Parsing apical oxalate exchange in Caco-2BBe1 monolayers: siRNA knockdown of SLC26A6 reveals the role and properties of PAT-1

The purpose of this investigation was to quantitate the contribution of the anion exchanger PAT-1 (putative anion transporter-1), encoded by SLC26A6, to oxalate transport in a model intestinal epithelium and to discern some characteristics of this exchanger expressed in its native environment. Control (Con) Caco-2 BBe1 monolayers, 6–8 days postseeding, were compared with those transfected with a small interfering RNA targeted to SLC26A6 (A6KD). Radiotracer and Ussing chamber techniques were used to determine the transepithelial unidirectional fluxes of Ox2−, Cl−, and SO42− whereas fluorometric/BCECF measurements of intracellular pH were used to assess HCO3− exchange. PAT-1 was functionally targeted to the apical membrane, and SLC26A6 knockdown reduced PAT-1 protein (>60%) and mRNA (>75%) expression in A6KD. No net flux of Ox2−, Cl−, or SO42− was detected in Con or A6KD monolayers, yet the unidirectional fluxes in A6KD were reduced 50, 35, and 15%, respectively. Cl−-dependent HCO3− efflux from A6KD was reduced 50% compared with Con. The difference between Con and A6KD properties represents that mediated solely by PAT-1, and by this approach we found that PAT-1-mediated oxalate influx and efflux are inhibited equally by mucosal DIDS (EC50 ∼5 μM) and that mucosal Cl− inhibits oxalate uptake with an EC50 < 20 mM. Transepithelial Cl− gradients supported large, DIDS-sensitive net absorptive or secretory fluxes of oxalate in a direction opposite that of the imposed Cl− gradient. The overall symmetry of PAT-1-mediated oxalate exchange suggests that vectorial oxalate transport observed in vivo is principally dependent on the magnitude and direction of counterion gradients.

Parameter estimation for mathematical models of NKCC2 cotransporter isoforms

An optimization problem, formulated using a nonlinear least-squares approach, was used to estimate parameters for kinetic models of the three isoforms of the kidney-specific Na-K-2Cl (NKCC2) cotransporter. Specifically, the optimization problem estimates the magnitude of model parameters (i.e., off-binding and translocation rate constants) by minimizing the distance between model unidirectional fluxes and published unidirectional 86Rb+ uptake curves for the A, B, and F isoforms of the NKCC2 cotransporter obtained in transfected Xenopus oocytes. By using different symmetry assumptions, NKCC2 models with five, six, seven, or eight parameters were evaluated. The optimization method identified parameter sets that yielded computed unidirectional fluxes consistent with the uptake data. However, the parameter values were not unique, in that systematic exploration of the parameter space revealed alternative parameter sets that fit the data with similar accuracy. Finally, we demonstrate that the optimization method can identify parameter sets for the three transporter isoforms that differ only in ion binding affinities, a result that is consistent with a published mutagenesis analysis of the molecular and structural bases for the differences in 86Rb+ uptake among the A, B, and F isoforms. These NKCC2 cotransporter models will facilitate the development of larger scale models of ion transport by thick ascending limb cells.

Energy Generators Driven by Nonlinear Transport Phenomena

We describe quantitatively effects of nonlinear transfer phenomena that drive energy generators (thermal engines) and heat pumps. It is shown that these transports can be treated either in a standard way or as peculiar rate processes (chemical reactions governed by appropriate affinities). An approach to nonlinear transports links heat fluxes with differences of temperature in certain power Ta. A more recent approach distinguishes in each elementary transfer step two competitive (unidirectional) fluxes and the resulting flux as their difference. We show how the kinetics of this sort can be implemented into the contemporary theory of thermal energy generators.