NHE3 and NHERF are targeted to the basolateral membrane in proximal tubules of colchicine-treated rats.
BACKGROUND
Depolymerization of microtubules in proximal tubule (PT) cells of colchicine-treated rats causes disruption of vesicle recycling and redistribution of some brush-border membrane (BBM) transporters into cytoplasmic vesicles. NHE3, an isoform of the Na+/H+ exchanger in the PT cell BBM, is acutely regulated by a variety of mechanisms, including protein trafficking and interaction with the PDZ protein, NHERF. The effects of microtubule disruption by colchicine on NHE3 trafficking in PT and the potential role of NHERF in this process have not been studied.


METHODS
Immunofluorescence and immunogold cytochemistry were performed on cryosections of kidney tissue, and immunoblotting of BBM isolated from the renal cortex and outer stripe of control and colchicine-treated (3.2 mg/kg, IP, a single dose 12 hours before sacrifice) rats.


RESULTS
In cells of the convoluted PT (S1/S2 segments) of control rats, NHE3 was located mainly in the BBM; subapical endosomes were weakly stained. In cells of the straight PT (S3 segment), NHE3 was present in the BBM and in lysosomes. In colchicine-treated rats, there was a marked redistribution of NHE3 from the BBM into intracellular vesicles and the basolateral plasma membrane in the S1/S2 segments. In the S3 segment, the abundance of BBM NHE3 was not visibly changed, but NHE3-positive intracellular organelles largely disappeared, and the antigen was detectable in the basolateral plasma membrane. The PDZ protein NHERF followed a similar pattern: in control animals, it was strong in the BBM and negative in the basolateral membrane in cells along the PT. After colchicine treatment, expression of NHERF in the basolateral membrane strongly increased in all PT segments, where it colocalized with NHE3.


CONCLUSIONS
The data indicate that: (a) microtubules are involved in the apical targeting of NHE3 and NHERF in renal PT cells, and (b) the parallel basolateral insertion of NHE3 and NHERF may represent an indirect targeting pathway that involves transient, microtubule-independent basolateral insertion of these proteins, followed by microtubule-dependent, vesicle-mediated transcytosis to the BBM.
Sabolić
Ivan
I
Unit of Molecular Toxicology, Institute for Medical Research and Occupational Health, Zagreb, Croatia. sabolic@imi.hr
Herak-Kramberger
Carol Mirna
CM
Ljubojević
Marija
M
Biemesderfer
Daniel
D
Brown
Dennis
D
D000818:Animals; D003078:Colchicine / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012016:Reference Values; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
D010750:Phosphoproteins; C000613387:Slc9a3 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D003078:Colchicine