Title: BONE MARROW-DERIVED CELLS CONTRIBUTE TO PARENCHYMAL REPAIR IN KIDNEY WARM ISCHEMIA/REPERFUSION AND TRANSPLANTATION-INDUCED COLD ISCHEMIA/REPERFUSION INJURY
Abstract: O418 Aims: Several recent studies show that bone marrow (BM)-derived cells migrate into the injured site and contribute to parenchymal repair. However, the extent of BM-derived cell involvement varies among studies from 0.6 to 40%, depending on the detection methods of BM-derived cells in the study. To precisely examine whether and to what extent BM-derived cells contribute to renal parenchymal repair, we employed 3D-fluorescent confocal microscopy/movie imaging in kidney warm ischemia/reperfusion (I/R) and transplantation-induced cold I/R injury models using GFP transgenic Sprague-Dawley (SD) rats. Method: In the warm I/R injury model, wild type SD rats were lethally (9.5Gy) irradiated and reconstituted with BM cells (200-250 x 106 cells) from GFP SD rats. At 8-12 weeks after BM reconstitution, renal warm ischemia was induced for 60 min by clamping the renal artery. For cold I/R injury, wild type SD to GFP SD kidney transplantation (KTx) was performed with or without 18 hrs of cold preservation in UW. Animals were sacrificed at 7 and 30 days after warm I/R injury or KTx. Cryostat sections of 6 and 50 μm prepared from kidney samples were stained with rhodamine-phalloidin (TRITC-phalloidin) for F-actin and Hoechst dye for nucleus. BM-derived GFP+ tubular epithelial cells were identified as GFP+/TRITC+ in fluorescent microscope and fluorescent confocal microscope. The frequency of tubules that contained GFP+/TRITC+ cells was determined in the outer medulla of the 6 μm section. For each animal, 200-750 tubules were counted from random fields. Results: At 8-12 weeks after BM reconstitution and before warm ischemia, no GFP+/TRITC+ tubular epithelial cells were detected, although >90% of leukocytes became GFP+ after BM reconstitution. Both warm and cold I/R induced substantial parenchyma damage with increased serum creatinine levels. In both kidney samples, GFP+/TRITC+ tubular epithelium was undeniably identified in several angles of 3D-confocal images. The frequency of tubules that had GFP+/TRITC+ cells was higher at 7 than 30 days after both warm I/R injury and KTx-induced cold I/R injury and increased depending on the severity of injury; cold I/R injury with highest creatinine levels showed the greatest frequency. Interestingly, however, GFP+/TRITC+ tubular epithelial cells were identified as isolated single cell integration or a cluster of maximum 2-3 cells. There was no tubule that was entirely organized with GFP+/TRITC+ cells. Conclusion: 3D-confocal imaging was useful in precisely detecting GFP+ BM-derived tubular epithelial cells after warm and cold I/R injury. The low frequency of GFP+ BM-derived cells in the repair process after I/R injury suggests that the involvement of BM-derived cells during renal parenchyma repair is transient and limited. However, an increase of damage appears to promote trans-differentiation of BM-derived cells into the tubular epithelium, suggesting that further study to enhance the process will improve parenchyma regeneration and repair. Table. The frequency of tubules that have GFP+ cells in outer medulla.Figure