Aquaporin 9 expression in the developing rat epididymis is modulated by steroid hormones

Fluid and solute transport across the epithelium of the male excurrent duct is important for sperm maturation and storage. Aquaporin 9 (AQP9), which allows permeation of water and neutral solutes, is abundant throughout the male reproductive tract, where it is expressed at the apical membrane of rat epididymal principal cells as early as at 1 week of age. We evaluated the effect of neonatal exposure to: 1) a GNRH antagonist (GNRHa); 2) diethylstilbestrol (DES); 3) ethinyl estradiol (EE); 4) DES plus testosterone (DES+TE); and 5) the anti-androgen flutamide on AQP9 expression in the epididymis of peripubertal rats. Control groups received the vehicle alone. In 25-day-old rats, quantification of the mean pixel intensity of immunofluorescence-stained sections showed a significant decrease in AQP9 staining in the apical membrane of epididymal principal cells after treatments with GNRHa, DES, or flutamide, compared to controls. These results were confirmed by western blotting. While EE induced a marked decrease in AQP9 levels by western blotting, the decrease in AQP9-associated fluorescence was not significant compared to controls. DES+TE-treated rats showed levels of AQP9 protein similar to controls, indicating maintenance of AQP9 expression by testosterone treatment in the presence of DES. Our data show that expression of AQP9 in the developing rat epididymis is downregulated by neonatal DES, GNRHa, EE, and flutamide, and that the effects mediated by estrogens can be prevented by testosterone administration

PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P2 mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis. © 2013 Szalinski et al

Çédille, revista de estudios franceses

Presentació

AMP-activated protein kinase regulates the vacuolar H+-ATPase via direct phosphorylation of the A subunit (ATP6V1A) in the kidney

The vacuolar H(+)-ATPase (V-ATPase) in intercalated cells contributes to luminal acidification in the kidney collecting duct and nonvolatile acid excretion. We previously showed that the A subunit in the cytoplasmic V1 sector of the V-ATPase (ATP6V1A) is phosphorylated by the metabolic sensor AMP-activated protein kinase (AMPK) in vitro and in kidney cells. Here, we demonstrate that treatment of rabbit isolated, perfused collecting ducts with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) inhibited V-ATPase-dependent H(+) secretion from intercalated cells after an acid load. We have identified by mass spectrometry that Ser-384 is a major AMPK phosphorylation site in the V-ATPase A subunit, a result confirmed by comparing AMPK-dependent phosphate labeling of wild-type A-subunit (WT-A) with that of a Ser-384-to-Ala A subunit mutant (S384A-A) in vitro and in intact HEK-293 cells. Compared with WT-A-expressing HEK-293 cells, S384A-A-expressing cells exhibited greater steady-state acidification of HCO3(-)-containing media. Moreover, AICAR treatment of clone C rabbit intercalated cells expressing the WT-A subunit reduced V-ATPase-dependent extracellular acidification, an effect that was blocked in cells expressing the phosphorylation-deficient S384A-A mutant. Finally, expression of the S384A-A mutant prevented cytoplasmic redistribution of the V-ATPase by AICAR in clone C cells. In summary, direct phosphorylation of the A subunit at Ser-384 by AMPK represents a novel regulatory mechanism of the V-ATPase in kidney intercalated cells. Regulation of the V-ATPase by AMPK may couple V-ATPase activity to cellular metabolic status with potential relevance to ischemic injury in the kidney and other tissues