In this study we investigate whether the apical sorting and the endocytosis of AQP2 is regulated by association with lipid rafts in MCD4 mouse collecting duct principal cells expressing human AQP2. Both intact cells and purified plasma membrane were solubilized in Triton X-100 or Lubrol WX at low temperature and lipid rafts were isolated on a dicontinuous sucrose gradient. Western blot analysis of the gradient fractions indicated that, in unstimulated MCD4 cells, almost all the AQP2 expressed within the cell was stably associated with Lubrol WX-insoluble/Triton X-100-soluble lipid rafts. FK stimulation, did not further increase the association of AQP2 with lipid rafts, but dramatically and specifically increased the abundance of rafts-associated AQP2 at the plasma membrane. In accordance with this observation, apically expressed AQP2 is strictly associated with the microvilli that are particularly rich in lipid rafts. Comparable results about the association of AQP2 with Lubrol-resistant rafts were obtained on kidney medulla from control versus water-deprived rats. Interestingly, both confocal scanning and apical surface biotinylation indicate that a mild reduction of endogenous cholesterol biosynthesis, achieved in MCD4 cells by a combination of lovastatin and mevalonate, promoted a dramatic increase of apical AQP2 in resting cells. This is of a particular interest in the view of a possible use of statins in the treatment of those NDI forms due to inactivation of the V2 receptor. A second important finding of this work concerns the possible role of the association of AQP2 with cholesterol/rafts in the progression from the ER and the apical sorting at the TGN. We followed the progression of newly synthesized AQP2 in BFA-treated MCD4 cells. Overnight treatment of MCD4 cells with BFA is able to accumulate AQP2 in the ER and deplete all the downstream intracellular compartments. In this condition, a significant amount of AQP2 was still Lubrol WX-insoluble indicating an early association with cholesterol in the ER. As the effect of BFA is reversible, after 1h and 30’ of BFA washout the bulk of AQP2 is transiting the Golgi apparatus and acquires the full association with the Lubrol-resistant rafts. Interestingly, when these experiments were performed in lovastatin/mevalonate-treated cells, the exit of AQP2 from both ER and Golgi was significantly slowed-down indicating that the presence of cholesterol plays a role in AQP2 sorting at both the ER and Golgi. Taken together, these findings suggest that in renal cells, AQP2 sorting into the apical route, localization into the microvilli and internalization require the association with cholesterol and Lubrol-rafts.

AQP2 association with Lubrol-rafts regulates both apical sorting and endocytosis.

PROCINO, Giuseppe;TAMMA, GRAZIA;SVELTO, Maria;
2008

Abstract

In this study we investigate whether the apical sorting and the endocytosis of AQP2 is regulated by association with lipid rafts in MCD4 mouse collecting duct principal cells expressing human AQP2. Both intact cells and purified plasma membrane were solubilized in Triton X-100 or Lubrol WX at low temperature and lipid rafts were isolated on a dicontinuous sucrose gradient. Western blot analysis of the gradient fractions indicated that, in unstimulated MCD4 cells, almost all the AQP2 expressed within the cell was stably associated with Lubrol WX-insoluble/Triton X-100-soluble lipid rafts. FK stimulation, did not further increase the association of AQP2 with lipid rafts, but dramatically and specifically increased the abundance of rafts-associated AQP2 at the plasma membrane. In accordance with this observation, apically expressed AQP2 is strictly associated with the microvilli that are particularly rich in lipid rafts. Comparable results about the association of AQP2 with Lubrol-resistant rafts were obtained on kidney medulla from control versus water-deprived rats. Interestingly, both confocal scanning and apical surface biotinylation indicate that a mild reduction of endogenous cholesterol biosynthesis, achieved in MCD4 cells by a combination of lovastatin and mevalonate, promoted a dramatic increase of apical AQP2 in resting cells. This is of a particular interest in the view of a possible use of statins in the treatment of those NDI forms due to inactivation of the V2 receptor. A second important finding of this work concerns the possible role of the association of AQP2 with cholesterol/rafts in the progression from the ER and the apical sorting at the TGN. We followed the progression of newly synthesized AQP2 in BFA-treated MCD4 cells. Overnight treatment of MCD4 cells with BFA is able to accumulate AQP2 in the ER and deplete all the downstream intracellular compartments. In this condition, a significant amount of AQP2 was still Lubrol WX-insoluble indicating an early association with cholesterol in the ER. As the effect of BFA is reversible, after 1h and 30’ of BFA washout the bulk of AQP2 is transiting the Golgi apparatus and acquires the full association with the Lubrol-resistant rafts. Interestingly, when these experiments were performed in lovastatin/mevalonate-treated cells, the exit of AQP2 from both ER and Golgi was significantly slowed-down indicating that the presence of cholesterol plays a role in AQP2 sorting at both the ER and Golgi. Taken together, these findings suggest that in renal cells, AQP2 sorting into the apical route, localization into the microvilli and internalization require the association with cholesterol and Lubrol-rafts.
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11586/64904
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact