J. Kanellis et al., Redistribution of cytoplasmic VEGF to the basolateral aspect of renal tubular cells in ischemia-reperfusion injury, KIDNEY INT, 57(6), 2000, pp. 2445-2456
Background. Vascular endothelial growth factor (VEGF) mRNA and protein expr
ession are increased by hypoxia in a variety of cell types and organs. In t
he kidney, however, chronic hypoxia does not up-regulate VEGF mRNA. This su
ggests 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 reve
rse transcription-polymerase chain reaction (RT-PCR). Immunohistochemical s
taining for VEGF was verified using two VEGF antibodies. To further substan
tiate the immunohistochemical findings, laser scanning confocal fluorescenc
e microscopy was used to demonstrate the distribution of VEGF protein in ra
t renal tubular epithelial cells (NRK52-E) subjected to hypoxia (40 min) an
d re-oxygenation (0, 5, 20, 40 and 80 min).
Results. Normal kidneys showed diffuse immunohistochemical staining for VEG
F in all tubules of the renal cortex and medulla. Following ischemia, stain
ing demonstrated a prominent shift of cytoplasmic VEGF to the basolateral a
spect of tubular cells with both VEGF antibodies. The distribution of cytop
lasmic 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 VEGF(164) compared with normal (
P < 0.01). VEGF(164) and VEGF(188) levels in the membrane fraction showed n
o change. Northern blots and semiquantitative RT-PCR showed no significant
up-regulation of VEGF mRNA or change in the splice pattern. NRK52-E cells s
ubjected to hypoxia and reoxygenation 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 du
ring ischemia reperfusion injury, preexisting VEGF in the tubular cell cyto
plasm redistributes to the basolateral aspect of the cells. These data sugg
est that the kidney may have evolved unique patterns of VEGF regulation to
cope with acute hypoxia.