Reduced Rab5 Prenylation Might Explain the Lovastatin-Induced Accumulation of AQP2 at the Apical Plasma Membrane in Renal Cells

We have previously shown that treatment with lovastatin accumulates the water channel AQP2 at the apical plasma membrane of cultured MCD4 renal cells by inhibiting its constitutive endocytosis. This observation might be of importance to rescue AQP2 apical expression in X-linked NDI. Here we propose a mechanism that might explain this effect. In addition to inhibiting cholesterol synthesis, statins also inhibit the synthesis of other sterol and non-sterol intermediate compounds produced by the mevalonate pathway including the isoprenoids, farnesyl- (FP) and geranylgeranyl-pyrophosphate (GGP). Proteins of the Rab GTPase family must be post-translationally prenylated by addition of two geranylgeranyl moieties in order to be properly targeted to membranes and to be active. Members of the Rab family, including Rab5, were found associated with immunoisolated AQP2 storage vesicles. Rab5 is expressed in early endosomes and in chlatrin-coated endocytic vesicles suggesting a role in regulating AQP2 endocytosis at the plasma membrane. In this study we found that, in MCD4 cells, mevalonate prevented apical accumulation of AQP2 induced by lovastatin incubation as demonstrated by confocal microscopy. Interestingly, similar results were obtained by provision of GGP, suggesting a role of prenylation in regulating AQP2 trafficking. Measurement of osmotic water permeability, obtained by a calcein-quenching-based assay on FlexStation, confirmed that the increase in water permeability induced by lovastatin treatment was completely abolished by mevalonate or GGP. In agreement with these results, analysis of Rab5 prenylation under lovastatin treatment, showed that its electrophoretic mobility on SDS-PAGE was slightly reduced, consistent with a reduction of protein isoprenylation. Conversely, addition of mevalonate or GGP prevented Rab5 electrophoretic shift. Taken together, these data suggest that statins might increase AQP2 apical expression by reducing Rab5 isoprenylation and its association to the apical plasma membrane where it regulates AQP2 endocytosis.