Growth Arrest Specific Protein 6/Axl Signaling in Human Inflammatory Renal Diseases Anette Fiebeler, MD, Joon-Keun Park, PhD, Dominik N. Muller, PhD, Carsten Lindschau, PhD, Michael Mengel, MD, Saskia Merkel, MD, Bernhard Banas, MD, Friedrich C. Luft, MD, and Hermann Haller, MD ● Background: Growth arrest-specific gene 6 (Gas6) and its binding partner, the receptor tyrosine kinase Axl, are important mediators in experimental nephritis. The authors tested whether the Gas6/Axl signaling pathway participates in human renal diseases. Methods: The authors compared 26 human renal specimens from patients with IgA nephritis, acute diffuse immune complex glomerulonephritis, acute lupus nephritis, antineutrophil cytoplasmic antibody—associated glomerulonephritis, acute transplant rejection, and normal renal tissue. Be- cause reactive oxygen species are pivotal in inflammation, the authors tested whether the Axl/Gas6 expression is influenced by NADPH oxidase in vitro. Results: Gas6 and Axl immunofluorescence was barely detectable in normal kidney. However, in disease Axl was copiously expressed in the small vessel media, glomeruli, distal tubules, and collecting ducts. Similarly, Gas6 was upregulated in the small vessel intima and media, all segments of the renal tubules, the brush border, and glomeruli. Gas6 and Axl upregulation was a prominent but nonspecific finding in these renal diseases. Cultured rat vascular smooth muscle cells and immortalized human mesangial cells were stimulated with angiotensin (Ang) II (1  10 7 mol/L) for 6 or 18 hours. Confocal microscopy and Western blot showed Ang II– dependent Gas6 and Axl expression. An antisense probe against the p22 phox unit of NADPH- oxidase suppressed Ang II–induced Gas6 and Axl expression. In addition, in p47 phox knockout cells Ang II–induced Gas6 and Axl expression were blocked. Conclusion: GAS6/Axl signaling is involved in human renal disease. The Ang II–induced Gas6 and Axl expression may be dependent on NADPH-oxidase. Gas6 and Axl are important signaling molecules in human renal disease and may be potential therapeutic targets. Am J Kidney Dis 43:286-295. © 2004 by the National Kidney Foundation, Inc. INDEX WORDS: Gas6; angiotensin; kidney; signal transduction; NADPH oxidase. T HE ABBREVIATION “Axl” comes from the Greek word anexelekto “the uncon- trolled.” The molecule, also known as Ark, Ufo, and Tyro7, was initially isolated from 2 patients with chronic myelogenous leukemia and is ex- pressed in monocytes and myeloic leuke- mias. 1-3 Together with Sky (also named Rse, Brt, Tif, Dtk, Etk, Tyro3) and Mer, Axl belongs to a group of 3 structurally related receptor tyrosine kinases. In triple knockout mice deficient in all 3 kinases, severe autoaggressive states are ob- served. The growth arrest specific protein 6 (Gas6) is a ligand for Axl, Sky, and Mer. Gas6 is structurally related to protein S and is a vitamin K–dependent protein. 4 Gas6 was identified as an upregulated protein in serum-starved NIH 3T3 cells. 5,6 Similarly, ip-E1A cells are protected from serum deprivation–induced apoptosis when they overexpress Axl. 7 Both Gas6 and Axl promote cell survival in different cell types. Recently, Gas6 and Axl were shown to be upregulated in an animal model of anti-Thy1–induced glomeru- lonephritis. Injection of the extracellular domain of Axl conjugated with the Fc portion of human IgG1 (Axl-Fc) abolished the typical glomerular changes of Thy1 glomerulonephritis. 8 In addi- tion, Gas6 was found to be overexpressed in a rat model of renal transplantation. 9 Even more com- pelling are recent findings showing that Gas6 gene–disrupted mice are resistant to nephrotoxic nephritis. 10 When these mice are outfitted with recombinant wild-type Gas6, the propensity for nephrotoxic nephritis is restored. These observa- tions suggest that Axl/Gas6 signaling plays a fundamental role in inflammatory renal disease. However, the relevance of this signaling path- From the Helios Klinikum-Berlin, Franz Volhard Clinic at the Max Delbru ¨ck Center, Medical Faculty of the Charite ´, Humboldt University of Berlin, Berlin; the Department of Pathology and Internal Medicine-Nephrology, Hannover University Medical School, Hannover; and the Department of Internal Medicine-Nephrology, University Medical School, Regensburg, Germany. Received July 7, 2003; accepted in revised form October 20, 2003. A.F. and J.-K.P. contributed equally to this article. Address reprint requests to Anette Fiebeler, MD, HELIOS Clinic Berlin, Franz Volhard Clinic at the Max Delbrueck Center, Wiltberg Str. 50, 13125 Berlin, Germany. E-mail: fiebeler@fvk-berlin.de © 2004 by the National Kidney Foundation, Inc. 0272-6386/04/4302-0005$30.00/0 doi:10.1053/j.ajkd.2003.10.016 American Journal of Kidney Diseases, Vol 43, No 2 (February), 2004: pp 286-295 286