The oxidative stress responsive kinase 1 (OSR1) contributes to WNK (with no K)-dependent regulation of renal tubular salt transport, renal salt excretion, and blood pressure. Little is known, however, about a role of OSR1 in the regulation of intestinal salt transport. The present study thus explored whether OSR1 is expressed in intestinal tissue and whether small intestinal Na+/H+ exchanger (NHE), small intestinal Na+-glucose cotransport (SGLT1), and/or colonic epithelium Na+ channel (ENaC) differ between knockin mice carrying one allele of WNK-resistant OSR1 (osr1+/KI) and wild-type mice (osr1+/+). OSR1 protein abundance was determined by Western blotting, cytosolic pH from BCECF fluorescence, NHE activity from Na+-dependent realkalinization following an ammonium pulse, SGLT1 activity from glucose-induced current, and colonic ENaC activity from amiloride-sensitive transepithelial current in Ussing chamber experiments. As a result, OSR1 protein was expressed in small intestine of both osr1+/KI mice and osr1+/+ mice. Daily fecal Na+, K+, and H2O excretion and jejunal SGLT1 activity were lower, whereas small intestinal NHE activity and colonic ENaC activity were higher in osr1+/KI mice than in osr1+/+ mice. NHE3 inhibitor S-3226 significantly reduced NHE activity in both genotypes but did not abrogate the difference between the genotypes. Plasma osmolarity, serum antidiuretic hormone, plasma aldosterone, and plasma corticosterone concentrations were similar in both genotypes. Small intestinal NHE3 and colonic α-ENaC protein abundance were not significantly different between genotypes, but colonic phospho-β-ENaC (ser633) was significantly higher in osr1+/KI mice. In conclusion, OSR1 is expressed in intestine and partial WNK insensitivity of OSR1 increases intestinal NHE activity and colonic ENaC activity.
- Na+/H+ exchanger
the oxidative stress responsive kinase 1 (OSR1) contributes to signaling of transport regulation during oxidative and osmotic stress (9, 25, 27, 40, 43, 47, 55). The kinase is phosphorylated and thus activated by the WNK (with no K) kinase isoforms WNK1 and WNK4, which are in turn activated by osmotic cell shrinkage (33, 40, 52, 59, 59), insulin (46), and antidiuretic hormone (ADH) (35). During evolution OSR1 was duplicated by the STE20/SPS1-related proline-alanine-rich kinase (SPAK) (7). Similar to SPAK (30), OSR1 upregulates the thiazide-sensitive Na+-Cl− cotransporter NCC (10) and the furosemide-sensitive Na+-K+-2Cl− cotransporter NKCC (2, 36), with both carriers contributing to increasing cell volume (29). By the same token, the kinases downregulate KCl cotransporters (15, 18, 27), i.e., carriers decreasing cell volume (23, 29). Moreover, OSR1 and SPAK increase the HCO3− permeability of the cystic fibrosis transmembrane conductance regulator CFTR (34). GCK-3, a Caenorhabditis elegans ortholog of mammalian SPAK and OSR1 (6), inhibits the ClC anion channel CLH-3b (11). Owing to their influence on the respective transport systems, the kinases participate in the regulation of cell volume, transepithelial transport, renal salt excretion, migration, and GABA neurotransmission (3, 7, 9, 16, 20, 22, 26, 27, 40, 53).
Certain mutations of WNK1 and WNK4 result in hypertension (13, 14, 27, 49, 56) and autonomic neuropathy (27). OSR1 is similarly implicated in the regulation of blood pressure (17, 50–52) and is a potential drug target in the treatment of hypertension (12, 17, 40, 50).
OSR1 and SPAK upregulate the Na+-Cl− cotransporter and the Na+-K+-2Cl− cotransporter by phosphorylation of the carriers (9, 10, 26, 27, 30, 31, 35, 40, 49). The kinases phosphorylate the amino acid sequence [S/G/V]RFx[V/I]xx[V/I/T/S]xx, where x represents any amino acid (8). The carriers are phosphorylated at the conserved Ser/Thr residues in the NH2-terminal domain of the carrier proteins (31, 32, 35).
In C. elegans, OSR1 is expressed in intestine (47). Surprisingly though, to the best of our knowledge, nothing is known on the potential role of OSR1 in intestinal transport regulation. Intestinal Na+ reabsorption is in large part accomplished by the small intestinal Na+/H+ exchanger NHE3 (21, 58), colonic Na+ reabsorption involves the amiloride-sensitive epithelial Na+ channel ENaC (28). Notably, in wnk4D561A/+ knockin mice the increased phosphorylation of the kinases OSR1 and SPAK and increased apical expression of phosphorylated Na+-Cl− cotransporter in the renal distal convoluted tubules was paralleled by an increased activity of ENaC in the kidney (57).
The present study thus explored the role of OSR1 in the regulation of small intestinal Na+/H+ exchanger (NHE) activity as well as amiloride-sensitive Na+ current in the terminal colon. To this end, experiments were performed in heterozygous OSR1 knockin mice resistant to WNK-mediated activation (osr1+/KI) and wild-type mice (osr1+/+) (38). The osr1+/KI mice may have decreased OSR1 activity; however, OSR1 protein expression is unaltered (48). As indicated earlier (38), homozygous OSR1 knockin mice (osr1KI/KI) are not viable.
MATERIALS AND METHODS
All animal experiments were conducted according to the German law for the welfare of animals and were approved by local authorities. Blood was drawn or tissue isolated from sex-matched 2- to 9-mo-old heterozygous OSR1 knockin mice (osr1+/KI) and wild-type mice (osr1+/+), kindly provided by Dario Alessi from the MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee, UK. As described earlier (38), in the knockin mice the T-loop Thr residue in OSR1 (Thr185) was mutated to Ala to prevent activation by WNK isoforms. Mice had free access to control diet (Walkermühle, Hechingen, Germany) and tap drinking water ad libitum. To obtain blood, mice were anesthetized with diethylether (Roth, Karlsruhe, Germany) and blood specimens were drawn from the retroorbital plexus into capillaries. To obtain tissue, the animals were anesthetized with diethylether and euthanized by cervical dislocation. Prior to further use, small intestinal segments were washed to remove fecal material.
Anti-OSR1 (1:900) (48), kindly provided by D. Alessi (University of Dundee); polyclonal anti-epithelial α-ENaC (1:500) (Abcam); polyclonal antibody against sodium channel protein (pan) 1:800 (Acris Antibodies), anti-phospho-β ENaC (SCNN1B; Ser633) polyclonal (1:400) (Bioss Antibodies), NHE3 (1:1,000) (Nobus Biologicals), β-actin (1:1,000) (Cell Signaling Technology, Frankfurt, Germany), GAPDH (1:3,000) (Cell Signaling Technology), horseradish peroxidase (HRP)-linked anti-rabbit (1:2,000) (Cell Signaling Technology), and anti-mouse secondary antibody (1:2,000) (GE Healthcare, Freiburg, Germany) were used.
Determination of serum ADH, aldosterone, and corticosterone concentrations.
Serum ADH concentration was determined by utilizing a commercial EIA kit (AVP EIA Kit, Phoenix Europe, Karlsruhe, Germany). The plasma aldosterone and corticosterone concentrations were determined by using a commercial radioimmunoassay kit (Demeditec, Kiel, Germany). All kits were used according to the manufacturer's instructions.
Mice were euthanized by cervical dislocation under ether anesthesia, and the abdomen was opened. The small intestine was cut longitudinally. The tissue was homogenized with an electric homogenizer at 4°C in lysis buffer [54.6 mM HEPES; 2.69 mM Na4P2O7; 360 mM NaCl; 10% (vol/vol) glycerol; 1% (vol/vol) Nonidet P-40] containing phosphatase and protease inhibitors (Roche, Mannheim, Germany). The small intestinal brush border membrane was prepared as described before (44).
Homogenates were clarified by centrifugation at 14,000 rpm for 20 min. The samples containing 100 μg protein were subjected to 10% SDS-PAGE and blotted onto nitrocellulose membranes (Schleicher & Schuell). The membranes were blocked for 2 h in Tris-buffered saline (TBS) containing 7% fat-free powder milk. Then, the membranes were incubated overnight with primary antibody against mouse OSR1 (residues 389–408 SAHLPQPAGQMPTQPAQVSL, S149C, detecting a molecule of ∼58 kDa; Ref. 48) and GAPDH followed by incubation with secondary HRP anti-mouse IgG. All antibodies were diluted in TBS containing 5% milk.
Small intestinal NHE3 activity.
For the isolation of ileal villi, animals were fasted for 6 h prior to the experiments. After euthanasia the terminal 2 cm of the ileum were removed and cut longitudinally. After wash with standard HEPES solution the small intestine was sliced into 0.3-cm2 sections. The tissues were transferred onto the cooled stage of a dissecting microscope and the individual villi were detached from the small intestine by snapping off the ileal base with sharpened microdissection tweezers. Care was taken not to touch the apical part of the villi. The villi were attached to a glass coverslip precoated with Cell-Tak adhesive (BD Biosciences, Heidelberg, Germany). For quantitative digital imaging of cytosolic pH (pHi), isolated individual villi were incubated in a HEPES-buffered Ringer solution containing 10 μM 2′,7′-bis-(carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethylester (BCECF-AM; Molecular Probes, Leiden, The Netherlands) for 15 min at 37°C. After loading, the chamber was flushed for 5 min with Ringer solution to remove any deesterified dye sticking to the outside of the villi (42). The perfusion chamber was mounted on the stage of an inverted microscope (Axiovert 135, Zeiss, Göttingen, Germany), which was used in the epifluorescence mode with a ×40 oil immersion objective (Neoplan, Zeiss). BCECF was successively excited at 490/10 and 440/10 nm, and the resultant fluorescent signal was monitored at 535/10 nm by use of an intensified charge-coupled device camera (Proxitronic, Bensheim, Germany) and specialized computer software (Metafluor, Puchheim, Germany). Individual cells from the brush border of the villi were outlined and monitored during the course of the measurement. Intensity ratio data (490/440) were converted into pH values by the high-K+/nigericin calibration technique (54). To this end, the cells were perfused at the end of each experiment for 5 min with standard high-K+/nigericin (10 μg/ml) solution (pH 7.0). The intensity ratio data thus obtained were converted into pH values by using the rmax, rmin, and pKa values previously generated from calibration experiments to generate a standard nonlinear curve (pH range 5 to 8.5).
For acid loading, cells were transiently exposed to a solution containing 20 mM NH4Cl leading to initial alkalinization of pHi due to entry of NH3 and binding of H+ to form NH4+ (41). The acidification of cytosolic pH upon removal of ammonia allowed calculating the mean intrinsic buffering power (β) of the cells (41). Assuming that NH4+ and NH3 are in equilibrium in cytosolic and extracellular fluid and that ammonia leaves the cells as NH3:
where ΔpHi is the decrease of pHi following ammonia removal and Δ[NH4+]i is the decrease of cytosolic NH4+ concentration, which is identical to the concentration of [NH4+]i immediately before the removal of ammonia. The pK for NH4+/NH3 is 8.9 (5) and at an extracellular pH (pHo) of 7.4 the NH4+ concentration in extracellular fluid ([NH4+]o) is 19.37 mM [20/(1+10 pHo−pK)]. The intracellular NH4+ concentration ([NH4]i) was calculated from
To calculate the ΔpH/min during realkalinization, a manual linear fit was placed over a narrow pH range (pH 6.7 to 6.9), which could be applied to all measured cells.
The solutions were composed (in mM) as follows: standard HEPES: 115 NaCl, 5 KCl, 1 CaCl2, 1.2 MgSO4, 2 NaH2PO4, 10 glucose, 32.2 HEPES; sodium-free HEPES: 132.8 N-methyl-d-glucamine (NMDG), 3 KCl, 1 CaCl2, 1.2 MgSO4, 2 KH2PO4, 32.2 HEPES, 10 mannitol, 10 glucose (for sodium-free ammonium chloride, 10 mM NMDG and mannitol were replaced with 20 mM NH4Cl); high K+ for calibration 105 KCl, 1 CaCl2, 1.2 MgSO4, 32.2 HEPES, 10 mannitol, 5 μM nigericin. The pH of the solutions was titrated to 7.4 or 7.0 with HCl/NaOH, HCl/NMDG, and HCl/KOH, respectively, at 37°C. The NHE3 inhibitor s3226 was a kind gift of Hoechst (Frankfurt, Germany).
Ussing chamber experiments.
ENaC activity was estimated from the amiloride-sensitive potential difference across the colonic epithelium. After removal of the outer serosal and the muscular layer of distal colon under a microscope, tissues were mounted onto a custom-made mini-Ussing chamber with an opening diameter of 0.99 mm and an opening area of 0.00769 cm2. The serosal and luminal perfusate contained (in mM) 145 NaCl, 1 MgCl2, 2.6 Ca-gluconate, 0.4 KH2PO4, 1.6 K2HPO4, 5 glucose. To assess ENaC-mediated transport, 50 μM amiloride (in ethanol; Sigma, Schnelldorf, Germany) was added to the luminal perfusate. Before mounting the tissue into the Ussing chamber, the empty chamber was placed into the apparatus, and the potential difference across the empty chamber was set to 0 mV. In all Ussing chamber experiments, the transepithelial potential difference (Vt) was determined continuously, and the apparent transepithelial resistance (Rt) was estimated from the voltage deflections (ΔVt) elicited by imposing test currents (It) of 1 μA. The resulting Rt was calculated according to Ohm's law. For the determination of the Rt the resistance of the empty chamber was subtracted.
Fecal weight, Na+, and K+.
Fecal Na+ was analyzed as described previously (39). Briefly, feces was collected in metabolic cages (Techniplast, Hohenpeissenberg, Germany). Fecal dry weight was taken after drying the sample at 80°C for ∼3 h. Then the dried feces was dissolved in 5 ml of 0.75 M nitric acid and shaken for 48 h to yield a homogenous creamy mass. The sample was then centrifuged at ∼2,500 g for 10 min, and 1 ml of the supernatant was again centrifuged at ∼10,000 rpm for 5 min. Aliquots from the second supernatant were stored at −20°C until analysis. To obtain the absolute electrolyte content of the feces in micromole, the measured electrolyte concentration (using a AFM 5051 flame photometer, Eppendorf, Germany) was multiplied by 5.
Electrogenic glucose transport.
For the analysis of electrogenic small intestinal glucose transport, jejunal segments were mounted into a custom-made mini-Ussing chamber with an opening of 0.00769 cm2. Under control conditions, the serosal and luminal perfusate contained (in mM) 115 NaCl, 2 KCl, 1 MgCl2, 1.25 CaCl2, 0.4 KH2PO4, 1.6 K2HPO4, 5 Na pyruvate, 25 NaHCO3, 20 mannitol (pH 7.4, NaOH). To induce current, glucose (20 mM) was added to the luminal perfusate at the expense of mannitol. Before mounting the tissue into the Ussing chamber, we placed the empty chamber into the apparatus and the potential difference across the empty chamber set to 0 mV. In all Ussing chamber experiments the Vt was determined continuously and apparent Rt was estimated from the ΔVt elicited by imposing It. The resulting Rt was calculated according to Ohm's law. For the determination of the Rt in the presence of mannitol or glucose, the resistance of the empty chamber was subtracted. The glucose-induced current was calculated as the difference between the current in the presence of 20 mM glucose and 20 mM mannitol.
The colons from osr1+/+ and osr1+/KI mice were perfused with 4% PFA/PBS, cryoprotected in 30% sucrose at 4°C overnight, and frozen in Tissue-Tek (Sakura Finetek). For immunohistochemistry, colon sections of 8 μm were dried at room temperature for 30 min and fixed for 15 min RT in 4% paraformaldehyde/PBS. Slides were rinsed three times in PBS, permeabilized with 0.5% Triton X-100/PBS for 10 min at room temperature, preblocked with 10% normal goat serum in PBS for 1 h at room temperature, and incubated overnight at 4°C with the primary rabbit polyclonal anti-α-ENaC antibody (1:500, Abcam). The primary antibody was detected with fluorescence-labeled secondary goat anti-rabbit-FITC conjugated antibody (1:1,000, Invitrogen) for 1 h at room temperature. Nuclei were stained with DRAQ-5 dye (1:2,000, Biostatus, Leicestershire, UK). The slides were mounted with ProLong Gold antifade reagent (Invitrogen). Images were taken on a Zeiss LSM5 EXCITER Confocal Laser Scanning Microscope (Carl Zeiss MicroImaging) with water immersion Plan-Neofluar×40/1.3 NA DIC.
Data are provided as means ± SE; n represents the number of independent experiments. All data were tested for significance by Student's unpaired two-tailed t-test. Only results with P < 0.05 were considered statistically significant.
To uncover a role of the OSR1 in the regulation of small intestinal Na+ transport, experiments were performed in OSR1 knockin mice (osr1+/KI) that were heterozygously carrying a WNK-insensitive Thr185AlaOSR1 mutant. The animals were compared with wild-type mice (osr1+/+). Because OSR1 participates in renal salt transport regulation, plasma osmolarity as well as serum ADH, plasma aldosterone, and plasma corticosterone concentration were determined in osr1+/+ and osr1+/KI mice to possibly reveal deranged regulation of electrolyte homeostasis. As listed in Table 1, no significant differences between the two genotypes were observed in serum ADH, plasma aldosterone, and plasma corticosterone concentrations.
A next series of experiments explored the expression of OSR1 in the small intestine from osr1+/+ and osr1+/KI mice. As shown in Fig. 1, OSR1 protein was expressed in both osr1+/KI mice and osr1+/+ mice. The band reflecting OSR1 protein is 58 kDa. The additional bands may reflect unspecific staining. OSR1 is expressed in small intestinal tissue of both osr1+/+ and osr1+/KI mice. The band density was similar in osr1+/+ and osr1+/KI mice; the Western blot does not, however, rule out minor differences between osr1+/+ and osr1+/KI mice. A recent study demonstrated unaltered expression of OSR1 even in homozygous osr1KI/KI embryonic stem cells (48).
In a first approach to estimate intestinal Na+ transport, the daily fecal Na+ excretion was determined. As a result, the fecal Na+ output was significantly lower in osr1+/KI mice than in osr1+/+ mice (Fig. 2). The daily fecal K+ excretion was similarly lower in osr1KI/+ mice than in osr1+/+ mice (Fig. 2). The weight of the feces was similar in osr1+/+ mice and in osr1+/KI mice (Fig. 2). The decreased fecal Na+ and K+ excretion in osr1+/KI mice was paralleled by a decrease of fecal water excretion. The fecal water content was significantly lower in osr1+/KI mice (43.1 ± 4.8%, n = 7) than in wild-type mice (56.2 ± 2.2%, n = 6).
To test whether increased small intestinal absorption of Na+ in osr1+/KI mice was due to increased activity of sodium glucose cotransporter (SGLT1), glucose-induced current in jejunal tissue was measured by use of an Ussing chamber. As a result, the glucose-induced current was significantly (P < 0.01) lower in osr1KI/+ mice (−1,622 ± 143 μA/cm2, n = 10) than in osr1+/+ mice (−2,226 ± 141 μA/cm2, n = 8).
The major small intestinal mechanisms accomplishing small intestinal Na+ reabsorption is the NHE. To estimate NHE activity, the pH recovery following an ammonium pulse was determined in ileum from osr1+/+ and osr1+/KI mice. Figure 3 illustrates the alterations of pHi during this maneuver. Prior to the maneuver, pHi was similar in osr1+/KI mice and osr1+/+ mice (Table 2). In both genotypes the application of 20 mM NH4Cl was followed by cytosolic alkalinization owing to entry of NH3 with subsequent binding of intracellular H+. The following NH4+ removal resulted in a sharp cytosolic acidification due to exit of NH3 with cellular retention of H+. The buffer capacity was similar in osr1+/KI mice and osr1+/+ mice. The cytosolic pH recovery in the absence of extracellular Na+ was negligible, indicating that small intestinal cells did not express functionally relevant Na+-independent H+ extrusion mechanisms. The addition of Na+ was, however, followed by rapid realkalinization, pointing to the activity of the Na+/H+ exchanger NHE3. The alkalinization was significantly more rapid in osr1+/KI mice than in osr1+/+ mice (Fig. 3, A and B), pointing to enhanced NHE activity in osr1+/KI mice.
To test for the contribution of NHE3 to the observed NHE activity, a further series of experiments was performed using the specific NHE3 inhibitor s3226 at a concentration of 10 μM. In the absence of s3226 the sodium-dependent pH recovery was significantly higher in osr1KI/+ mice than in osr1+/+ mice (Fig. 4). The addition of s3226 significantly reduced the sodium-dependent pH recovery in both osr1+/+ mice and osr1KI/+ mice (all values: n = 9) but did not abolish the difference between the genotypes. Accordingly, the sodium-dependent pH recovery was still significantly higher in osr1+/KI mice after S3226 treatment (Fig. 4B). Furthermore, Western blot analysis of small intestinal brush border membrane revealed that membrane abundance of NHE3 was not different between the genotypes (n = 12, Fig. 5).
The fecal Na+ excretion is also modified by colonic Na+ reabsorption via the amiloride-sensitive apical Na+ channel ENaC. Amiloride was used at 50 μM, a concentration fully inhibiting ENaC but too low to inhibit NHE3 activity (IC50 > 100 μM; Ref. 45). The amiloride-sensitive Na+ transport through this channel generates a lumen-negative transepithelial potential and a transepithelial current. As shown in Fig. 6, the amiloride-sensitive equivalent short-circuit current in distal colonic epithelium was significantly higher in colonic tissue from osr1+/KI mice than in colonic tissue from osr1+/+ mice.
Additional experiments addressed the expression, localization, and phosphorylation of ENaC protein by utilizing Western blot and immunofluorescence analysis. As shown in Fig. 7, α-ENaC abundance (Fig. 7, A and B) and localization (Fig. 7C) was not different between osr1+/+ and osr1+/KI mice (n = 3). As shown in Fig. 7, D and E, phospho-β-ENaC (ser 633) was significantly higher in osr+/KI (n = 7).
The present study reveals that WNK resistance of OSR1 affects small intestinal and colonic Na+ reabsorption. Both small intestinal NHE and colonic ENaC activity were significantly higher in OSR1 knockin mice carrying one allele of WNK-insensitive Thr185AlaOSR1 mutant (osr1+/KI) than in wild-type mice (osr1+/+). Accordingly, fecal Na+ excretion was significantly lower in osr1+/KI mice than in osr1+/+ mice.
In theory, the NHE activity in osr1+/KI small intestine could result from cytosolic acidification, which is known to stimulate NHE activity (19). However, the cytosolic pH was similar in osr1+/KI mice and in osr1+/+ mice, indicating that the NHE is stimulated by some other mechanism. Alternatively, the stimulation of the NHE in osr1+/KI could result from cell shrinkage, which stimulates the NHE in parallel to the Cl−/HCO3− exchanger (24, 29). Activation of those two carriers allows the entry of NaCl, followed by osmotically obliged water. The H+ and HCO3− extruded in exchange for Na+ (NHE) and Cl− (Cl−/HCO3− exchanger) are replenished in the cell by cytosolic formation from CO2, which easily crosses the cell membrane (24, 29). Volume regulatory stimulation of the NHE may result from defective OSR1-dependent stimulation of the Na+-K+-2Cl− cotransporter in osr1+/KI cells, because OSR1 is known to stimulate the Na+-K+-2Cl− cotransporter and to participate in cell volume regulation (3, 7, 9, 16, 26, 27, 40, 53). However, NHE1 rather than NHE3 is implicated in the regulation of cell volume (1).
Besides activation by cell shrinkage, OSR1 may influence NHE activity and ENaC more directly. However, evidence for an inhibitory effect of OSR1 on NHE activity or ENaC has not been published. At least in theory, the enhanced activity of NHE activity and ENaC in osr1+/KI mice could result from renal salt loss due to compromised activation of thiazide-sensitive Na+-Cl− cotransporter and the furosemide-sensitive Na+-K+-2Cl− cotransporter in the kidney. We did not observe significant differences in water- and electrolyte-regulating hormones between osr1+/KI mice and osr1+/+ mice. This does not, however, rule out minor changes of hormone release that affect small intestinal transport.
In contrast to NHE and ENaC activity, the glucose-induced current is lower in osr1+/KI mice than in osr1+/+ mice. In theory, this effect could again reflect an influence of OSR1 on SGLT1 activity or an effect on other carriers and channels indirectly influencing glucose-induced current.
The present observations reveal differences between heterozygous mice and wild-type mice. Homozygous OSR1 knockin mice (osr1KI/KI) are not viable (38), highlighting the physiological importance of WNK-sensitive OSR1-dependent regulation. The substantial difference of fecal salt excretion, small intestinal NHE, and colonic ENaC activity between osr1+/KI mice and osr1+/+ mice underscores the power of WNK-sensitive OSR1-dependent regulation.
In conclusion, small intestinal NHE and colonic ENaC activity are significantly higher in osr1+/KI than in osr1+/+ mice. The observations thus point to a novel functional role of the OSR1.
This work was supported by the Deutsche Forschungsgemeinschaft.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: V.P., G.P., A.F., R.R., D.M., T.P., and I.A. performed experiments; V.P., G.P., A.F., R.R., D.M., A.R., M.F., and F.L. approved final version of manuscript; D.M., A.R., and M.F. analyzed data; A.R., M.F., and F.L. edited and revised manuscript; M.F. interpreted results of experiments; M.F. prepared figures; F.L. conception and design of research; F.L. drafted manuscript.
The authors are indebted to Rebecca Lam, Max Planck Institute Frankfurt for critically reading the manuscript. They are further indebted to Dario Alessi from the Medical Research Council Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee, UK, who kindly provided the mice and the antibodies. The authors further gratefully acknowledge the meticulous preparation of the manuscript by Lejla Subasic, Tanja Loch, and Sari Rübe.
- Copyright © 2012 the American Physiological Society