Impaired degradation of WNK1 and WNK4 kinases causes PHAII in mutant KLHL3 knock-in mice

Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia and metabolic acidosis, and genes encoding with-no-lysine kinase 1 (WNK1) and WNK4 kinases are known to be responsible. Recently, Kelch-like 3 (KLHL3) and Cullin3, components of KLHL3-Cullin3 E3 ligase, were newly identiﬁed as responsible for PHAII. We have reported that WNK4 is the substrate of KLHL3-Cullin3 E3 ligase-mediated ubiquitination. However, WNK1 and Na–Cl cotransporter (NCC) were also reported to be a substrate of KLHL3-Cullin3 E3 ligase by other groups. Therefore, it remains unclear which molecule is the target(s) of KLHL3. To investigate the pathogenesis of PHAII caused by KLHL3 mutation, we generated and analyzed KLHL3 R528H/ 1 knock-in mice. KLHL3 R528H/ 1 knock-in mice exhibited salt-sensitive hypertension, hyperkalemia and metabolic acidosis. Moreover, the phosphorylation of NCC was increased in the KLHL3 R528H/ 1 mouse kidney,indicating thattheKLHL3 R528H/ 1 knock-inmouseisanidealmouse modelofPHAII.Interestingly,the protein expression of both WNK1 and WNK4 was signiﬁcantly increased in the KLHL3 R528H/ 1 mouse kidney, conﬁrming that increases in these WNK kinases activated the WNK-OSR1/SPAK-NCC phosphorylation cascade in KLHL3 R528H/ 1 knock-in mice. To examine whether mutant KLHL3 R528H can interact with WNK kinases, we measuredthebindingofTAMRA-labeledWNK1andWNK4peptidestofull-lengthKLHL3usingﬂuorescencecor-relation spectroscopy, and found that neither WNK1 nor WNK4 bound to mutant KLHL3 R528H. Thus, we found that increased proteinexpression levels ofWNK1 and WNK4 kinases causePHAIIbyKLHL3 R528Hmutation due to impaired KLHL3-Cullin3-mediated ubiquitination.


INTRODUCTION
Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia, and metabolic acidosis (1,2). Mutations in with-no-lysine kinase 1 (WNK1) and WNK4 genes were reported to be responsible for PHAII (3). It was previously demonstrated that the WNK kinase family phosphorylates and activates oxidative stress-responsive kinase 1 (OSR1) and STE20/ SPS1-related proline/alanine-rich kinase (SPAK) (4,5), and that activated OSR1/SPAK kinases could phosphorylate and activate Na-Cl cotransporter (NCC), constituting the WNK-OSR1/SPAK-NCC phosphorylation that KLHL3 interacts with Cullin3 and WNK4, induces WNK4 ubiquitination and reduces WNK4 protein levels in cultured cells and Xenopus laevis oocytes (21)(22)(23)(24). Interestingly, it was also reported that WNK1 could be a substrate of KLHL3-Cullin3 E3 ubiquitin ligase (21,24). Moreover, another group reported that KLHL3 was able to bind to NCC and regulate its intracellular localization in cultured cells (16). Therefore, it remains unclear which molecule involved in the pathogenesis of PHAII is the in vivo target of KLHL3. In addition, the above experiments were performed in cultured cells. Thus, it is necessary to clarify the role of KLHL3 mutation in PHAII pathogenesis in vivo.
In this study, to answer these questions, we generated KLHL3 R528H/+ knock-in mice that carry the same mutation as autosomal dominant type PHAII patients. This KLHL3 R528H/+ knock-in PHAII model mouse revealed that increased protein expression levels of WNK1 and WNK4 kinases, due to impaired binding of KLHL3 with WNK kinases, cause PHAII in vivo. These results also indicated that both WNK1 and WNK4 are physiologically regulated by KLHL3-Cullin3-mediated ubiquitination in vivo.

RESULTS
Generation of KLHL3 R528H/1 knock-in mice KLHL3 R528H/+ knock-in mice were generated using homologous recombination in Baltha1 embryonic stem (ES) cells to create a mutant allele (25). Exon 15 of the Klhl3 gene was replaced by a cassette expressing a neomycin selective marker flanked by loxP sites, which was followed by the mutant exon 15 (R528H) (Fig. 1A). Recombinant ES cell clones were injected into morula to generate chimeric mice. The neo cassette was deleted by crossing the mutant KLHL3 flox/+ mice with CAG promoter-Cre transgenic mice. Successful generation of KLHL3 R528H/+ knock-in mice was confirmed by genomic sequencing (Fig. 1B-E). In addition to the generation of KLHL3 R528H/+ heterozygous mice, we generated KLHL3 R528H/R528H homozygous mice to more readily detect the pathological effects of mutant KLHL3 R528H.
PHAII phenotypes of KLHL3 R528H/1 knock-in mice There were no significant differences in body weight and physical appearance between KLHL3 R528H/+ and wild-type mice. To confirm the KLHL3 R528H/+ mouse as an accurate model of PHAII, we measured systolic blood pressure of mice fed a normalsalt diet. The systolic blood pressure in the KLHL3 R528H/+ mice fed a normal-salt diet did not differ from that of wild-type mice ( Table 1). Since PHAII shows salt-sensitive hypertension, we then measured blood pressure in mice fed a high-salt diet, which revealed that a high-salt diet produced significantly higher systolic blood pressure in KLHL3 R528H/+ mice compared with wild-type mice (133.5 + 1.6 mmHg versus 120.1 + 5.1 mmHg, respectively; n ¼ 5 and 4, P , 0.05). Moreover, as shown in Table 2, KLHL3 R528H/+ mice exhibited hyperkalemia and metabolic acidosis similar to PHAII patients. These data clearly indicate that the KLHL3 R528H/+ mouse is an ideal model of PHAII caused by KLHL3 mutation. The severity of hyperkalemia and metabolic acidosis was not changed under a high-salt diet (Supplementary Material, Fig. S1). We also performed blood pressure measurement and analysis of blood biochemical characteristics of KLHL3 R528H/R528H homozygous knock-in mice (Tables 1 and 2). Although KLHL3 R528H/R528H mice also exhibited salt-sensitive hypertension, hyperkalemia and metabolic acidosis, the blood pressure and blood biochemistries did not significantly differ from those of KLHL3 R528H/+ mice. Increased protein expression levels of WNK1 and WNK4 in KLHL3 R528H/1 mouse kidney To investigate the pathogenesis of PHAII caused by the R528H mutation of KLHL3 in vivo, we examined the protein expression and phosphorylation of molecules constituting the WNK signaling pathway. As shown in Figure 2A and B, protein expression levels of WNK1 and WNK4 were significantly increased 1.8and 1.4-fold, respectively, in the kidney of KLHL3 R528H/+ mice compared with those of wild-type mice. Accordingly, phosphorylation of OSR1, SPAK and NCC was also increased in KLHL3 R528H/+ mice. The KLHL3 R528H/R528H homozygous mouse showed obvious increases of WNK1 and WNK4 protein levels (6.9-and 2.4-fold, respectively, compared with wild-type mice) and increased phosphorylation of OSR1 and SPAK. However, we also found that the protein level and the phosphorylation status of NCC in KLHL3 R528H/R528H homozygous knock-in mice were not significantly increased compared with those in KLHL3 R528H/+ heterozygous knock-in mice, suggesting that the levels of increased WNK1 and WNK4 in the KLHL3 R528H/+ heterozygous knock-in mice might be high enough to fully phosphorylate and activate NCC. Considering that constitutive activation of NCC is the cause of PHAII, this saturated phosphorylation status of NCC could explain why the blood pressure and blood chemistries in KLHL3 R528H/ R528H homozygous knock-in mice did not differ from those of KLHL3 R528H/+ mice (Tables 1 and 2).
In addition, we confirmed that mRNA levels of WNK1 and WNK4 were not increased in the KLHL3 R528H/+ heterozygous mouse kidney (Fig. 2C), indicating that the increased protein levels of WNK1 and WNK4 were due to impaired degradation rather than transcriptional activation. To confirm that protein expression levels of WNK1 and WNK4 were increased in distal convoluted tubules (DCTs) where NCC is present, we performed double immunofluorescence of mouse kidney. As shown in Figure 3A and B, most of the KLHL3, WNK1 and WNK4 signals were colocalized with NCC, and signal intensities of WNK1 and WNK4 at DCT were apparently higher in the KLHL3 R528H/+ mouse kidney. Considering that both the WNK1 and WNK4 transgenic mice were reported to cause activation of the WNK-OSR1/SPAK-NCC phosphorylation cascade (9,21), these data clearly indicated that the essential pathogenesis of PHAII caused by KLHL3 mutation is due to increased WNK1 and WNK4 in DCT, leading to activation of the WNK-OSR1/ SPAK-NCC phosphorylation signaling cascade.

Defective binding between the acidic motif of WNK1/WNK4 and mutant KLHL3 R528H
We had previously reported that KLHL3 mutation in the Kelch domain decreased its binding to the acidic domain of WNK4, leading to impaired ubiquitination and reduced WNK4 protein levels in HEK 293 cells (21). To confirm that the increased protein levels of WNK1 and WNK4 in KLHL3 R528H/+ mouse kidney were caused by impaired binding between WNK kinases and the mutant KLHL3 R528H, we measured the diffusion time of the TAMRA-labeled acidic motif of WNK1 or WNK4 peptide using fluorescence correlation spectroscopy (FCS) in the presence of different concentrations of GST-fusion proteins of wild-type and mutant KLHL3 R528H; the binding of KLHL3 to these peptides is observed as increased diffusion time of the fluorescent peptide (21,26,27). Wild-type KLHL3 increased the diffusion time of the TAMRA-labeled WNK1 and WNK4 peptides, confirming that wild-type KLHL3 can interact with the acidic motif of WNK1 as well as that of WNK4 (Fig. 4). On the Regulation of epithelial Na 1 channels and ROMK in KLHL3 R528H/1 mice We investigated epithelial Na + channels (ENaC) and renal outer medullary K + channels (ROMK), two important channels for Na + reabsorption and K + secretion in cortical collecting ducts (CCD), in KLHL3 R528H/+ mice. As shown in Figure 5, although KLHL3 R528H/+ mice did not show a significant change in the protein levels of total ENaC a subunit (85 kDa) compared  with wild-type mice, the levels of cleaved ENaC a subunit (35 kDa) were increased in KLHL3 R528H/+ mice. The level of ENaC b subunit was also increased in KLHL3 R528H/+ mice. Similar to ENaC a subunit, KLHL3 R528H/+ mice showed no significant change in the total protein level of ENaC g subunit (85 kDa). However, a significant increase of cleaved ENaC g subunit (70 kDa) was found in the KLHL3 R528H/+ mouse kidney. Similar to WNK4 D561A/+ knock-in mice (7), these results indicate that ENaC is activated in the KLHL3 R528H/+ mouse kidney. On the other hand, KLHL3 R528H/+ mice did not show a significant difference in protein levels of ROMK in immunoblot analysis of whole kidney (Fig. 5).

DISCUSSION
Investigation of the pathophysiology of PHAII is extremely important, not only to increase knowledge of this rare inherited disease, but also for the discovery of novel mechanisms of salt handling in the kidney. Although KLHL3 was identified as responsible for PHAII, several molecules have recently been reported as substrates that interact with KLHL3 in cultured cells. It was demonstrated that the loss of interaction between KLHL3 and WNK4 induced impaired ubiquitination of WNK4 and increased protein levels of WNK4 (21)(22)(23)(24). In addition, WNK1 was also reported to be bound to KLHL3 (21,24). In contrast, Louis-Dit-Picard et al. (16) reported that KLHL3 is responsible for direct regulation of NCC membrane expression. Therefore, the pathophysiological role of KLHL3 in PHAII required investigation of in vivo kidney. For this purpose, we generated KLHL3 R528H/+ knock-in mice that carry a mutation found in human PHAII patients (15,16). This KLHL3 R528H/+ knock-in mouse exhibited salt-sensitive hypertension, hyperkalemia and metabolic acidosis, which are characteristic symptoms of PHAII patients. Moreover, increased NCC phosphorylation was also observed. These results clearly confirmed that our KLHL3 R528H/+ mouse is an ideal model of mutant KLHL3-induced PHAII.
To investigate the mechanisms of PHAII in KLHL3 R528H/+ mice, we assessed the protein levels and phosphorylation status of the WNK-OSR1/SPAK-NCC phosphorylation signaling cascade. Interestingly, KLHL3 R528H/+ heterozygous mice showed increased protein levels of both WNK1 and WNK4 in the kidney, and KLHL3 R528H/R528H homozygous mice more

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Human Molecular Genetics, 2014, Vol. 23,No. 19 clearly demonstrated increased WNK1 and WNK4 protein levels. We further demonstrated using an immunofluorescence assay that protein levels of both WNK1 and WNK4 are increased in DCT in KLHL3 R528H/+ mice. We also confirmed by FCS assay that mutant KLHL3 R528H did not bind to the acidic motif of either WNK1 or WNK4. Considering these observations, we have demonstrated for the first time that the essential mechanism of mutant KLHL3-induced PHAII involves impaired ubiquitination and increased protein levels of both of WNK1 and WNK4 in DCT in vivo (Fig. 6). Importantly, these facts suggest that Cullin3-KLHL3 E3 ligase complexes physiologically regulate WNK-OSR1/ SPAK-NCC phosphorylation cascades in vivo, indicating that KLHL3 plays an important role in the physiological mechanisms of sodium handling in the kidney. To date, physiological regulators of the WNK-OSR1/SPAK-NCC phosphorylation signal cascade, such as insulin (28 -31), angiotensin II (10,(32)(33)(34) and aldosterone (32,(35)(36)(37), have been reported. However, the regulatory mechanism of these factors in WNK signaling remains unknown. The novel KLHL3-mediated regulation of WNK signals might be involved in these mechanisms. Further investigation is required to clarify this issue.
It was reported that KLHL3 was able to bind to NCC and regulate its intracellular localization in cultured cells (16). However, WNK and OSR1/SPAK were activated in KLHL3 R528H/+ mice, but were not down-regulated by increased NCC phosphorylation. Moreover, it was reported that simple over-expression of NCC did not produce the PHAII phenotype in NCC transgenic mice (38), indicating that increased phosphorylation, but not increased protein expression, of NCC is required for the PHAII phenotype in vivo. Considering these in vivo observations, the essential pathogenesis of PHAII caused by KLHL3 mutation is not due to impaired ubiquitination or regulation of NCC, but to the impaired ubiquitination of WNK kinases. This discrepancy could be due to the experimental system, the genetically engineered mouse model or cultured cells.
Boyden et al. (15) reported that symptoms of human PHAII patients caused by KLHL3 mutation are more severe than those caused by mutation in WNK1 or WNK4. WNK4 D561A/+ knock-in (7) and WNK1 +/FHHt (9) PHAII mouse models exhibited increases of only a single kind of WNK kinase that carries a mutation. The increased severity of KLHL3 mutation-induced PHAII in human patients may be explained by the physiological difference between the increase of 'both WNK1 and WNK4 kinases' and 'a single WNK kinase'. Accumulation of both WNK1 and WNK4 kinases by KLHL3 mutation could result in further increases in phosphorylation of downstream components compared with the accumulation of a single WNK kinase (Fig. 6). However, the KLHL3 R528H/+ mouse shows a less severe phenotype than other mouse models of PHAII previously reported (7,9), which could be explained by the difference of origin of ES cells used for the generation of the knock-in mice, i.e. the difference of genetic background of these mice. In this study we utilized Baltha1 ES cells derived from C57BL/6 mouse, which is a one-renin-gene mouse strain. However, the other mouse models of PHA II were established with ES cells derived from two-renin-gene mouse strains, 129Sv (7,9). It was reported that 129Sv mice showed increased blood pressure response to salt intake, compared with C57BL/6 mice (39). Further investigation to compare the phenotypes of three PHAII model mice under the same genetic background will be required. Finally, we discuss the involvement of ENaC and ROMK in PHAII caused by KLHL3 mutation. ENaC was activated in KLHL3 R528H/+ heterozygous knock-in mice, similar to WNK4 D561A/+ mice (7). However, inconsistent with the KLHL3 R528H/+ and WNK4 D561A/+ mice, WNK1 +/FHHt mice are reported to show no activation of ENaC (9). This difference might be explained by whether WNK4 is increased or not. On the other hand, in the case of ROMK, we did not find differences in ROMK expression in the kidney between wild-type and KLHL3 R528H/+ mice. However, we were unable to perform microdissection of kidney, and the available anti-ROMK antibody detects all ROMK variants. Therefore, to clarify the in vivo effect of KLHL3 on ROMK in CCD, further investigation is required.
In summary, we established and analyzed KLHL3 R528H/+ knock-in mice, and clarified the essential pathogenesis of mutant KLHL3-induced PHAII. Mutant KLHL3 causes accumulation of both WNK1 and WNK4 at DCT due to loss of binding ability of KLHL3 to WNK kinases. This regulation of the WNK-OSR1/SPAK-NCC phosphorylation cascade by Cullin3-KLHL3 E3 ligase complexes plays important physiological and pathophysiological roles for sodium handling in the kidney in vivo.

Generation of KLHL3 R528H/1 knock-in mice
To generate KLHL3 R528/+ knock-in mice, the targeting vector was prepared using the BAC recombineering system (40). The point mutation (R528H) was introduced into exon 15 of the targeting vector by galK selection system (41). The targeting vector was then transfected into Baltha1 ES cells (25), which are derived from C57BL/6 mice, by electroporation as previously reported (42). After selection with 150 mg/ml G418 and 2 mM ganciclovir, targeted ES clones were selected by PCR with a sense primer F1 (5 ′ -ATA GCA GAG CCG TCT CTG TG-3 ′ ) located within the neo cassette and an antisense primer R1 (5 ′ -ACT TGT GTA GCG CCA AGT GC-3 ′ ) located following exon 15, Southern blotting and sequencing of the mutation site. Selected ES clones were injected into C57BL/6 morula. Chimeric males were bred with C57BL/6 females to produce mutant KLHL3 flox/+ (R528H) mice, and the neo cassette was then deleted by crossing these mutant KLHL3 flox/+ mice with transgenic mice expressing Cre recombinase under the control of the CAG promoter (43). Offsprings were genotyped by PCR with sense primer F2 (5 ′ -CAC AGG GTA ACT GGG GCT GGT-3 ′ ) and antisense primer R2 (5 ′ -GGA AGA ACT GTG ACC CCC GC-3 ′ ) flanking the remaining loxP site and exon 15.

Animals
Studies were performed using 10-week-old mice that had free access to food and water. Mice of each genotype were placed on a normal-salt diet (NaCl 0.4% w/w) or a high-salt diet (8.0% w/w) for 1 week. All experiments were performed 1 week after dietary change. The Animal Care and Use Committee of Tokyo Medical and Dental University approved the experimental protocol.

Measurement of blood pressure
We measured blood pressure by using a radiotelemetric method in which a blood pressure transducer (Data Sciences International, St. Paul, MN, USA) was inserted into the left carotid artery. Seven days after transplantation, each mouse was housed individually in a standard cage on a receiver under a 12 h lightdark cycle. Systolic and diastolic blood pressure was recorded every minute via radiotelemetry. For each mouse, we measured blood pressure values for more than 3 consecutive days and calculated the mean + SE of all values. These experiments were performed under a normal-salt (NaCl 0.4% w/w) or high-salt (8.0% w/w) diet.

Blood analysis
Blood was drawn from the retro-orbital sinus under light ether anesthesia. Serum data were determined using the i-STAT system (FUSO Pharmaceutical Industries, Osaka, Japan).

Quantitative PCR analysis
Quantitative PCR analysis was performed on kidney as previously described (46). Total RNA from mouse kidneys was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. Total RNA was reverse transcribed using Omniscript reverse transcriptase (Qiagen, Hilden, Germany). Quantitative real-time PCR by Thermal Cycler Dice (Takara Bio, Otsu, Japan) was performed using the primer sets shown in a previous report (47).

Fluorescence correlation spectroscopy
Fluorescent TAMRA-labeled WNK1 and WNK4 peptides covering the acidic motif were prepared (Hokkaido System Science Co., Ltd., Hokkaido, Japan). Human full-length KLHL3 (wildtype and R528H mutant) was cloned into pGEX6P-1 vectors. The recombinant GST-fusion KLHL3 protein expressed in BL21 Escherichia coli cells was purified using glutathione Sepharose beads. The TAMRA-labeled WNK peptides were incubated at room temperature for 30 min with different concentrations of GST-KLHL3 (0 -2 mM) in 1× PBS containing 0.05% Tween 20 (reaction buffer). FCS measurements using the FluoroPoint-light analytical system (Olympus, Tokyo, Japan) were performed as previously described (26,27). The measurements were repeated five times per sample.

Statistics
Data are presented as means + SE. A student's t-test was used for comparisons between groups. ANOVA and Tukey's test were used for multiple comparisons.