engleski

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 aim of this work is to study the effect of microtubule disruption on NHE3 trafficking in PT, and the potential role of NHERF in this process. Methods. Immunofluorescence and immunogold cytochemistry was performed on cryosections of kidney tissue, whereas immunoblotting was performed with BBM isolated from the renal cortex and outer stripe of control and colchicine-treated rats (3.2 mg/kg, i.p., a single dose 12 h before sacrifice). Results. In cells of the convoluted PT (CPT) of control rats, NHE3 was located mainly in the BBM; subapical endosomes were weakly-stained. In cells of the straight PT (SPT), 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 (BLM) in the CPT. In the SPT, the abundance of BBM NHE3 was not visibly changed, but NHE3-positive intracellular organelles were no longer detectable, and the antigen was detectable in the BLM. The PDZ protein NHERF followed similar pattern: in control animals, it was strong in the BBM and negative-to-weak in the BLM in cells along the PT. After colchicine treatment, expression of NHERF in the BLM 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.

cytoskelton; membrane polarity; microtubules; sodium-proton exchanger; kidney

nije evidentirano