Background: Vascular endothelial growth factor (VEGF) mRNA and protein expression are increased by hypoxia in a variety of cell types and organs. In the kidney, however, chronic hypoxia does not up-regulate VEGF mRNA. This suggests that VEGF may be regulated by unique mechanisms in the kidney.
Methods: Unilateral ischemia was induced in rats by vascular cross-clamping (40 min) followed by reperfusion (0, 20, 40, and 80 min). The distribution of VEGF protein was determined by immunohistochemical staining and Western blotting. mRNA was detected by Northern blotting and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemical staining for VEGF was verified using two VEGF antibodies. To further substantiate the immunohistochemical findings, laser scanning confocal fluorescence microscopy was used to demonstrate the distribution of VEGF protein in rat renal tubular epithelial cells (NRK52-E) subjected to hypoxia (40 min) and re-oxygenation (0, 5, 20, 40 and 80 min).
Results: Normal kidneys showed diffuse immunohistochemical staining for VEGF in all tubules of the renal cortex and medulla. Following ischemia, staining demonstrated a prominent shift of cytoplasmic VEGF to the basolateral aspect of tubular cells with both VEGF antibodies. The distribution of cytoplasmic VEGF returned to normal following 40 and 80 minutes of reperfusion. Western blots of cytoplasmic samples from ischemic kidneys reperfused for 0 and 20 minutes showed decreased levels of VEGF164 compared with normal (P < 0.01). VEGF164 and VEGF188 levels in the membrane fraction showed no change. Northern blots and semiquantitative RT-PCR showed no significant up-regulation of VEGF mRNA or change in the splice pattern. NRK52-E cells subjected to hypoxia and re-oxygenation for 0 and 5 minutes showed increased staining for VEGF compared with normal, with prominent VEGF staining at the periphery of the cell, similar to the appearance in ischemic kidneys. VEGF staining became more diffuse with further re-oxygenation.
Conclusion: Although synthesis of VEGF mRNA and protein is not increased during ischemia reperfusion injury, pre-existing VEGF in the tubular cell cytoplasm redistributes to the basolateral aspect of the cells. These data suggest that the kidney may have evolved unique patterns of VEGF regulation to cope with acute hypoxia.