Regulation of ROMK Channel and K+ Homeostasis by Kidney-specific WNK1 Kinase*

WNK kinases are serine-threonine kinases with an atypical placement of the catalytic lysine. WNK1, the first member discovered, has multiple alternatively spliced isoforms, including a ubiquitously expressed full-length long form (L-WNK1) and a kidney-specific form (KS-WNK1) predominantly expressed in the kidney. Intronic deletions of WNK1 that increase WNK1 transcript cause pseudohypoaldosteronism type 2, an autosomal-dominant disease characterized by hypertension and hyperkalemia. L-WNK1 inhibits renal K+ channel ROMK, likely contributing to hyperkalemia in PHAII. Previously, we reported that KS-WNK1 by itself has no effect on ROMK1 but antagonizes L-WNK1-mediated inhibition of ROMK1. Amino acids 1–253 of KS-WNK1 (KS-WNK1(1–253)) are sufficient for reversing the inhibition of ROMK1 caused by L-WNK1(1–491). Here, we further investigated the mechanisms by which KS-WNK1 counteracts L-WNK1 regulation of ROMK1. We reported that two regions of KS-WNK1(1–253) are involved in the antagonism of L-WNK1; one includes the first 30 amino acids unique for KS-WNK1 encoded by the alternatively spliced initiating exon 4A, and the other is equivalent to the autoinhibitory domain (AID) of L-WNK1. Mutations of two phenylalanine residues known to be critical for autoinhibitory function of AID abolish the ability of the AID region of KS-WNK1 to antagonize L-WNK1. To examine the physiological role of KS-WNK1 in the regulation of renal K+ secretion, we generated transgenic mice that overexpress amino acids 1–253 of KS-WNK1 under the control of a kidney-specific promoter. Transgenic mice have lower serum K+ levels and higher urinary fractional excretion of K+ compared with wild type littermates despite the same amount of daily urinary K+ excretion. Moreover, transgenic mice (compared with wild type littermates) displayed a higher abundance of ROMK on the apical membrane of distal nephron. Thus, KS-WNK1 is an important physiological regulator of renal K+ excretion, likely through its effects on the ROMK1 channel.

. ROMK K ϩ channels are expressed in the connecting tubule and the cortical collecting duct and are important for base-line (non-flow-stimulated) renal K ϩ secretion (14). Thus, a decrease in K ϩ secretion by the kidney resulting from the inhibition of ROMK by L-WNK1 may contribute to hyperkalemia in patients of PHAII with WNK1 mutations. However, there are multiple alternatively spliced WNK1 isoforms differentially expressed in tissues (4,5). The effects of deletions of the first intron on splice variants of WNK1 and the effects of individual isoforms on K ϩ transport remain largely unknown.
Recently, we and others reported that kidney-specific WNK1, by itself, has no effect on ROMK1 but antagonizes the inhibition of ROMK1 caused by L-WNK1 (12,15). K ϩ secretion by kidney is critical for controlling serum K ϩ levels and overall K ϩ homeostasis (14). As an important pathway for K ϩ secretion in kidney, the abundance of ROMK on the apical membrane of distal nephron is regulated by dietary K ϩ intake (14). The apical ROMK abundance decreases or increases during low or high dietary K ϩ intake, respectively (16,17). The decrease in the apical abundance of ROMK in response to dietary K ϩ restriction involves an increase in the clathrin-mediated endocytosis and subsequent degradation of the channel protein (18,19). We reported that dietary K ϩ restriction in rats increases the expression of L-WNK1 and decreases that of KS-WNK1 (12). The increase in the L-WNK1 to KS-WNK1 ratio would be expected to cause inhibition of ROMK1. These results suggest that KS-WNK1 is an important physiological antagonist of L-WNK1, and the ratio of L-WNK1 to KS-WNK1 regulates surface abundance of ROMK1 and renal K ϩ secretion during changes in dietary K ϩ intake.
In the present study, we further examined the mechanism by which KS-WNK1 antagonizes L-WNK1 regulation of ROMK1. We identified two regions within amino acids 1-253 of KS-WNK1 that are involved in binding to and antagonism of L-WNK1. Furthermore, to examine the physiological role of KS-WNK1 in the regulation of K ϩ secretion in vivo, we generated transgenic mice overexpressing amino acids 1-253 of KS-WNK1 and found that they display lower serum K ϩ levels and increased tubular excretion of K ϩ relative to wild type littermates despite a similar K ϩ intake. These results further support the important physiological role of KS-WNK1 in the regulation ROMK1 activity and renal K ϩ excretion.
Cell Culture, Immunoprecipitation, and Western Blot Analysis-HEK 293 cells were cultured, transfected, and harvested as described previously (12). For coimmunoprecipitation, the proteins were immunoprecipitated from cell lysates by using monoclonal anti-FLAG antibody (1:100 dilution; Sigma) and followed by protein A-Sepharose beads. The precipitates were washed three time with 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Triton X-100. For Western blot analysis, total lysates, immunoprecipitates, or kidney homogenate were resolved by SDS-PAGE gel electrophoresis, and proteins were transferred onto nitrocellulose membranes. The membranes were incubated with the indicated antibodies and developed using enhanced chemiluminescence.
Whole Cell Patch-Clamp Recording of ROMK1 Channels-HEK 293 cells were cotransfected with cDNAs encoding GFP-ROMK1 and a fragment of L-WNK1 and/or KS-WNK1. In each experiment, the total amount of DNA for transfection was balanced by using empty vectors. Approximately 36 -48 h after transfection, whole cell currents were recorded by using an Axopatch 200B amplifier as previous described (12). Transfected cells were identified by using epifluorescent microscopy. The bath and pipette solution contained 145 mM KCl, 2 mM MgCl 2 ,2 mM CaCl 2 , 10 mM Hepes (pH 7.4), and 145 mM KCl, 2 mM EDTA, 10 mM Hepes (pH 7.4), respectively. Capacitance and access resistance were monitored and 75% compensated. The voltage protocol consists of 0-mV holding potential and 400-ms steps from Ϫ100 to 100 mV in 20-mV increments.
Generation of Transgenic Mice-The FLAG-KS-WNK1(1-253) fusion fragment was generated by PCR using plasmid pIRES-hrGFP-KS-WNK1(1-253) as template. The ϳ0.8-kb restriction fragment was isolated and cloned into unique SbfI and SmaI sites downstream to the Ksp-cadherin promoter of pKsp-BGH plasmid (provided by Dr. Peter Igarashi, University of Texas Southwestern Medical Center at Dallas). The plasmid insert was verified by DNA sequencing. The ϳ2.6-kb transgene fragment Ksp-FLAG-KS-WNK1(1-253) was isolated by digestion with NdeI and KpnI followed by agarose gel electrophoresis, electroelution, and purification by anion exchange chromatography (Elutip-d, NH). Purified DNA was concentrated in Microcon 30 filters (Millipore, MA), resuspended at a concentration of 80 ng/l in microinjection buffer (10 mM Tris-Cl, pH 7.4, 0.25 mM EDTA), and sterilized by filtration through 0.2-m filters. Transgene DNA was microinjected into the pronuclei of fertilized oocytes by standard pronuclear injection. Fertilized oocytes were from C57BL/6 crosses. Microinjection was performed by the University of Texas Southwestern Transgenic Mouse Core Facility. The microinjected embryos were transferred into the oviducts of pseudopregnant foster mothers and were permitted to develop to term.
Genotyping of Transgenic Mice-Founder (G0) mice were identified by PCR analysis. Genomic DNA was isolated from tails of transgenic mice using a standard method. Two pairs of primers were used for genotyping by PCR. One is specific for endogenous mouse WNK1 (forward, 5Ј-AAA ATA CTC TGT CAG GCT TAA GTG T-3Ј, and reverse, 5Ј-TGA AGC CAG GCA TTA AGC ACT C-3Ј), which would produce a 266-bp fragment in both wild type and transgenic mice. The other is specific for transgenic fragment (forward, 5Ј-GCA GAT CAG CAT CAA CAG CTG-3Ј, and reverse, 5Ј-CAA TGC GAT GCA ATT TCC TC-3Ј), which would produce a 320-bp fragment only in transgenic mice. The condition for PCR includes 35 cycles of 95°C for 20 s, 60°C for 30 s, and 72°C for 45 s. PCR products were detected by electrophoresis on 2.0% agarose gels.
Quantitative Real Time PCR Analysis-Total RNA was isolated from whole kidney with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Gene expression by quantitative real time PCR was carried out on ABI 7000 as described in Applied Biosystems User Bulletin no. 2 using the TagMan assay. Primer from exon 4A (RP forward, 5Ј-GCT GCT GTT CTC AAA AGG ATT GTA T-3Ј), from exon 5 (RP reverse, 5Ј-CAG GAA TTG CTA CTT TGT CAA AAC TG-3Ј), and from TagMan probe (5Ј-TGA GGG AGT GAA GCC A-3Ј) were used to amplify the kidney-specific isoform (12). Relative KS-WNK1 mRNA levels were calculated with 18 S rRNA as the internal control. Kidney cDNA was prepared from three to five animals as described above, and each sample was assayed in triplicate.
Immunofluorescent Staining-The mice were anesthetized by Avertin and perfused via the heart with 15 ml of PBS followed by 15 ml of 4% paraformaldehyde in PBS. The kidneys were harvested and postfixed for 4 h in 4% paraformaldehyde in PBS at 4°C, dehydrated by immersion in 30% sucrose in PBS overnight at 4°C, and mounted in OTC (Tissue-Tek) for sectioning. The sections (4 -5-m thickness) were stained with primary antibodies: rabbit polyclonal anti-FLAG antibody (1:300, Sigma) or anti-ROMK (1:1000), followed by secondary antibodies: Alexa Fluor 488 goat anti-rabbit IgG (1:500) or Alexa Fluor 568 goat antirabbit IgG (1:500). The fluorescent images were obtained using Zeiss LSM510 confocal microscope as described (19).
Blood and Urine Measurements-Under anesthesia by Avertin, blood was drawn from mice by retro-orbital bleeding into heparinized tubes. Electrolytes were measured using a flame photometer. Creatinine was measured by capillary electrophoresis (P/ACE MDQ; Beckman Coulter). Spot urine samples were collected by catching spontaneous voids. 24-h urine samples were collected using metabolic cages (Hatteras Instruments). All of the experiments involving animals were performed in compliance with relevant laws and institutional guidelines and were approved by the University of Texas Southwestern Medical Center at Dallas Institutional Animal Care and Use Committee.
Statistical Analysis-Statistical comparisons between two groups of data were made using two-tailed unpaired Student's t tests. Multiple comparison were made using oneway analysis of variance followed by two-tailed Student's t tests adjusted for multiple comparisons. p values less than 0.05 and 0.01 were considered significant for single and multiple comparisons, respectively. Experiments shown in each panel of the figures were repeated at least three times with similar results.  Are Involved in the Antagonism of L-WNK1-We have reported that KS-WNK1(1-253) antagonizes inhibition of ROMK1 by L-WNK1 by binding to amino acids 1-491 of L-WNK1 (12). To further define the molecular determinant(s) of KS-WNK1 involved in antagonism of L-WNK1 regulation of ROMK1, we generated several overlapping and nonoverlapping smaller fragments of KS-WNK1(1-253) and examined their effects on WNK1(1-491)mediated inhibition of ROMK1. These fragments of KS-WNK1 include amino acids 1-196, 1-137, 1-77, and 31-253, respectively (Fig. 1A). To examine the effects of these fragments on antagonism of L-WNK1 regulation of ROMK1, HEK cells were cotransfected with plasmids expressing green fluorescent protein (GFP)-tagged ROMK1, WNK1(1-491) and one each of KS-WNK1 fragment and recorded for ROMK1 current density using whole cell patch-clamp recording. We have shown that WNK1(1-491) fully recapitulates the effect of full-length L-WNK1 on ROMK1 (12,13). As shown in Fig. 1B, we confirmed that WNK1(1-491) inhibits ROMK1 (compare bars 1 and 2) and that KS-WNK1(1-253) reverses WNK1(1-491)mediated inhibition of ROMK1 (bar 3) as reported previously by us (12). Expression of KS-WNK1(1-253) exerts no effect on ROMK1 in the absence of WNK1(1-491) (not shown in Fig. 1B; see Ref. 12). Here, we found that each of the smaller KS-WNK1 constructs generated could reverse the inhibition of ROMK1 caused by WNK1(1-491) (Fig. 1B, bars 4 -7).
Role of AID Domain of KS-WNK1 in the Interaction with L-WNK1 and Regulation ROMK1 Channel-In our recent study reporting that AID reverses WNK1(1-491) inhibition of ROMK1 (21), we did not examine whether the effect was mediated by binding of AID to WNK1(1-491). Here, we examined the binding interaction between WNK1(1-491) and AID domain (as in "FLAG-WNK1(491-555)"). Two phenylalanine residues within the AID domain of L-WNK1 are critical for its regulation of the kinase activity (20). We have shown that mutations of these two conserved phenylalanines abolished the ability of AID to reverse WNK1(1-491)-mediated inhibition of ROMK1 (21). We therefore used AID carrying double phenylalanine mutations (FLAG-WNK1(491-555)/FFAA) (  2 and 3, respectively). Notably, WNK1(491-555) exhibits as doublets in SDS-PAGE gel and double mutations of phenylalanine (WNK1(491-555)/FFAA) slowed the migration of doublets in gel electrophoresis (Fig. 2B). Thus, the AID domain probably exists in two different conformations in SDS-PAGE gel electrophoresis buffer, and double mutations of phenylalanine to alanine may cause further conformational change(s). The WNK1 fragment containing amino acids 1-555, however, does not exist as doublets in SDS-PAGE gel (12), suggesting that amino acids 1-491 affect the conformation of AID domain. These results support the idea that AID interacts with WNK1(1-491).
Transgenic Mice Overexpressing KS-WNK1(1-253)-To investigate the in vivo physiological role of KS-WNK1 in the regulation of K ϩ secretion, we generated transgenic mice overexpressing amino acids 1-253 of KS-WNK1 under the control of a kidney-specific Ksp-cadherin gene promoter, which directs gene expression in the renal tubules from the thick ascending limb to the collecting duct (23). The Ksp-KS-WNK1 transgenic plasmid construct contains FLAG-tagged rat KS-WNK1(1-253) cDNA placed behind the Ksp-cadherin promoter in a pKsp-BGH vector (23). A ϳ2.6-kb restriction fragment containing Ksp-cadherin promoter, the KS-WNK1(1-253)-FLAG fusion gene, and the polyadenylation signal was purified (Fig.  4A) and used for pronuclear microinjection to generate transgenic founder lines. Genotyping of founder mice was performed by polymerase chain reaction of tail genomic DNA using two sets of PCR primers, one specific for transgene (and produces a 320 bp fragment) and the other specific for endogenous WNK1 (and produces a 266-bp fragment). Fig. 4B shows an example of positive transgene expression in one of founder lines. Founders were mated with wild-type mice of the same genetic background to produce offspring. The relative expression of KS-WNK1 transgene versus endogenous KS-WNK1 in transgenic offspring were examined by quantitative real time PCR using primers containing nucleotide sequence identical for rat and mouse. Fig. 4C shows that the abundance of KS-WNK1 message RNA in mice homozygous for TG(KS-WNK1) is ϳ3-fold higher than that for wild type mice. Expression of transgenic KS-WNK1(1-253) protein in transgenic but not wild type mice was verified by Western blot analysis of whole kidney homogenates (Fig. 4D).
We further examined the expression and localization of transgenic FLAG-KS-WNK1(1-253) protein in the kidney of homozygous TG(KS-WNK1) mice by immunofluorescent staining using polyclonal anti-FLAG antibodies. Previously, Shao et al. (23) reported that Ksp promoter directs protein expression in renal tubules from the thick ascending limbs to the collecting duct. More recently, Lin and Igarashi 3 reported that expression of protein driven by the Ksp promoter (examined using a reporter) is detected in ϳ20% of proximal tubules and in Ͼ90% of tubules from the thick ascending limb to collecting ducts. Consistent with these reports on tubular expression of proteins directed by Ksp promoter, we found that transgenic KS-WNK1(1-253) is abundantly expressed in distal tubular segments of TG(KS-WNK1) (Fig. 5, A and B) but not of wild type mice (Fig. 5, C and D). Importantly, transgenic KS-WNK1(1-253) is abundantly expressed in connecting tubules and cortical collecting ducts, tubular segments where K ϩ secretion occurs predominantly (Fig. 5A, labeled C). Compared with distal tubular segments, the expression in the proximal convoluted tubule is much lower (Fig. 5A, labeled P). Also, the expression of transgenic KS-WNK1(1-253) protein driven by Ksp promoter is abundant in the outer medulla (Fig. 5B), because of a high density of thick ascending limbs and collecting ducts in this region.  . C and D, kidney sections of cortex (C) and outer medulla (D) from wild-type littermates. The microscopic images were obtained using 20ϫ objective lens. Fluorescent images were merged with differential interference contract images to better illustrate subcellular distribution. Scale bars, 100 m. C indicates connecting tubules or cortical collecting ducts based on the morphology (longitudinal tubules and merging) and cell heterogeneity (principal versus intercalated cells); G indicates glomerulus; and P indicates proximal convoluted tubules. The experiments were repeated four times with similar results.

Increased ROMK Expression in TG(KS-WNK1) Mice-We
next examined the abundance of ROMK in the transgenic mice and wild-type littermates by immunofluorescent staining. ROMK channels are expressed in tubular segments from the thick ascending limb of Henle's loop to cortical collecting ducts (14,17). Using an antibody previously characterized by us (19,24), we found that the expression of ROMK on the apical membrane of tubules is increased in the transgenic mice compared with the wild type (Fig. 6). ROMK channels on the apical membrane of principal cells of connecting tubules and cortical collecting ducts are important exit pathways for base-line (nonflow-stimulated) K ϩ secretion (14,17). Higher magnification images revealed that the expression of ROMK on the apical membrane of connecting tubules and cortical collecting ducts is indeed increased in transgenic mice compared with wild type mice (Fig. 6, B and D, labeled C). We do not have a reliable monoclonal anti-FLAG antibody with a low background in the immunofluorescent staining. We therefore used sequential sections (ϳ4 -5-m thickness) for immunofluorescent staining of ROMK and transgenic KS-WNK1 using polyclonal anti-ROMK and anti-FLAG antibodies, respectively. As shown in cortical sections from homozygous TG(KS-WNK1) mice, ROMK (Fig. 6E) and transgenic KS-WNK1 (Fig. 6F) were coexpressed in many distal nephron segments including connecting tubules/cortical collecting ducts (indicated by arrows) and the distal convoluted tubule (indicated by arrowheads).
Renal K ϩ Excretion in TG(KS-WNK1) Transgenic Mice-We used the transgenic model to study the role of KS-WNK1 in K ϩ homeostasis and renal K ϩ secretion. We found that serum K ϩ levels were significantly lower in homozygous TG (KS-WNK1) than in wild type littermates (3.9 Ϯ 0.2 mM versus 4.9 Ϯ 0.2 mM, p ϭ 0.02) (Fig. 7A). A decrease in serum K ϩ levels may be due to decreased dietary intake, increased intracellular shift, and/or increased net secretion by renal tubules. To examine these possibilities, we measured 24-h urinary excretion and fractional excretion of K ϩ (FE K ) in homozygous TG(KS-WNK1) and wild type mice. As shown, the steady-state 24-h urinary K ϩ excretion was not different between TG and wild type mice (Fig. 7B), suggesting that the two groups have equal dietary K ϩ intake. Fractional excretion of K ϩ (FE K ), however, were much higher in homozygous TG(KS-WNK1) than in wild type (Fig. 7C, 11.5 Ϯ 0.84% versus 5.1 Ϯ 1.67%, p ϭ 0.01). 24-h urinary volume, creatinine excretion, and creatinine clearance were not different between TG and wild type littermates (not shown). These results support the idea that the decrease in serum K ϩ levels in TG mice is due to increased net secretion of K ϩ by renal tubules. For comparison, steady-state serum Na ϩ (155 Ϯ 3 mM versus 155 Ϯ 4 mM), 24-h urinary Na ϩ excretion (147 Ϯ 12 M versus 177 Ϯ 15 M), and fractional excretion of Na ϩ (1.7 Ϯ 0.3% versus 2.2 Ϯ 0.4%) were not significantly different between homozygous TG mice and wild type littermates (n ϭ 8 each group; p Ͼ 0.1 for all).

DISCUSSION
WNK kinases comprise a recently identified group of serine and threonine kinases in which the location of the lysine required for ATP binding is unique (1). Mutations in the genes encoding two members, WNK1 and WNK4, cause PHAII, a disease characterized by hypertension and hyperkalemia (3,7). WNK4 is expressed primarily in epithelial tissues including the kidney (25). WNK1 has multiple isoforms including a ubiquitously expressed L-WNK1 and a kidney-specific KS-WNK1 (4,5). Many studies have reported that L-WNK1 and WNK4 play important roles in regulating renal electrolyte homeostasis (8 -11). The function and the physiological role of KS-WNK1 are relatively less understood.
We have recently shown that KS-WNK1 is an antagonist of L-WNK1 regulation of ROMK1 and that amino acids 1-253 of KS-WNK1 are sufficient for the antagonism of L-WNK1 (12).  wild-type mice. A, serum K ϩ levels in homozygous TG(KS-WNK1) and wildtype mice. The mice were on regular rodent chow. The results are the means Ϯ S.E.; n ϭ 10 for each group. An asterisk denotes p Ͻ 0.01 versus wild type by unpaired Student's t test. In separate experiments, we found that serum K ϩ levels of homozygous TG(KS-WNK1) (4.0 Ϯ 0.12 mM, n ϭ 4) were significantly lower than that of heterozygous TG(KS-WNK1) (4.58 Ϯ 0.13 mM, n ϭ 10), which were significantly lower than that of wild type littermates (5.09 Ϯ 0.24 mM, n ϭ 7) (p Ͻ 0.05 between each group). B, 24-h urinary excretion of K ϩ in homozygous TG(KS-WNK1) and wild-type mice. C, fractional excretion of K ϩ (FE K ) in homozygous TG(KS-WNK1) and wild-type mice.
In the present study, we further report that two regions within amino acids 1-253 of KS-WNK1 are critical for antagonism of L-WNK1. One is the region encoded by the alternative initiating exon 4A. This region is unique to KS-WNK1. The other is the region equivalent to the AID domain of L-WNK1. The AID domain of WNK1 contains a FXF motif that suppresses the catalytic activity by direct binding to the kinase domain (20). Mutation of phenylalanine residues releases the inhibition of WNK1 kinase activity by the AID domain (20). The FXF motif is reminiscent of one type of ERK2 docking domain found in several proteins (26). Interestingly, the AID of WNK1 inhibits kinase activity of ERK2 (20).
The regulation of ROMK1 by WNK1 involves protein-protein interactions that are independent of WNK1 kinase activity (13,21). The kinase and AID domains of WNK1, nevertheless, play critical roles in its regulation of ROMK1 (13,21). To understand the role of WNK1 kinase domain in the regulation of ROMK1, we have found that proper folding of the WNK1 kinase domain (not its kinase activity) is important for the amino-terminal proline-rich motifs of WNK1 to bind the endocytic scaffold protein intersectin (13,21). Binding of WNK1 to intersectin leads to enhanced endocytosis (and thus inhibition) of ROMK. We have further shown that AID domain contributes to WNK1 inhibition of ROMK1 by binding and interfering with the function of the kinase domain (12,13,21). Amino acids 84 -148 of KS-WNK1 are identical to the AID domain of L-WNK1 (amino acids 491-556) (Fig. 1A). Thus, it is not surprising that this AID-equivalent region of KS-WNK1 can antagonize L-WNK1 inhibition of ROMK1. These findings, however, are interesting in light of the fact that KS-WNK1 does not contain a kinase domain. This fact supports the idea that the physiological role of AID domain of KS-WNK1 is to regulate other WNK kinases that contain the kinase domain, such as L-WNK1. It would be interesting to investigate in the future whether KS-WNK1, through its AID domain, also regulates other WNKs and/or other protein kinases with a FXF motif in the kinase-inhibitory domain as in ERK2.
To examine the in vivo role of KS-WNK1 in the regulation of K ϩ secretion, we generated transgenic mice overexpressing KS-WNK1(1-253) in kidney tubules. KS-WNK1(1-253)transgenic mice have lower serum K ϩ levels and increased fractional excretion of K ϩ compared with wild type littermates despite the same level of dietary K ϩ intake. Compared with the wild type mice, the expression of ROMK in the apical membrane of renal tubules including connecting tubules and cortical collecting ducts is markedly increased in the transgenic mice. These results support the hypothesis that KS-WNK1 antagonizes the L-WNK1-mediated enhancement of endocytosis of ROMK1. Overall, these results support the idea that KS-WNK1 is a physiological antagonist of L-WNK1 with respect to renal K ϩ excretion. Yet, there are limitations in our study that deserve caution. First, endogenous KS-WNK1 transcript is detected in the thick ascending limb, the distal convoluted tubule, and the cortical collecting duct but not in the proximal convoluted tubule (6). As above, TG-KS-WNK1(1-253) driven by the exogenous Ksp promoter is weakly expressed in the proximal tubule in addition to its abundant expression in the distal nephron segments. The possibility that a low level expression of TG-KS-WNK1(1-253) in the proximal tubule may affect K ϩ transport via nonspecified mechanism cannot be excluded. Second, we used amino acids 1-253 of KS-WNK1 for transgenic expression in mice because this region is sufficient for antagonism of L-WNK1. Our results, although supporting the idea that amino acids 1-253 are sufficient for antagonism of L-WNK1 inhibition of ROMK-mediated K ϩ secretion, cannot exclude the possibility that full-length KS-WNK1 may have a different effect in vivo. Finally, KS-WNK1 may also regulate other K ϩ transporters besides ROMK.
Studies using Xenopus oocytes and cultured cells have also suggested that KS-WNK1 may regulate renal Na ϩ transport (27,28). In the present study, we found that steady-state serum Na ϩ and urinary excretion of Na ϩ are not different between mice overexpressing TG(KS-WNK1(1-253) and wild type. These results, however, do not exclude the physiological role of KS-WNK1 in the regulation of Na ϩ transport in vivo. Because of compensatory responses, steady-state serum and urinary Na ϩ measurement may not reflect renal tubular Na ϩ transport ability. Moreover, amino acids of KS-WNK1 involved in regulation of Na ϩ transporters may reside outside the region of amino acids 1-253. Our results, nevertheless, support the idea that the effect of KS-WNK1(1-253) on K ϩ transport is not secondary to increased urinary excretion of Na ϩ .