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Immunofluorescent localization of the Rab-GAP protein TBC1D4 (AS160) in mouse kidney

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Abstract

TBC1D4 (or AS160) was identified as a Rab-GTPase activating protein (Rab-GAP) that controls insulin-dependent trafficking of the glucose transporter GLUT4 in skeletal muscle cells and in adipocytes. Recent in vitro cell culture studies suggest that TBC1D4 may also regulate the intracellular trafficking of kidney proteins such as the vasopressin-dependent water channel AQP2, the aldosterone-regulated epithelial sodium channel ENaC, and the Na+-K+-ATPase. To study the possible role of TBC1D4 in the kidney in vivo, we raised a rabbit polyclonal antibody against TBC1D4 to be used for immunoblotting and immunohistochemical studies. In immunoblots on mouse kidney homogenates, the antibody recognizes specific bands at the expected size of 160 kDa and at lower molecular weights, which are absent in kidneys of TBC1D4 deficient mice. Using a variety of nephron-segment-specific marker proteins, immunohistochemistry reveals TBC1D4 in the cytoplasm of the parietal epithelial cells of Bowman’s capsule, the thin and thick limbs of Henle’s loop, the distal convoluted tubule, the connecting tubule, and the collecting duct. In the latter, both principal as well as intercalated cells are TBC1D4-positive. Thus, with the exception of the proximal tubule, TBC1D4 is highly expressed along the nephron and the collecting duct, where it may interfere with the intracellular trafficking of many renal transport proteins including AQP2, ENaC and Na+-K+-ATPase. Hence, TBC1D4 may play an important role for the control of renal ion and water handling and hence for the control of extracellular fluid homeostasis.

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Acknowledgments

The study is part of the M.D. thesis of Natascha Lier. The study was supported by Grants from the Swiss National Science Foundation 310000-122243/1 (to JL) and the Novartis Foundation (to JL). The laboratory of JL is also supported by the National Centre of Competence in Research (NCCR) Kidney.CH and the Zurich Center for Integrative Human Physiology (ZIHP). We gratefully acknowledge the expert technical help of Monique Carrel. Initial immunohistochemical studies were also performed with antibodies against TBC1D4 (AS160), which have been kindly provided by Dr. Gustav E. Lienhard (Dartmouth Medical School, Hanover, NH, USA).

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  1. Institute of Anatomy and Zurich Center for Integrative Human Physiology, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland

    Natascha Lier, Nikolay Gresko, Marianna Di Chiara, Dominique Loffing-Cueni & Johannes Loffing

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  1. Natascha Lier
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  2. Nikolay Gresko
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  3. Marianna Di Chiara
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  4. Dominique Loffing-Cueni
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  5. Johannes Loffing
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Correspondence to Johannes Loffing.

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Lier, N., Gresko, N., Di Chiara, M. et al. Immunofluorescent localization of the Rab-GAP protein TBC1D4 (AS160) in mouse kidney. Histochem Cell Biol 138, 101–112 (2012). https://doi.org/10.1007/s00418-012-0944-1

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