Shear stress-induced volume decrease in C11-MDCK cells by BK-alpha/beta4

Abstract

Large-conductance, calcium-activated potassium channels (BK) are expressed in principal cells (PC) and intercalated cells (IC) in mammalian nephrons as BK-alpha/beta1 and BK-alpha/beta4, respectively. IC, which protrude into the lumens of tubules, express substantially more BK than PC despite lacking sufficient Na-K-ATPase to support K secretion. We previously showed in mice that IC exhibit size reduction when experiencing high distal flows induced by a high-K diet. We therefore tested the hypothesis that BK-alpha/beta4 are regulators of IC volume via a shear stress (tau)-induced, calcium-dependent mechanism, resulting in a reduction in intracellular K content. We determined by Western blot and immunocytochemical analysis that C11-Madin-Darby canine kidney cells contained a predominance of BK-alpha/beta4. To determine the role of BK-alpha/beta4 in tau-induced volume reduction, we exposed C11 cells to tau and measured K efflux by flame photometry and cell volume by calcein staining, which changes inversely to cell volume. With 10 dynes/cm(2), calcein intensity significantly increased 39% and monovalent cationic content decreased significantly by 37% compared with static conditions. Furthermore, the shear-induced K loss from C11 was abolished by the reduction of extracellular calcium, addition of 5 mM TEA, or BK-beta4 small interfering (si) RNA, but not by addition of nontarget siRNA. These results show that BK-alpha/beta4 plays a role in shear-induced K loss from IC, suggesting that BK-alpha/beta4 regulate IC volume during high-flow conditions. Furthermore, these results support the use of C11 cells as in vitro models for studying BK-related functions in IC of the kidney.

Fig. 1.

Potassium efflux from Madin-Darby canine kidney (MDCK) cells exposed to variable shear stresses (τ). MDCK cells were exposed to either 0 (static), 1, 2.5, 5, or 10 dynes/cm² in a parallel-plate flow chamber (PPFC). Compared with the static condition, 5 and 10 dynes/cm² produced K efflux. Values are means ± SE (n = 5). *Significant difference (P < 0.05).

Fig. 2.

Expression of calcium-activated potassium channel BK-α and -β subunits in C7 and C11 cells by immunocytochemistry. A: yz-plane stacked view of multiple 1-μm slices of cells where C7 stained with rabbit IgG control or BK-α (green) and BK-β1 (red) antibodies. B: C11 stained with rabbit IgG control or BK-α (green) and BK-β4 (red) antibodies. C: Western blot of BK-α, BK-β4, BK-β4 with blocking peptide, and actin. D: Western blot band density was normalized to actin. All symbols are same as in Fig. 1 (n = 3).

Fig. 3.

Calcein responses to hypotonic (HYPO) and hypertonic (HYPER) conditions. Calcein assay was verified by exposing C11 cells to isotonic (ISO), HPO, or HYPER solutions for 30 min. Calcein fluorescent intensity was plot for each buffer and normalized to ISO conditions. All symbols are same as in Fig. 1 (n = 5).

Fig. 4.

Volume status measured by calcein intensity of C11 cells exposed to either static (A) or flow (10 dynes/cm²; B) in a PPFC for 5 and 30 min. Compared with baseline, calcein intensity in static conditions decreased after 5 and 30 min by 17 and 25%, respectively; however, with shear stress, calcein intensity increased after 5 (25%) and 30 min (14%). *Significant difference (P < 0.05) from corresponding static condition (n = 6).

Fig. 5.

Potassium (n = 6, A), Na (n = 6; B), and K+Na efflux (C) from C11 exposed to 10 dynes/cm² compared with static in a PPFC at 30 min and 5 h. All symbols are same as in Fig. 1.

Fig. 6.

Potassium efflux from C11 cells exposed to static or 10 dynes/cm² in a PPFC for 5 h. Compared with static, flow-induced K efflux in control (n = 6), but not with reduced (45 μM) extracellular calcium (n = 6) or 5 mM tetraethylammonium (TEA; n = 7). All symbols are same as in Fig. 1.

Fig. 7.

Effects of BK-β4 small interfering (si) RNA on shear-stress (10 dynes/cm²)-induced K efflux from C11. A: C11 were untreated (normal), transfected with green fluorescent protein (control), 4 nontarget siRNA oligos (siRNA nontarget), or 6 siRNA oligos for the BK-β4 subunit (siRNA BK-β4). As assessed by Western blot analysis, BK-β4 siRNA silenced ∼80% of BK-β4 compared with control. There was no effect of siRNA on BK-α expression. B: flow-induced K efflux at 5 h was blocked by BK-β4 siRNA (n = 4), but not by nontarget siRNA (n = 4). All symbols are same as in Fig. 1.

Fig. 8.

Shear stress (10 dynes/cm²)-dependent, transient calcium increases in C11. A: shear stress transiently increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), which returned to near baseline levels. B and C: bar plots summarizing shear stress-induced average (B) and peak [Ca²⁺]_i (C). All symbols are same as in Fig. 1.