Claudin-8 modulates paracellular permeability to acidic and basic ions in MDCK II cells

Abstract

Renal net acid excretion requires tubular reabsorption of filtered bicarbonate, followed by secretion of protons and ammonium in the collecting duct, generating steep transtubular gradients for these ions. To prevent passive backleak of these ions, the tight junctions in the collecting duct must be highly impermeable to these ions. We previously generated a Madin-Darby canine kidney (MDCK II) cell line with inducible expression of claudin-8, a tight junction protein expressed in the collecting duct. In these cells, claudin-8 was shown to function as a paracellular barrier to alkali metal and divalent cations. We have now used this model to test the hypothesis that claudin-8 also functions as a paracellular barrier to acidic or basic ions involved in renal acid excretion. We developed a series of precise and unbiased methods, based on a combination of diffusion potential, short-circuit current, and pH stat measurements, to estimate paracellular permeability to protons, ammonium and bicarbonate in MDCK II cells. We found that under control conditions (i.e. in the absence of claudin-8), these cells are highly permeable to the acidic and basic ions tested. Interestingly, proton permeation exhibited an unusually low activation energy similar to that in bulk solution. This suggests that paracellular proton transfer may occur by a Grotthuss mechanism, implying that the paracellular pores are sufficiently wide to accommodate water molecules in a freely mobile state. Induction of claudin-8 expression reduces permeability not only to protons, but also to ammonium and bicarbonate. We conclude that claudin-8 probably functions to limit the passive leak of these three ions via paracellular routes, thereby playing a permissive role in urinary net acid excretion.

Figure 1. Typical data from a pH gradient experiment

I_SC, short-circuit current; pH, apical chamber pH; I_H, short-circuit current attributable to transepithelial pH gradient. Arrows indicate time points at which NaOH was added to the apical chamber.

Figure 2. Determination of passive, transepithelial H⁺ permeability by three independent methods

A, HCl diffusion potential, short-circuit current, and pH stat-titrated net proton flux measured after imposing a 10⁴-fold transepithelial H⁺ gradient, in claudin-8-induced (Dox−) and uninduced (Dox+) MDCK II cells. B, comparison of the H⁺ permeability determined by the three methods. Columns in A and B represent mean ±s.e.m. (n = 3). *P < 0.05; **P < 0.00005.

Figure 3. Properties of the H⁺ short-circuit current (I_SC)

A, magnitude of I_SC is similar when the H⁺ gradient is imposed in different directions. B, net H⁺ flux (J), calculated from I_SC, is proportional to the H⁺ concentration difference (Δ[H⁺]). Lines were fitted by linear regression to the equation, J =P×Δ[H⁺]. The indicated estimates for the permeability P_H (× 10⁻⁶ cm s⁻¹) were derived from the slopes.

Figure 4. Effect of amiloride in H⁺ gradient experiment

The cis compartment was acidified, and I_SC (positive values indicate current flow from basolateral to apical side) and pH in the trans compartment were monitored. Amiloride (1 mm) was then added (arrows), either to the apical chamber (AP), basolateral chamber (BL), or to both (AP + BL).

Figure 5. Effect of ZnCl₂ in H⁺ gradient experiment

The cis compartment was acidified, and I_SC and pH in the trans compartment monitored. ZnCl₂ (1 mm) was then added (arrows) to the cis compartment (AP, apical; BL, basolateral).

Figure 6. Effect of induction of claudin-8 expression on HCO₃⁻ and NH₄⁺ permeability

A, determination of NH₄⁺ permeability by three different methods. B, determination of HCO₃⁻ permeability, concurrently with Na⁺ and Cl⁻ permeability, by dilution potential. *P < 0.005; **P < 0.0005 (n = 4).

Figure 7. Effect of claudin-8 induction on permeabilities to various cations

Comparison of the effect of claudin-8 induction on permeabilities to various cations, expressed as the ratio of the mean permeability in Dox− (induced) cells to the mean permeability in Dox+ (control) cells. Error bars represent 95% confidence intervals. *P < 0.05 versus Na⁺, Cs⁺ and NH₄⁺. Data are representative of three independent experiments.

Figure 8. Temperature dependence of paracellular cation permeability

A, Arrhenius plots from Dox+ (•) and Dox− (○) monolayers. The ordinate represents permeability to Na⁺, Cs⁺ and H⁺ on a logarithmic scale. The abscissa represents the reciprocal of the absolute temperature. Data points represent measurements performed at (from left to right) 37°C, 24°C and 16°C. All three plots are scaled to the same proportions so that the slopes of the lines can be directly compared. The lines were obtained by fitting the data by non-linear regression to the equation: ln(y) = ln(K) −E_a/RT, where y is the ordinate variable, K is a pre-exponential constant, and E_a is the activation energy (n = 3). B, estimates of activation energy, derived from the slopes of the lines in A. *P < 0.0005 for H⁺ (Dox+) versus H⁺ (Dox−), and for H⁺ (Dox+) versus Na⁺ (Dox+).