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J. Biol. Chem., Vol. 282, Issue 9, 6644-6652, March 2, 2007

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Expression of the Calcium-binding Protein S100A4 Is Markedly Up-regulated by Osmotic Stress and Is Involved in the Renal Osmoadaptive Response^*

Christopher J. Rivard, Lewis M. Brown, Nestor E. Almeida, Arvid B. Maunsbach, Kaarina Pihakaski-Maunsbach, Ana Andres-Hernando, Juan M. Capasso, and Tomas Berl¹

From the Division of Renal Diseases and Hypertension, School of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262 and the Water and Salt Research Center, Department of Anatomy, University of Aarhus, DK-8000 Aarhus, Denmark

Proteomic analysis of Inner Medullary Collecting Duct (IMCD3) cells adapted to increasing levels of tonicity (300, 600, and 900 mosmol/kg H₂O) by two-dimensional difference gel electrophoresis and mass spectrometry revealed several proteins as yet unknown to be up-regulated in response to hypertonic stress. Of these proteins, one of the most robustly up-regulated (22-fold) was S100A4. The identity of the protein was verified by high pressure liquid chromatography-mass spectrometry. Western blot analysis confirmed increased expression with increased tonicity, both acute and chronic. S100A4 protein expression was further confirmed by immunocytochemical analysis. Cells grown in isotonic conditions showed complete absence of immunostaining, whereas chronically adapted IMCD3 cells had uniform cytoplasmic localization. The protein is also regulated in vivo as in mouse kidney tissues S100A4 expression was many -fold greater in the papilla as compared with the cortex and increased further in the papilla upon 36 h of thirsting. Increased expression of S100A4 was also observed in the medulla and papilla, but not the cortex of a human kidney. Data from Affymetrix gene chip analysis and quantitative PCR also revealed increased S100A4 message in IMCD3 cells adapted to hypertonicity. The initial expression of message increased at 8-10 h following exposure to acute sublethal hypertonic stress (550 mosmol/kg H₂O). Protein and message half-life in IMCD3 cells were 85.5 and 6.8 h, respectively. Increasing medium tonicity with NaCl, sucrose, mannitol, and choline chloride stimulated S100A4 expression, whereas urea did not. Silencing of S100A4 expression using a stable siRNA vector (pSM2; Open Biosystems) resulted in a 48-h delay in adaptation of IMCD3 cells under sublethal osmotic stress, suggesting S100A4 is involved in the osmoadaptive response. In summary, we describe the heretofore unrecognized up-regulation of a small calcium-binding protein, both in vitro and in vivo, whose absence profoundly delays osmoadaptation and slows cellular growth under hypertonic conditions.

Received for publication, October 5, 2006 , and in revised form, December 29, 2006.

^* This work was supported by National Institutes of Health Grants DK-19928 and DK-66544 (to T. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Division of Renal Diseases and Hypertension, University of Colorado Health Sciences Center, 4200 E. 9th Ave., MS C-281, Denver, CO 80262. Tel.: 303-315-7204; Fax: 303-315-4852; E-mail: tomas.berl{at}uchsc.edu.

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