Proteolytic systems modulating collecting duct sodium transport
Description
Serine proteases such as furin and prostasin have been shown to activate epithelial sodium channels (ENaC) in the collecting duct. Inhibition of apical proteases with aprotinin decreases Na+ current (Ieq), and subsequent application of the protease trypsin increases Ieq. However, transepithelial resistance (Rte) in M-1 mouse cortical collecting duct cells also decreases with aprotinin and increases with trypsin; Rte effects are opposite to those expected with inhibition and activation respectively of ENaC. The purpose of present studies was to characterize the regulation of Na+ transport and transepithelial permeability by proteolytic systems in the cortical collecting duct, the site of final regulation of Na+ excretion by the kidney Prostasin knockdown M-1 cells had no significant decrease in baseline Ieq and Rte. However, prostasin or trypsin significantly increased Rte in prostasin knockdown cells. These results suggest that prostasin is essential for resistance, but may be not critical for Na + current. Apical addition of ENaC inhibitory peptides LPHPLQRL (alpha-8) or PRFLNLIPLLVFNEN (gamma-15) abruptly inhibited Ieq. In contrast to trypsin and aprotinin, alpha-8 or gamma-15 had little or no effect on Rte. Apical addition of aprotinin after application of alpha-8 or gamma-15 inhibited Rte just as in cells untreated with inhibitory peptides. To elucidate if effects on Rte require Na+ transport, M-1 cells were treated with basolateral ouabain or apical amiloride (in the latter case after aprotinin pretreatment). Subsequent addition of trypsin increased Rte in both cases even in absence of transport. Basolateral application of TGF-beta1 significantly decreased Ieq. TGF-beta1 also increased paracellular permeability (indicated by the decrease in Rte), a response that is not expected based on an effect solely on ENaC. The lack of response to trypsin in TGF-beta1 treated cells implies that the downregulation of prostasin is not a major factor in the inhibition of Ieq by TGF-beta1 In conclusion, these studies indicate that proteolytic enzymes including endogenous prostasin alter Na+ transport in collecting duct cells both via the release of ENaC inhibitory peptides and in addition via the effects on Rte, probably reflecting paracellular permeability, independent of the effects on Na+ channels and Na+ transport