoshida, Yasuaki Shirayoshi & Ichiro Hisatome

  2. Department of Cardiology and Vascular Medicine, Faculty of Medicine, Diponegoro University, Semarang, Indonesia

    Sulistiyati Bayu Utami & Udin Bahrudin

  3. Division of Medicine and Clinical Science, Department of Multidisciplinary Internal Medicine, School of Medicine, Tottori University Faculty of Medicine, Yonago, Japan

    Chishio Munemura

  4. Department of Human Physiology and Pathology, Faculty of Pharma-Sciences, Teikyo University, Tokyo, Japan

    Makoto Hosoyamada

  5. Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, Yonago, Japan

    Yuji Nakayama, Katsumi Higaki & Eiji Nanba

  6. Department of Biological Regulation, Tottori University Faculty of Medicine, Yonago, Japan

    Haruaki Ninomiya

  7. Department of Pathophysiology, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan

    Kimiyoshi Ichida

  8. Division of Cardiovascular Medicine, Department of Molecular Medicine and Therapeutics, Tottori University, Yonago, Japan

    Kazuhiro Yamamoto

  9. Department of Pathophysiology and Therapy in Chronic Kidney Disease, Jikei University School of Medicine, Minato-ku, Tokyo, Japan

    Tatsuo Hosoya

Authors
  1. Sulistiyati Bayu Utami
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  2. Endang Mahati
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  3. Peili Li
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  14. Haruaki Ninomiya
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  16. Kimiyoshi Ichida
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  17. Kazuhiro Yamamoto
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  18. Tatsuo Hosoya
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  19. Ichiro Hisatome
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Corresponding author

Correspondence to Peili Li.

Additional information

S. B. Utami and E. Mahati have equally contributed to this manuscript.

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Utami, S.B., Mahati, E., Li, P. et al. Apoptosis induced by an uromodulin mutant C112Y and its suppression by topiroxostat. Clin Exp Nephrol 19, 576–584 (2015). https://doi.org/10.1007/s10157-014-1032-8

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  • DOI: https://doi.org/10.1007/s10157-014-1032-8

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