Lymphangiogenesis in renal inflammation and transplantation
Vass, David George
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The lymphatic system plays an important role in both tissue homeostasis and inflammation. During the surgical procedure there is complete disruption of lymphatic drainage of the allograft kidney. The time course and nature of lymphatic reconnection following transplantation is poorly understood. In addition to the extra-renal lymphangiogenesis required for lymphatic reconnection, some patients may develop de novo lymphatic vessels within the renal parenchyma during acute rejection or chronic allograft damage. This work sought to examine the time course and mechanism of lymphangiogenesis and the role of macrophages in this process. Injection of carbon black and Evan’s blue into the rat kidney resulted in rapid transit to the draining hilar renal lymph node. Surgical disruption of the lymphatic drainage of the kidney prevented trafficking of carbon black to the renal lymph node at 24 hours. At day 6 there was macroscopic and microscopic evidence of carbon black localisation in the renal lymph node suggesting functional reconnection. Careful histological analysis of hilar renal tissue indicated that the large lymphatic trunks were replaced by a network of small proliferating lymphatic vessels. Assessment of intra-renal lymphangiogenesis was undertaken in 2 distinct experimental models of renal transplantation. In a murine model of acute allograft rejection there was no evidence of increased lymphatic vessel number at day 7. In a collaboration with Sheffield University, tissue from a rat model of interstitial fibrosis and tubular atrophy was examined. The rat tissue exhibited a prominent macrophage and T-cell infiltration at 12 months but there was no difference in the number of perivascular lymphatic vessels. In contrast, there were numerous lymphatic vessels evident in the interstitium that were absent in control isograft tissue. Interestingly, the number of lymphatic vessels correlated with the extent of fibrosis. Analysis of vascular endothelial cell growth factor-C (VEGF-C) mRNA expression did not show any increase in allografts. The model of unilateral ureteric obstruction (UUO) was employed as a model of rapidly progressive inflammatory fibrosis. UUO was associated with rapid and prominent interstitial lymphangiogenesis. This was associated with a marked increase in macrophage and T-lymphocyte infiltration and increased whole kidney mRNA expression of VEGF-C. The role of macrophages in lymphangiogenesis was explored by administration of macrophage depleting liposomal clodronate. No effect upon lymphangiogenesis was found but liposomal clodronate failed to deplete ED-1 positive macrophages in the kidney. A macrophage isolation strategy was thus employed using the myeloid CD11b marker cells and flow cytometric cell sorting and immunomagnetic bead sorting. Although gene expression studies demonstrated increased ED1 mRNA expression by CD11b enriched cells, no difference in VEGF-C mRNA expression between CD11b cells obtained from obstructed kidneys versus cells from sham controls was evident. Lastly, despite extensive efforts, immunostaining for VEGF-C was unsuccessful. In summary, lymphangiogenesis can reconstitute the lymphatic drainage of the kidney and is prominent in both chronic allograft injury and the acutely obstructed kidney in the rat. Although VEGF-C is the likely driver of lymphangiogenesis direct evidence of macrophage VEGF-C production was not found.