Cyclosporine stimulates the renal epithelial sodium channel by elevating cholesterol

Abstract

Cyclosporine A (CsA) is an efficient immunosuppressant used for reducing allograft rejection but with a severe side effect of causing hypertension. We hypothesize that the renal epithelial sodium channel (ENaC) may participate in CsA-induced hypertension. In the present study, we used the patch-clamp cell-attached configuration to examine whether and how CsA stimulates ENaC in A6 distal nephron cells. The data showed that CsA significantly increased ENaC open probability. Since CsA is an inhibitor of the ATP-binding cassette A1 (ABCA1) transporter, we employed 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), another ABCA1 inhibitor, and found that DIDS mimicked the effects of CsA on ENaC basal and cholesterol-induced activity but without any additive effect if combined with CsA. CsA and DIDS also had an identical effect on reduced ENaC activity caused by cholesterol extraction. ABCA1 protein was detected in A6 cells by Western blot analysis. Confocal microscopy data showed that both CsA and DIDS facilitated A6 cells to uptake cholesterol. Since enhanced ENaC activity is known to cause hypertension, these data together suggest that CsA may cause hypertension by stimulating ENaC through a pathway associated with inhibition of ABCA1 and consequent elevation of cholesterol in the cells.

Fig. 1.

Cyclosporine A (CsA) increases epithelial sodium channel (ENaC) open probability (P_O) in A6 distal nephrons cells. A: representative ENaC single-channel currents recorded from two cell-attached patches; one shows that addition of 10 μM CsA to the basolateral bath stimulated ENaC (top), whereas the other shows that CsA had no effect on ENaC activity (bottom). The mean ENaC P_O during each 3 min before and after CsA was calculated, as shown at the bottom. B: representative ENaC single-channel current recorded under control conditions (top) or after 1 h of pretreatment with 10 μM CsA applied to the basolateral bath (bottom). C: summary plots show that after CsA ENaC P_O was significantly increased. C- and O₁- indicate the channel at either closed or open state, respectively.

Fig. 2.

4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) mimics the effect of CsA on ENaC P_O. A: representative single-channel current of ENaC recorded under control conditions (top), after 1 h of treatment with 100 μM DIDS applied to the basolateral bath (middle), or after 1 h of treatment with both 100 μM DIDS and 10 μM CsA from basolateral membrane (bottom). B: summary plots show that ENaC P_O was significantly increased after 100 μM DIDS treatment. Addition of CsA to DIDS did not further increase ENaC P_O compared with DIDS alone. C: Western blotting analysis of A6 cell lysate detected by an antibody to mouse ABCA1 transporter. The two lanes represent two loadings: 25 μg (left) and 50 μg (right) of protein from the same sample showing consistent ABCA1 labeling in all three experiments.

Fig. 3.

Confocal microscopy optical cross (XZ) section of A6 cell monolayer cultured on filters, basolaterally treated with either 10 μM CsA (A) or 100 μM DIDS (B) for 1 h and then incubated with 100 μg/ml NBD cholesterol for 10 min. Images on the left show that NBD cholesterol was hardly observed in untreated (control) A6 cells. Images on the right show that a significant amount of NBD cholesterol was found in A6 cells pretreated with either CsA or DIDS. These images represent three experiments showing consistent results. The exact same parameters were used when each section was scanned.

Fig. 4.

A: basolateral addition of 100 μg/ml cholesterol alone had no effect on ENaC activity. Cholesterol significantly enhanced ENaC activity after basolateral pretreatment with either 10 μM CsA (B) or 100 μM DIDS (C). Representative ENaC currents are shown at left, whereas summary plots are presented at right.

Fig. 5.

A: extraction of cholesterol by basolateral addition of 20 mM MβCD significantly decreased ENaC P_O. MβCD had no effect on ENaC activity in cells pretreated with either basolateral 10 μM CsA (B) or 100 μM DIDS (C). Representative ENaC currents are shown at left, whereas summary plots are presented at right.

Fig. 6.

A hypothetical model of pathways for CsA stimulation of ENaC. In polarized distal nephron cells, ABCA1 is responsible for flopping (outwardly transporting) cholesterol (CHO) from the inner leaflet to the outer leaflet of the cell membrane. Abolishment of ABCA1 function by CsA or DIDS (1) causes cholesterol accumulation in the inner leaflet of the basolateral membrane (2) and probably also elevates cytosolic cholesterol levels. CsA or DIDS also facilitates the cells to uptake exogenous CHO by blocking ABCA1-mediated CHO flopping. The elevated CHO in the basolateral membrane migrates toward the apical membrane possibly via either intracellular trafficking (3a) or lateral diffusion along the inner leaflet passing through the tight junction (TJ) (3b). Subsequently, the increased Cho in the cytoplasm and/or in the inner leaflet of the apical membrane enhances ENaC activity.