Prostaglandin E2 stimulates the epithelial sodium channel (ENaC) in cultured mouse cortical collecting duct cells in an autocrine manner.

Prostaglandin E2 (PGE2) is the most abundant prostanoid in the kidney, affecting a wide range of renal functions. Conflicting data have been reported regarding the effects of PGE2 on tubular water and ion transport. The amiloride-sensitive epithelial sodium channel (ENaC) is rate limiting for transepithelial sodium transport in the aldosterone-sensitive distal nephron. The aim of the present study was to explore a potential role of PGE2 in regulating ENaC in cortical collecting duct (CCD) cells. Short-circuit current (ISC) measurements were performed using the murine mCCDcl1 cell line known to express characteristic properties of CCD principal cells and to be responsive to physiological concentrations of aldosterone and vasopressin. PGE2 stimulated amiloride-sensitive ISC via basolateral prostaglandin E receptors type 4 (EP4) with an EC50 of ∼7.1 nM. The rapid stimulatory effect of PGE2 on ISC resembled that of vasopressin. A maximum response was reached within minutes, coinciding with an increased abundance of ß-ENaC at the apical plasma membrane and elevated cytosolic cAMP levels. The effects of PGE2 and vasopressin were nonadditive, indicating similar signaling cascades. Exposing mCCDcl1 cells to aldosterone caused a much slower (∼2 h) increase of the amiloride-sensitive ISC. Interestingly, the rapid effect of PGE2 was preserved even after aldosterone stimulation. Furthermore, application of arachidonic acid also increased the amiloride-sensitive ISC involving basolateral EP4 receptors. Exposure to arachidonic acid resulted in elevated PGE2 in the basolateral medium in a cyclooxygenase 1 (COX-1)-dependent manner. These data suggest that in the cortical collecting duct, locally produced and secreted PGE2 can stimulate ENaC-mediated transepithelial sodium transport.

Association of Plasminuria with Overhydration in Patients with CKD.

BACKGROUND AND OBJECTIVES: Hypervolemia is a common feature of patients with CKD and associated with hypertension. Recent work has shown stimulation of sodium retention by urinary plasmin during nephrotic syndrome. However, it is unclear whether plasminuria plays a role in patients with stable CKD and non-nephrotic proteinuria. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: In this cross-sectional study, we analyzed the fluid status of 171 patients with CKD consecutively presenting to our outpatient clinic from 2012 to 2013 using bioimpedance spectroscopy (Body Composition Monitor [BCM]; Fresenius Medical Care, Germany) and its associations to the urinary excretion of plasminogen and plasmin from a spot urine sample. Two-electrode voltage clamp measurements were performed in Xenopus laevis oocytes expressing human epithelial sodium channel to investigate whether plasmin in concentrations found in urine can activate the channel. RESULTS: Overhydration >5% and overhydration >10% of the extracellular volume were found in 29% and 17% of the patients, respectively, and overhydration was associated with edema, hypertension, higher stages of CKD, and proteinuria. Proteinuria was the strongest independent predictor for overhydration (+0.58 L/1.73 m(2) per 10-fold increase; P<0.001). Urinary excretion of plasmin(ogen) quantified by ELISA correlated strongly with proteinuria (r=0.87) and overhydration (r=0.47). Using a chromogenic substrate, active plasmin was found in 44% of patients and correlated with proteinuria and overhydration. Estimated urinary plasmin concentrations were in a range sufficient to activate epithelial sodium channel currents in vitro. In multivariable analysis, urinary excretion of plasmin(ogen) was associated with overhydration similar to proteinuria. CONCLUSIONS: Hypervolemia in patients with CKD is strongly associated with proteinuria, even in the non-nephrotic range. Protein-rich urine contains high amounts of plasminogen and active plasmin, rendering plasminuria as a possible link between proteinuria and hypervolemia.

Proteolytic activation of the human epithelial sodium channel by trypsin IV and trypsin I involves distinct cleavage sites.

Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK(178)), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases.