Manuel Schlimpert1,2,3, Simon Lagies1,2,3, Barbara Müller4, Vadym Budnyk4, Kelly Daryll Blanz2,3,5, Gerd Walz4, Bernd Kammerer6,7. 1. Center for Biological Systems Analysis, Albert-Ludwigs-University of Freiburg, Freiburg, Germany. 2. Spemann Graduate School of Biology and Medicine, Albert-Ludwigs-University of Freiburg, Freiburg, Germany. 3. Faculty of Biology, Albert-Ludwigs-University of Freiburg, Freiburg, Germany. 4. Renal Division, Department of Medicine, Albert-Ludwigs-University of Freiburg, Medical Center, Freiburg, Germany. 5. Center for Chronic Immunodeficiency (CCI), Albert-Ludwigs-University of Freiburg, Medical Center, Freiburg, Germany. 6. Center for Biological Systems Analysis, Albert-Ludwigs-University of Freiburg, Freiburg, Germany. bernd.kammerer@zbsa.uni-freiburg.de. 7. BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany. bernd.kammerer@zbsa.uni-freiburg.de.
Abstract
INTRODUCTION: Nephronophthisis (NPH) is an inherited form of cystic kidney disease with various extrarenal manifestations accounting for the largest amount of endstage renal disease in childhood. Patient mutations of Anks6 have also been found to cause NPH like phenotypes in animal models. However, little is known about functionality of Anks6. OBJECTIVES/ METHODS: We investigated the impact of Anks6 depletion on cellular metabolism of inner medullary collecting duct cells by GC-MS profiling and targeted LC-MS/MS analysis using two different shRNA cell lines for tetracycline-inducible Anks6 downregulation, namely mIMCD3 krab shANKS6 i52 and mIMCD3 krab shANKS6 i12. RESULTS: In combination, we could successfully identify 158 metabolites of which 20 compounds showed similar alterations in both knockdown systems. Especially, large neutral amino acids, such as phenylalanine, where found to be significantly downregulated indicating disturbances in amino acid metabolism. Arginine, lysine and spermidine, which play an important role in cell survival and proliferation, were found to be downregulated. Accordingly, cell growth was diminished in tet treated mIMCD3 krab shANKS6 i52 knockdown cells. Deoxynucleosides were significantly downregulated in both knockdown systems. Hence, PARP1 levels were increased in tet treated mIMCD3 krab shANKS6 i52 cells, but not in tet treated mIMCD3 krab shANKS6 i12 cells. However, yH2AX was found to be increased in the latter. CONCLUSION: In combination, we hypothesise that Anks6 affects DNA damage responses and proliferation and plays a crucial role in physiological amino acid and purine/pyrimidine metabolism.
INTRODUCTION:Nephronophthisis (NPH) is an inherited form of cystic kidney disease with various extrarenal manifestations accounting for the largest amount of endstage renal disease in childhood. Patient mutations of Anks6 have also been found to cause NPH like phenotypes in animal models. However, little is known about functionality of Anks6. OBJECTIVES/ METHODS: We investigated the impact of Anks6 depletion on cellular metabolism of inner medullary collecting duct cells by GC-MS profiling and targeted LC-MS/MS analysis using two different shRNA cell lines for tetracycline-inducible Anks6 downregulation, namely mIMCD3 krab shANKS6 i52 and mIMCD3 krab shANKS6 i12. RESULTS: In combination, we could successfully identify 158 metabolites of which 20 compounds showed similar alterations in both knockdown systems. Especially, large neutral amino acids, such as phenylalanine, where found to be significantly downregulated indicating disturbances in amino acid metabolism. Arginine, lysine and spermidine, which play an important role in cell survival and proliferation, were found to be downregulated. Accordingly, cell growth was diminished in tet treated mIMCD3 krab shANKS6 i52 knockdown cells. Deoxynucleosides were significantly downregulated in both knockdown systems. Hence, PARP1 levels were increased in tet treated mIMCD3 krab shANKS6 i52 cells, but not in tet treated mIMCD3 krab shANKS6 i12 cells. However, yH2AX was found to be increased in the latter. CONCLUSION: In combination, we hypothesise that Anks6 affects DNA damage responses and proliferation and plays a crucial role in physiological amino acid and purine/pyrimidine metabolism.
Authors: Lucas Willmann; Manuel Schlimpert; Sebastian Halbach; Thalia Erbes; Elmar Stickeler; Bernd Kammerer Journal: J Chromatogr B Analyt Technol Biomed Life Sci Date: 2015-07-14 Impact factor: 3.205