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1 Physiology and Biophysics, USC Keck School of Medicine, Los Angeles, CA, USA
2 The Water and Salt Research Center, Department of Cell Biology, Institute of Anatomy, University of Aarhus, Aarhus C, DK-8000, Denmark
* To whom correspondence should be addressed. E-mail: mcdonoug{at}usc.edu.
We previously reported that NHE3 and NaPi2 are acutely retracted from the proximal tubule (PT) microvilli (MV) during acute hypertension (high BP) or 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 microvilli 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 blood pressure was increased 50-60 mmHg by increasing peripheral resistance for 20 min; PTH protocol: rats were infused i.v. with 6.6 µg/kg of PTH followed by 0.1 µg/kg.min infusion for 1hr. For light microscopy, rats were infused with 25 mg horseradish peroxidase (HRP) 10 min before kidney fixation. Kidney slices were dual labeled with either NHE3 or NaPi2, and either CCV 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 In comparison, NaPi2 was retracted from MV to below the AP2 stained region in both models, a little co-localizing with HRP staining. At the EM level with immunogold labeling, during high BP NHE3 was concentrated in a distinct domain in the base of the microvilli 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.
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