Simvastatin enhances aquaporin-2 surface expression and urinary concentration in vasopressin-deficient Brattleboro rats through modulation of Rho GTPase

Abstract

Statins are 3-hydroxyl-3-methyglutaryl-CoA reductase inhibitors that are commonly used to inhibit cholesterol biosynthesis. Emerging data have suggested that they also have "pleotropic effects," including modulating actin cytoskeleton reorganization. Here, we report an effect of simvastatin on the trafficking of aquaporin-2 (AQP2). Specifically, simvastatin induced the membrane accumulation of AQP2 in cell cultures and kidneys in situ. The effect of simvastatin was independent of protein kinase A activation and phosphorylation at AQP2-Ser(256), a critical event involved in vasopressin (VP)-regulated AQP2 trafficking. Further investigation showed that simvastatin inhibited endocytosis in parallel with downregulation of RhoA activity. Overexpression of active RhoA attenuated simvastatin's effect, suggesting the involvement of this small GTPase in simvastatin-mediated AQP2 trafficking. Finally, the effect of simvastatin on urinary concentration was investigated in VP-deficient Brattleboro rats. Simvastatin acutely (3-6 h) increased urinary concentration and decreased urine output in these animals. In summary, simvastatin regulates AQP2 trafficking in vitro and urinary concentration in vivo via events involving downregulation of Rho GTPase activity and inhibition of endocytosis. Our study provides an alternative mechanism to regulate AQP2 trafficking, bypassing the VP-vasopressin receptor signaling pathway.

Figures

Fig. 1.

Simvastatin-induced membrane accumulation of aquaporin-2 (AQP2) in AQP2-expressing LLCPK1 cells (LLC-AQP2) and in principal cells of the collecting duct (CD) from kidney slices in vitro. A: LLC-AQP2 cells treated with ethanol (EtOH, control), vasopressin (VP, 10−8 M) for 30 min, and with simvastatin at various concentrations for 1 h. Membrane accumulation of AQP2 is seen in cells treated with VP and with simvastatin in a dose-dependent manner. Specifically, AQP2 membrane accumulation is detectable at 10 μM of simvastatin and peaks at 100 μM of simvastatin. Simvastatin-induced membrane accumulation of AQP2 was further investigated in Brattleboro rat kidney slices in vitro as shown in B. Similar to results in cell culture, treatment with simvastatin (100 μM) for 1 h causes significant membrane accumulation of AQP2 in principal cells of the CD in kidney slices (indicated by arrows), whereas in the control unstimulated state, AQP2 distributes throughout the cytoplasm. C: quantification of apical accumulation of AQP2 in kidney slices treated with simvastatin (ST). A strong and significant apical accumulation of AQP2 is seen in CD located in the cortex and outer medulla, whereas a weaker apical staining is seen in the inner medulla. Cont, control. Error bars represent means ± SE. *P < 0.01. Data were obtained from 3 experiments. Bar = 10 μm in A and B.

Fig. 2.

Simvastatin-induced membrane accumulation of AQP2 is not mediated through cAMP/protein kinase type A (PKA) activation and is independent of phosphorylation of AQP2 at Ser256. The level of cAMP inside cells (LLC-AQP2) after treatment with VP or simvastatin was measured (A). There is no significant elevation of intracellular cAMP in response to treatment with simvastatin, even at 200 μM. In contrast, intracellular cAMP is elevated >6-fold in cells treated with VP (10−8 M). Simvastatin-induced membrane accumulation was next examined in the presence of PKA inhibitors, H-89 and myristoylated PKA inhibitor (mPKI) (B). LLC-AQP2 cells were pretreated with 1 μM H-89 or 1 μM mPKI for 1 h before addition of VP or simvastatin. H-89 or mPKI alone do not cause any change of baseline AQP2 distribution (d and g), nor do they affect simvastatin-induced membrane accumulation of AQP2 (c, f, and i). In contrast, the VP-induced membrane accumulation of AQP2 is markedly inhibited by blocking PKA activity both with H-89 or mPKI (b, e, and h). The effect of simvastatin on AQP2 trafficking was also investigated in LLC-AQP2-S256A mutant cells expressing Ser256A, mimicking unphosphorylated AQP2 at Ser256 using both immunofluorescence staining (C) and cell surface biotinylation (D). Even though to a lesser degree compared with LLC-AQP2 cells [expressing wild-type (wt) AQP2], simvastatin induces membrane accumulation of AQP2 in cells expressing AQP2-S256A while VP does not (C and D). The histogram in D represents quantification of surface-biotinylated AQP2 signal from 3 independent experiments (percentage increase of signal intensity over background). stat, Statin; MW, molecular weight. Error bars represent means ± SE. *P < 0.01 in B. Scale bar = 10 μm in B and C.

Fig. 3.

Simvastatin inhibits endocytosis but has little effect on exocytosis. Endocytosis of the fluid phase marker fluorescein isothiocyanate (FITC)-dextran (10 kDa) in cells treated with simvastatin is significantly reduced by acute treatment with simvastatin in LLC-AQP2 cells (A). There is a significant 75% reduction of the uptake of FITC-dextran by cells treated with 100 μM simvastatin. Similarly, VP and methyl-β-cyclodextrin (mβCD) also cause significant reduction of the endocytosis of FITC-dextran (*P < 0.01). The specific effect of simvastatin on clathrin-medicated endocytosis was also investigated using rhodamine-labeled transferrin (TrfRh), which is endocytosed via the clathrin-mediated pathway (B). Simvastatin greatly reduces the endocytosis of rhodamine-transferrin compared with the untreated control. Much of the rhodamine-transferrin remains on the cell surface without being endocytosed in cells treated with 100 μM simvastatin. In contrast to its dramatic effect on endocytosis, simvastatin causes a minor (∼10%, *P < 0.05) reduction of overall exocytosis in our soluble, secreted yellow fluorescent protein fluorimetric exocytosis assay even at the highest concentration of 200 μM, but not at 100 μM, whereas VP treatment increases overall exocytosis (C), which is consistent with our previous report (9). Error bars represent means ± SE. Data were obtained from 3 independent experiments. Scale bar = 10 μm in B.

Fig. 4.

Simvastatin downregulates Rho GTPase in cells, and expression of constitutively active RhoA attenuates the effect of simvastatin-induced membrane accumulation of AQP2. LLC-AQP2 cells were treated with various concentrations of simvastatin for 1 h. The activity of RhoA inside cells was evaluated using a RhoA activity assay kit (Cytoskeleton) to detect the Rhotekin-RBD-RhoA-GTP complex. There is a significant, dose-dependent downregulation of RhoA activity in simvastatin-treated cells (A). Simvastatin causes significant downregulation of RhoA activity at a concentration of 50 μM and more dramatically at 200 μM. Total RhoA in simvastatin-treated and control cells was unchanged by simvastatin (data not shown). A, bottom: actin loading control. B: quantification of the percentage of change of RhoA activity from simvastatin-treated samples vs. control. Data were obtained from 3 independent experiments. Error bars represent means ± SE. *P <0.05. The involvement of RhoA in simvastatin-induced AQP2 trafficking was further investigated in cells by immunofluorescence staining and cell surface biotinylation (C and D). Expression of constitutively active RhoA [RhoA CA in C, indicated by coexpression of green fluorescent protein (GFP)] attenuates membrane accumulation of AQP2 induced by simvastatin (arrows in C, middle), whereas expression of dominant-negative RhoA (RhoA DN) has no obvious effect on simvastatin-induced AQP2 trafficking (arrowheads in C, bottom). This is clearly seen in surface biotinylation experiments (D). While there is no significant effect on the level of surface AQP2 at baseline, expression of the constitutively active RhoA attenuates simvastatin-induced surface accumulation of AQP2 (biotinylated AQP2). Expression of the dominant-negative RhoA results in membrane accumulation of AQP2, even without any further stimulation. D: quantification of surface-biotinylated AQP2 signal from 3 independent experiments. Error bars represent means ± SE. Scale bar = 10 μm in C.

Fig. 5.

Simvastatin increases urinary concentration and induces membrane accumulation of AQP2 in the CD of kidneys from Brattleboro (BB) rats. After treatment, the urine output from each animal (3 animals in the control group and 3 animals in the treated group) was collected and measured every hour during the first 6 h and then every 12 h after injection. The total urine volume of pooled 6-h urine from individual animals was measured and photographed (A). Clearly, the simvastatin-treated group produces less than half of the urine volume compared with the control animals. An increase in urinary osmolality is detectable as soon as 1 h after injection (B). The percentage increase or decrease of hourly urinary osmolality in individual animals compared with its own baseline after injection is shown in B. There is a clear stimulatory effect on urinary osmolality in treated animals (statin#) compared with the controls (cont#) within 6 h postinjection. AQP2 distribution in CD of treated and control BB animals was examined by immunofluorescence staining of kidneys (C). In rats treated with simvastatin for 6 h, there is a significant membrane accumulation of AQP2 in CD principal cells in the cortex and medullary outer stripe but not in the papilla. The fluorescence signal resulting from apical AQP2 in principal cells was acquired and quantified using IPlab software as detailed in the text (D). There is a significant increase of apical AQP2 staining in CD from cortex and outer medulla in treated animals. Error bars represent means ± SE. *P < 0.01. Scale bar = 10 μm in C. Arrows indicate membrane accumulation of AQP2.