Bone Marrow Contribution to Renal Repair Following Ischemic Injury

BONE MARROW CONTRIBUTION TO RENAL REPAIR FOLLOWING ISCHEMIC INJURY. Matthew C. Egalka, Sujata Kale, , Lloyd G. Cantley. Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT. Repair of the mammalian renal tubular epithelium following ischemia/reperfusion injury is largely effected by surviving epithelial cells and other cells resident in the kidney interstitium; however, the precise nature of the contribution of circulating extrarenal cells is unknown. The purpose of this study is to determine whether bone marrow cells have the capacity to differentiate into renal tubular epithelium. This was investigated using both an ex vivo transplantation model as well as an attempt to grow epithelial cells from bone marrow in a liquid culture system. Recipient female C57BL/6 mice were myeloablated via irradiation and transplanted intravenously with lineage-depleted syngeneic male bone marrow. Following this, renal ischemia was induced by surgical interruption of the renal vascular pedicle. Thin sections were assayed for donor contribution by examination by fluorescence in situ hybridization (FISH) for the Ychromosome. Tubules incorporating donor (Y-chromosome positive) cells were only rarely found (< 2%) in the outer medulla of damaged kidneys; far more frequent were donor-derived interstitial cells, which appear not to be entirely inflammatory in phenotype. For the tissue culture experiments, collagenase-released marrow stromal cells (CR-MSC) were obtained by collagenase treatment of bone chips following mechanical dissociation of bones harvested from C57BL/6 mice. These cells were grown in culture and examined via immunostaining and reverse-transcription polymerase chain reaction for epithelial markers. Unfortunately, the resultant cells, although appearing to form epithelial colonies, were found to not express the epithelial markers cytokeratin and ZO-1 by RT-PCR and immunostaining; instead they appeared to be fibroblasts with a novel morphology. In sum, bone marrow contribution to the renal epithelium appears to be a rare phenomenon at best and as of now cannot be replicated in vitro. ACKNOWLEDGEMENTS None of this research would have been possible without the support of my mentor, Dr. Lloyd Cantley. I am extremely lucky to have such a sympathetic and encouraging physicianscientist as my principal investigator. Through the four years that I have been pursuing research in Dr. Cantley’s laboratory, I have been supremely fortunate to be able to rely on him for technical and scientific advice, clinical education, and moral support. Additionally, all the current and former members of the Cantley Lab have been instrumental in keeping my project going and providing me with whatever help I need. Specifically, Dr. Sujata Kale deserves special thanks for her role in the genesis of this project, and Dr. Malika Israilovna for her serendipitous discovery of collagenase-released MSC. I especially appreciate the scientific support lent me by Dr. Anil Karihaloo, Dr. Shuta Ishibe, Dr. Roland Schmidt, and Dr. Akashi Togawa, who were always available to bounce whatever nonsensical idea was floating around my head. Additional thanks for outstanding technical advice and support go to Dr. Baoyuan Bi, Dr. Jiankan Guo, and Feng Wei, as well as our stellar preundergraduate student Marie Louise Frevert. My sincere gratitude goes out to Dr. Dan Biemesderfer in the Section of Nephrology and the Biemesderfer lab, specifically Sue Ann Mentone for reagents and technical assistance. I would also be remiss without acknowledging the contribution of Dr. Diane Krause (Department of Laboratory Medicine) to my project, both for serving as my extra-departmental reader as well as providing the FISH technique that is a cornerstone of my project. Finally, none of medical school would have been possible without the help of my family and friends for supporting me through my nascent medical career. My parents Joanne Chandler and Ron Egalka, my sister Kerrin Egalka and especially my wonderful and highly tolerant fiancée Susan Jun have dealt with my alternating elation and frustration that co-vary with biological research of any bent. I could not ask for a more understanding group of loved ones.