Coupling of epithelial Na+ and Cl- channels by direct and indirect activation by serine proteases.

The mammalian collecting duct (CD) is continuously exposed to urinary proteases. The CD expresses an epithelial Na(+) channel (ENaC) that is activated after cleavage by serine proteases. ENaC also exists at the plasma membrane in the uncleaved form, rendering activation by extracellular proteases an important mechanism for regulating Na(+) transport. Many exogenous and a small number of endogenous extracellular serine proteases have been shown to activate the channel. Recently, kallikrein 1 (KLK1) was shown to increase γENaC cleavage in the native CD indicating a possible direct role of this endogenous protease in Na(+) homeostasis. To explore this process, we examined the coordinated effect of this protease on Na(+) and Cl(-) transport in a polarized renal epithelial cell line (Madin-Darby canine kidney). We also examined the role of native urinary proteases in this process. Short-circuit current (I(sc)) was used to measure transport of these ions. The I(sc) exhibited an ENaC-dependent Na(+) component that was amiloride blockable and a cystic fibrosis transmembrane conductance regulator (CFTR)-dependent Cl(-) component that was blocked by inhibitor 172. Apical application of trypsin, an exogenous S1 serine protease, activated I(ENaC) but was without effects on I(CFTR). Subtilisin an exogenous S8 protease that mimics endogenous furin-type proteases activated both currents. A similar activation was also observed with KLK1 and native rat urinary proteases. Activation with urinary proteases occurred within minutes and at protease concentrations similar to those in the CD indicating physiological significance of this process. ENaC activation was irreversible and mediated by enhanced cleavage of γENaC. The activation of CFTR was indirect and likely dependent on activation of an endogenous apical membrane protease receptor. Collectively, these data demonstrate coordinated stimulation of separate Na(+) and Cl(-) transport pathways in renal epithelia by extracellular luminal proteases. They also indicate that baseline urinary proteolytic activity is sufficient to modify Na(+) and Cl(-) transport in these epithelia.

Methods for stable recording of short-circuit current in a Na+-transporting epithelium.

Epithelial Na(+) transport as measured by a variety of techniques, including the short-circuit current technique, has been described to exhibit a "rundown" phenomenon. This phenomenon manifests as time-dependent decrease of current and resistance and precludes the ability to carry out prolonged experiments aimed at examining the regulation of this transport. We developed methods for prolonged stable recordings of epithelial Na(+) transport using modifications of the short-circuit current technique and commercial Ussing-type chambers. We utilize the polarized MDCK cell line expressing the epithelial Na(+) channel (ENaC) to describe these methods. Briefly, existing commercial chambers were modified to allow continuous flow of Ringer solution and precise control of such flow. Chamber manifolds and associated plumbing were modified to allow precise temperature clamp preventing temperature oscillations. Recording electrodes were modified to eliminate the use of KCl and prevent membrane depolarization from KCl leakage. Solutions utilized standard bicarbonate-based buffers, but all gasses were prehydrated to clamp buffer osmolarity. We demonstrate that these modifications result in measurements of current and resistance that are stable for at least 2 h. We further demonstrate that drifts in osmolarity similar to those obtained before prior to our modifications can lead to a decrease of current and resistance similar to those attributed to rundown.