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1Department of Physiology and Biophysics, University of Southern California Keck School of Medicine, Los Angeles, California 90089-9142; and 2The Water and Salt Research Center, Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark
Submitted 3 May 2004 ; accepted in final form 16 July 2004
We previously reported that Na+/H+ exchanger type 3 (NHE3) and NaPi2 are acutely retracted from the proximal tubule (PT) microvilli (MV) during acute hypertension [high blood pressure (BP)] or parathyroid hormone (PTH) treatment. By subcellular membrane fractionation, NHE3 and NaPi2 show indistinguishable redistribution patterns out of light-density into heavy-density membranes in response to either treatment consistent with a retraction from the apical MV to the intermicrovillar cleft region. This study aimed to examine the redistribution of PT NHE3 vs. NaPi2 by confocal and electron microscopy during high BP and during PTH treatment to determine whether their respective destinations overlap or are distinct. High-BP protocol: systolic BP was increased 50–60 mmHg by increasing peripheral resistance for 20 min; PTH protocol: rats were infused with 6.6 µg/kg iv of PTH followed by 0.1 µg·kg–1·min–1 infusion for 1 h. For light microscopy, rats were infused with 25 mg of horseradish peroxidase (HRP) 10 min before kidney fixation. Kidney slices were dual labeled with either NHE3 or NaPi2 and either clathrin-coated vesicle adaptor protein AP2 or endosome marker HRP. The results demonstrate retraction of NHE3 from the MV to the base of MV during either high-BP or PTH treatment: NHE3 staining did not retract below the AP2-stained domain or to HRP-labeled endosomes in either model. In comparison, NaPi2 was retracted from MV to below the AP2-stained region in both models, a little colocalizing with HRP staining. At the electron microscopic level with immunogold labeling, during high BP NHE3 was concentrated in a distinct domain in the base of the MV while NaPi2 moved to endosomes. The results demonstrate that there are divergent routes of retraction of PT NHE3 and NaPi2 from the MV during acute hypertension or PTH treatment: NHE3 is not internalized but remains at the base of the MV while NaPi2 is internalized.
kidney; sodium transporter; pressure diuresis; immunoelectron microscopy
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