Plasma kallikrein activates the epithelial sodium channel in vitro but is not essential for volume retention in nephrotic mice.

AIM: Recent work has demonstrated that activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases causes sodium retention in nephrotic syndrome. The aim of this study was to elucidate a potential role of plasma kallikrein (PKLK) as a candidate serine protease in this context. METHODS: We analysed PKLK in the urine of patients with chronic kidney disease (CKD, n = 171) and investigated its ability to activate human ENaC expressed in Xenopus laevis oocytes. Moreover, we studied sodium retention in PKLK-deficient mice (klkb1-/- ) with experimental nephrotic syndrome induced by doxorubicin injection. RESULTS: In patients with CKD, we found that PKLK is excreted in the urine up to a concentration of 2 µg mL-1 which was correlated with albuminuria (r = .71) and overhydration as assessed by bioimpedance spectroscopy (r = .44). PKLK increased ENaC-mediated whole-cell currents, which was associated with the appearance of a 67 kDa γ-ENaC cleavage product at the cell surface consistent with proteolytic activation. Mutating a putative prostasin cleavage site in γ-ENaC prevented channel stimulation by PKLK. In a mouse model for nephrotic syndrome, active PKLK was present in nephrotic urine of klkb1+/+ but not of klkb1-/- mice. However, klkb1-/- mice were not protected from ENaC activation and sodium retention compared to nephrotic klkb1+/+ mice. CONCLUSION: Plasma kallikrein is detected in the urine of proteinuric patients and mice and activates ENaC in vitro involving the putative prostasin cleavage site. However, PKLK is not essential for volume retention in nephrotic mice.

Impact of sacropelvic fixation on the development of postoperative sacroiliac joint pain following multilevel stabilization for degenerative spine disease.

OBJECTIVE: We hypothesised, that the inclusion of the ilium for multilevel lumbosacral fusions reduces the incidence of postoperative sacroiliac joint (SIJ) pain. The primary objective of this study was to compare the frequency of postoperative SIJ pain in patients undergoing multilevel stabilization with and without sacropelvic fixation for multilevel degenerative spine disease. In addition, we aimed at identifying factors that may predict the worsening or new onset of postoperative SIJ pain. METHODS: A total of 63 patients with multisegmental fusion surgery with a minimum follow up of 12 months were evaluated. 34 patients received sacral fixation (SF group) and 29 patients received an additional sacropelvic fixation device (SPF group). Primary outcome parameters were changes in SIJ pain between the groups and the influence of pelvic parameters, the patient́s age, the patient́s body mass index (BMI) and the length of the stabilization on the SIJ pain. RESULTS: Between the two surgical groups there were no differences concerning age (p=0.3), BMI (p=0.56), length of follow up (p=0.96), length of the construct (p=0.56). In total 31.7% of the patients had a worsening/new onset of SIJ pain after surgery. An additional fixation of the SIJ with iliac screws or iliosacral plate did not have an influence on the SIJ pain (p=0.67). Likewise, pelvic parameters were not predictive for the outcome of the SIJ pain. Only an increased preoperative BMI correlated with a higher chance of a new onset of SIJ pain (p=0.037). CONCLUSION: In our retrospective study there was no influence of a sacropelvic fixation techniques on the SIJ pain in patients with multilevel degenerative spine disease after multilevel stabilization surgeries. The patients' BMI is the only preoperative factor that correlated with a higher incidence to develop postoperative SIJ pain, independently of the implantation of a sacropelvic fixation device.

Segmental heterogeneity of swelling-induced Cl- transport in rat small intestine.

The effect of cell swelling induced by hypotonic media was studied in segments of rat small intestine. In the Ussing chamber, exposure to a hypotonic medium caused a decrease in short-circuit current (Isc) and potential difference (Vms) in the jejunum, whereas the ileum responded with an increase in Isc and Vms. The transition from one pattern to the other was located about in the middle of the small intestine. Tissue conductance decreased in both segments, probably due to a reduction of paracellular shunt conductance induced by the cell swelling. Voltage scanning experiments revealed that the observed decrease in total tissue conductance in the ileum was caused solely by a decrease in local conductance in the villus region while the crypt conductance did not change, suggesting that the decrease in paracellular conductance of the crypts is compensated by an increase in cellular conductance. The response in both segments was dependent on the presence of Cl- and was blocked by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). It was not affected by the neurotoxin tetrodotoxin. In the jejunum the swelling-induced decrease in Isc was reduced in the presence of the cyclooxygenase inhibitor, indomethacin, or the lipoxygenase inhibitor, nordihydroguaiaretic acid. In the ileum the Cl- secretion induced by hypotonicity was blocked by the K+ channel blocker quinine and was reversed into a decrease in Isc when serosal Ca2+ was zero. We conclude that the observed volume regulatory changes are initiated in the jejunum by an eicosanoid-mediated opening of basolateral Cl- channels and in the ileum by a Ca2+-mediated opening of K+ channels which enhances apical Cl- efflux.

Measurement of paracellular epithelial conductivity by conductance scanning.

A new method, conductance scanning, allows determination of local para- and transcellular conductivities in flat epithelia. Experiments were performed on kidney distal tubule cells, MDCK clone C11, which form monolayers on permeable supports. Above the apical surface, local voltage drops generated by a sinusoidal current clamp were recorded by means of a scanning microelectrode. Data were collected above cell centres and tight junctions. The scanning signal was always significantly higher above the tight junctions, but was uniformly distributed along the junctions. For determination of conductivities two procedures were applied. Method 1: the supraepithelial potential distribution was computed for given trans- and paracellular currents at all positions of the electrode. In a fit algorithm, the currents were varied until the calculated potential difference equalled the voltage measured. Method 2: after collecting scanning data in control Ringer's, intercellular space width was reduced by mucosal addition of 40 mM sucrose and a second set of data was obtained at decreased paracellular, but presumably unchanged transcellular, conductivity. From these data, trans- and paracellular conductivities were calculated. Results of both methods were in excellent agreement. Confluent MDCK-C11 monolayers exhibited a transepithelial conductivity of 13 mS/cm2. The transcellular pathway contributed 2.6 mS/cm2 (20%) and the paracellular pathway 10. 5 mS/cm2 (80%) to the total conductivity. Collapse of the lateral intercellular spaces decreased the paracellular conductivity to 4 mS/cm2 (60%). Confluent MDCK-C11 monolayers constitute true "leaky" epithelia with homogeneously distributed trans- and paracellular conductivities. In conclusion, conductance scanning fills a methodical gap, which hitherto impeded the functional characterization of tight junctions.

Localization of cAMP- and aldosterone-induced K+ secretion in rat distal colon by conductance scanning.

1. Aldosterone- and adrenaline-induced K+ secretion were investigated in rat late distal colon using conductance scanning and Ussing chamber techniques. K+ secretion was unmasked by the K+ channel blocker tetraethylammonium (TEA). Electrogenic Na+ absorption was inhibited by amiloride. Rb+ net fluxes consistently measured about 80% of K+ secretion estimated using change in short-circuit current (delta ISC) measurements. 2. Partial block of K+ absorption by mucosal ouabain did not change TEA-sensitive K+ secretion. Thus, K+ absorption and K+ secretion are not coupled. 3. Additivity of Rb+ fluxes as well as delta ISC caused by 3 nM aldosterone (6 h in vitro incubation) and, subsequently, adrenaline suggested additivity of aldosterone-induced and cAMP-mediated K+ secretion in the presence of amiloride. 4. Conductance scanning under control conditions revealed a small TEA-sensitive K+ conductivity in surface epithelium (0.3 +/- 0.2 mS cm-2) but not in crypts, as well as a small basal K+ secretion in surface epithelium (delta ISC = 0.3 mumol h-1 cm-2), which increased during sham incubation. 5. Aldosterone (3 nM, 6 h in vitro incubation) resulted, after correction for the basal K+ secretion, in a K+ secretion of delta ISC = 0.9 mumol h-1 cm-2. Aldosterone induced a TEA-sensitive conductivity of 1.1 +/- 0.3 mS cm-2 in surface epithelium, but not in crypts. 6. Adrenaline (5 microM) caused, in fresh tissue, a K+ secretion of delta ISC = 1.2 mumol h-1 cm-2 and equal conductivity changes in crypts (0.7 +/- 0.2 mS cm-2) and surface epithelium (0.7 +/- 0.1 mS cm-2). 7. We conclude that K+ secretion induced by aldosterone in physiological concentration is restricted to surface epithelium, whereas cAMP-mediated K+ secretion is located equally in crypts and surface epithelium.

Ovine male genital duct epithelial cells differentiate in vitro and express functional CFTR and ENaC.

To investigate the biology of the male genital duct epithelium, we have established cell cultures from the ovine vas deferens and epididymis epithelium. These cells develop tight junctions, high transepithelial electrical resistance, and a lumen-negative transepithelial potential difference as a sign of active transepithelial ion transport. In epididymis cultures the equivalent short-circuit current (I(sc)) averaged 20.8+/-0.7 microA/cm(2) (n = 150) and was partially inhibited by apical application of amiloride with an inhibitor concentration of 0.64 microM. In vas deferens cultures, I(sc) averaged 14.4+/-1.1 microA/cm(2) (n = 18) and was also inhibited by apical application of amiloride with a half-maximal inhibitor concentration (K(i)) of 0.68 microM. The remaining amiloride-insensitive I(sc) component in epididymis and vas deferens cells was partially inhibited by apical application of the Cl(-) channel blocker diphenylamine-2-carboxylic acid (1 mM). It was largely dependent on extracellular Cl(-) and, to a lesser extent, on extracellular HCO(-)(3). It was further stimulated by basolateral application of forskolin (10(-5) M), which increased I(sc) by 3.1+/-0.3 microA/cm(2) (n = 65) in epididymis and 0.9+/-0.1 microA/cm(2) (n = 11) in vas deferens. These findings suggest that cultured ovine vas deferens and epididymis cells absorb Na(+) via amiloride-sensitive epithelial Na(+) channels (ENaC) and secrete Cl(-) and HCO(-)(3) via apical cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. This interpretation is supported by RT-PCR data showing that vas deferens and epididymis cells express CFTR and ENaC mRNA.

Basolateral proteinase-activated receptor (PAR-2) induces chloride secretion in M-1 mouse renal cortical collecting duct cells.

1. Using RT-PCR, Northern blot analysis, and immunocytochemistry, we confirmed renal expression of proteinase-activated receptor (PAR-2) and demonstrated its presence in native renal epithelial and in cultured M-1 mouse cortical collecting duct (CCD) cells. 2. We investigated the effects of a PAR-2 activating peptide (AP), corresponding to the tethered ligand that is exposed upon trypsin cleavage, and of trypsin on M-1 cells using patch-clamp, intracellular calcium (fura-2) and transepithelial short-circuit current (ISC) measurements. 3. In single M-1 cells, addition of AP elicited a concentration-dependent transient increase in the whole-cell conductance. Removal of extracellular Na+ had no effect while removal of Cl- prevented the stimulation of outward currents. The intracellular calcium concentration increased significantly upon application of AP while a Ca2+-free pipette solution completely abolished the electrical response to AP. 4. In confluent monolayers of M-1 cells, apical application of AP had no effect on ISC whereas subsequent basolateral application elicited a transient increase in ISC. This increase was not due to a stimulation of electrogenic Na+ absorption since the response was preserved in the presence of amiloride. 5. The ISC response to AP was reduced in the presence of the Cl- channel blocker diphenylamine-2-carboxylic acid on the apical side and abolished in the absence of extracellular Cl-. 6. Trypsin elicited similar responses to those to AP while application of a peptide (RP) with the reverse amino acid sequence of AP had no effect on whole-cell currents or ISC. 7. In conclusion, our data suggest that AP or trypsin stimulates Cl- secretion by Ca2+-activated Cl- channels in M-1 CCD cells by activating basolateral PAR-2.