Ostrosky-Frid M et al. (2021), Role of KLHL3 and dietary K+ in regulating KS-W...

XB-ART-58080

Am J Physiol Renal Physiol 2021 May 01;3205:F734-F747. doi: 10.1152/ajprenal.00575.2020.

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Role of KLHL3 and dietary K+ in regulating KS-WNK1 expression.

Ostrosky-Frid M , Chávez-Canales M , Zhang J , Andrukhova O , Argaiz ER , Lerdo-de-Tejada F , Murillo-de-Ozores A , Sanchez-Navarro A , Rojas-Vega L , Bobadilla NA , Vazquez N , Castañeda-Bueno M , Alessi DR , Gamba G .

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The physiological role of the shorter isoform of with no lysine kinase (WNK)1 that is exclusively expressed in the kidney (KS-WNK1), with particular abundance in the distal convoluted tubule, remains elusive. KS-WNK1, despite lacking the kinase domain, is nevertheless capable of stimulating the NaCl cotransporter, apparently through activation of WNK4. It has recently been shown that a less severe form of familial hyperkalemic hypertension featuring only hyperkalemia is caused by missense mutations in the WNK1 acidic domain that preferentially affect cullin 3 (CUL3)-Kelch-like protein 3 (KLHL3) E3-induced degradation of KS-WNK1 rather than that of full-length WNK1. Here, we show that full-length WNK1 is indeed less impacted by the CUL3-KLHL3 E3 ligase complex compared with KS-WNK1. We demonstrated that the unique 30-amino acid NH2-terminal fragment of KS-WNK1 is essential for its activating effect on the NaCl cotransporter and recognition by KLHL3. We identified specific amino acid residues in this region critical for the functional effect of KS-WNK1 and KLHL3 sensitivity. To further explore this, we generated KLHL3-R528H knockin mice that mimic human mutations causing familial hyperkalemic hypertension. These mice revealed that the KLHL3 mutation specifically increased expression of KS-WNK1 in the kidney. We also observed that in wild-type mice, the expression of KS-WNK1 was only detectable after exposure to a low-K+ diet. These findings provide new insights into the regulation and function of KS-WNK1 by the CUL3-KLHL3 complex in the distal convoluted tubule and indicate that this pathway is regulated by dietary K+ levels.NEW & NOTEWORTHY In this work, we demonstrated that the kidney-specific isoform of with no lysine kinase 1 (KS-WNK1) in the kidney is modulated by dietary K+ and activity of the ubiquitin ligase protein Kelch-like protein 3. We analyzed the role of different amino acid residues of KS-WNK1 in its activity against the NaCl cotransporter and sensitivity to Kelch-like protein 3.

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Species referenced: Xenopus laevis
Genes referenced: cul3 myc nkd1 osr1 slc12a3 stk39 wnk1 wnk3 wnk4

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	Figure 1. Kelch-like protein 3 (KLHL3)+/R528H and KLHL3R528H/R528H mice display the expected changes in the expression and phosphorylation levels of components of the with no lysine kinase 4 (WNK4)-STE20/SPS1-related proline-alanine-rich protein kinase (SPAK)/oxidative stress response-1 (OSR1)-NaCl cotransporter (NCC) pathway. A: total kidney extracts from wild-type (WT), KLHL3+/R528H [heterozygrous (HET)] and KLHL3R528H/R528H [homozygous (HOM)] mice were subjected to Western blot analysis with the indicated antibodies. Each sample was derived from a separate littermate animal. B: band intensities were quantified using ImageJ, and the results are presented relative to the expression of GAPDH. Increased expression of NCC and WNK4 as well as increased phosphorylation of SPAK (Ser373) and NCC (Thr60) were observed in KLHL3+/R528H mice. Such differences were more dramatic in KLHL3R528H/R528H mice, in which an increase in the expression of SPAK and OSR1 was also observed. ENaC, epithelial Na+ channel; ns, not significant.
	Figure 2. Immunofluorescent staining of kidney sections was performed. The primary antibodies used are indicated. Representative images from cortical and medullary regions are shown. Fluorescence intensity was quantified using ImageJ, and the results are presented in the corresponding bar graphs. ENaC, epithelial Na+ channel; KLHL3, Kelch-like protein 3; pSPAK, phospho-SPAK; SPAK, STE20/SPS1-related proline-alanine-rich protein kinase; WNK1, with no lysine kinase 1.
	Figure 3. Kidney-specific with no lysine kinase (WNK)1 (KS-WNK1) protein levels are undetectable in kidney tissue of wild-type mice but are high in mice in which cullin 3-Kelch-like protein 3 (KLHL3) E3-mediated degradation is prevented. Total kidney lysates from KLHL3R528H/R528H mice, KS-WNK1−/− mice, and double mutants (KLHL3R528H/R528H; KS-WNK1−/− mice) were analyzed by Western blot to assess the expression of WNK1 isoforms (as measured by the pan-WNK1 antibody). The robust band observed in KLHL3R528H/R528H mice (arrow) that was absent in wild-type mice corresponds to KS-WNK1, as corroborated by its absence in the double mutants. The WNK4 blot is presented at the bottom. The expected increase in WNK4 expression was observed in KLHL3R528H/R528H samples and in KLHL3R528H/R528H; KS-WNK1−/− mice.
	Figure 4. Kidney-specific with no lysine kinase (WNK)1 (KS-WNK1), WNK3, and WNK4, but not full-length WNK1 (L-WNK1), are degraded when coexpressed with Kelch-like protein 3 (KLHL3) in oocytes. A: thiazide-sensitive 22Na+ uptake was assessed in Xenopus laevis oocytes injected with the indicated cRNAs. Uptake values observed in the control group (NCC only) were set to 100%, and the other groups were normalized accordingly. While both L-WNK1 and KS-WNK1 increased Na+ uptake, KLHL3 coexpression prevented NaCl cotransporter (NCC) activation by KS-WNK1 but not by L-WNK1. Dots represent uptake values for individual oocytes. At least three independent experiments were performed with >10 oocytes per group (****P < 0.0001 vs. NCC; three points outside graphic limits). B: representative Western blots showing the effect of KLHL3 coexpression on L-WNK1, KS-WNK1, WNK3, and WNK4 levels. Oocytes were injected with cRNAs encoding for c-Myc-tagged L-WNK1, KS-WNK1, or WNK3 or Flag-tagged WNK4 with or without Flag-tagged KLHL3. All kinases except L-WNK1 were degraded in the presence of KLHL3. C: densitometric analysis of the Western blots presented in B. Two independent experiments were performed with similar results. c-Myc-tagged L-WNK1, KS-WNK1, or WNK3 or Flag-tagged WNK4 were normalized to 100% and compared with those observed in groups expressing KLHL3.
	Figure 5. The segment encoded by exon 4a in kidney-specific with no lysine kinase 1 (KS-WNK1) is needed to activate NaCl cotransporter (NCC) and to be targeted for degradation by cullin-3 (CUL3)–Kelch-like protein 3 (KLHL3) E3. A: thiazide-sensitive Na+ uptake of NCC cRNA-injected oocytes was set to 100%, and uptake values of additional groups were normalized accordingly. KS-WNK1 coexpression increased NCC activity, but the KS-WNK1-Δ4a mutant failed to activate (n = 3 transport assays, **P < 0.0001 vs. NCC; one point outside graphic limits). B: representative Western blots showing KS-WNK1 and KS-WNK1-Δ4a expression in the absence or presence of KLHL3. CUL3-KLHL3 E3-induced degradation of KS-WNK1 is observed regardless of proteosome inhibition, whereas KS-WNK1-Δ4a is expressed but resistant to CUL3-KLHL3 E3-induced degradation. C: densitometric analysis of the Western blots presented in B. Results from four different experiments were included. Expression of KS-WNK1 or KS-WNK1-Δ4a in the absence of KLHL3 were arbitrarily set to 100% and compared with expression levels observed in the presence of KLHL3 (n = 4 Western blots, *P > 0.001 vs. control without KLHL3).
	Figure 6. Mutation of the six conserved cysteines encoded in exon 4a impairs the ability of kidney-specific with no lysine kinase 1 (KS-WNK1) to activate NaCl cotransporter (NCC) but does not prevent cullin-3 (CUL3)-Kelch-like protein 3 (KLHL3) E3-induced degradation. A: amino acid sequence encoded by exon 4a. Different KS-WNK1 mutants were generated for this work with variations in the sequence of this region. The modifications introduced in each mutant are indicated. B: NCC was coexpressed in oocytes with KS-WNK1, KS-6CxS (in which all six cysteines were mutated to serine), or KS-2CxS (in which the two peripheral cysteines were mutated to serine). Thiazide-sensitive Na+ uptake of NCC-expressing oocytes was set to 100% and compared with all other groups, which were normalized accordingly. KS-WNK1-6CxS did not activate NCC, whereas KS-WNK1-2CxS did activate NCC, albeit at a lower level than wild-type KS-WNK1 (n = 3 transport assays, **P < 0.0001 vs. NCC; 3 points outside graphic limits). C: representative Western blots showing the expression of KS-WNK1-6CxS and KS-WNK1-2CxS. Both mutant proteins are targeted for degradation by CUL3-KLHL3 E3, whereas treatment with MG132 could prevent KS-WNK1-2CxS degradation. D: compiled results of densitometric analysis from at least two different Western blot experiments like that presented in (B). Expression levels of KS-WNK1, KS-WNK1-6CxS, and KS-WNK1-2CxS in the absence of KLHL3 were normalized to 100% and compared with groups expressing KLHL3 (n = 2−4 Western blots, P < 0.01 and ****P < 0.0001 vs. control without KLHL3).
	Figure 7. Mutation of the five conserved hydrophobic residues encoded in exon 4a impairs the ability of kidney-specific with no lysine kinase 1 (KS-WNK1) to activate NaCl cotransporter (NCC) and prevent cullin-3-Kelch-like protein 3 (KLHL3) E3-induced degradation. A: NCC was coexpressed in oocytes with KS-WNK1 or KS-5Q (in which all five hydrophobic residues were mutated to glutamine), and thiazide-sensitive Na+ uptake was assessed. Uptake levels observed for NCC-expressing oocytes were normalized to 100% and compared with those observed for groups expressing KS-WNK1 and KS-5Q. The strong activation of NCC induced by KS-WNK1 was not observed in the KS-5Q mutant (n = 5 transport assays, **P < 0.0001 vs. NCC; 2 points outside graphic limits). B: representative Western blots showing the expression of KS-WNK1 and KS-5Q. KS-WNK1 was degraded in the presence of KLHL3, whereas KS-5Q was not degraded. Treatment with MG132 increased KS-5Q expression, probably by impairing degradation even more. C: compiled results of densitometric analysis from three different experiments like that presented in B. Expression levels of KS-WNK1 and KS-5Q in the absence of KLHL3 were normalized to 100% and compared with those observed in groups expressing KLHL3 (n = 3 Western blots, P < 0.01 vs. control without KLHL3).
	Figure 8. Mutation of valine 11 or valine 13 located in the hydrophobic motif of exon 4a impairs the ability of kidney-specific with no lysine kinase 1 (KS-WNK1) to activate NaCl cotransporter (NCC) and cullin-3-Kelch-like protein 3 (KLHL3) E3-induced degradation of KS-WNK1. A: NCC was coexpressed in oocytes with KS-WNK1 or with KS-WNK1 mutants containing one of the following single amino acid substitutions: V11A, F12A, V13A, I14A, or I15A. Uptake levels observed for NCC-expressing oocytes were normalized to 100% and compared with those observed for groups expressing KS-WNK1 mutants. All mutants except KS-WNK1-V11A were capable of activating NCC (n = 3−5 transport assays, P < 0.05, P < 0.001, and *P < 0.0001 vs. NCC; 16 points outside graphic limits). B: representative Western blots showing the expression of KS-WNK1 and of each single-residue mutant in the absence or presence of KLHL3. Compared with wild-type (WT) KS-WNK1, there was significantly less degradation of the V11A and V13A mutants in the presence of KLHL3. Other mutants degraded similarly to the WT. C: compiled results of densitometric analysis from at least five different experiments like that presented in B. Expression levels of KS-WNK1 and single-residue mutants in the absence of KLHL3 were normalized to 100%. Degradation of single-residue mutants were compared with WT KS-WNK1 in the presence of KLHL3 (n = 5−8 Western blots, P < 0.05 and ***P < 0.001 vs. control with KLHL3).
	Figure 9. Renal kidney-specific with no lysine kinase 1 (KS-WNK1) expression is induced by a low-K+ diet (LKD). Total kidney lysates from wild-type (WT) and Kelch-like protein 3 (KLHL3)+/R528H mice maintained on a normal-K+ diet (NKD) or LKD were analyzed by Western blot to assess the expression of KS-WNK1. Compared with WT mice, moderately higher expression was observed in KLHL3+/R528H mice. In addition, a robust increase in KS-WNK1 expression was observed in WT mice but also in KLHL3+/R528H mice, suggesting that KLHL3-targeted degradation was further affected under this condition. Results of quantitation of the band corresponding to KS-WNK1 are shown at the bottom. Band intensity values of mice on a NKD were normalized to 100%. A WNK4 blot is also shown. The expected increase in WNK4 expression was observed in KLHL3+/R528H samples. Expression was further increased when mice were placed on a LKD, as previously reported (10). n = 9 for WT mice on the LKD and NKD, n = 3 for KLHL3+/R528H mice on the NKD, and n = 4 for KLHL3+/R528H mice on the LKD. *P < 0.05 versus the NKD. WNK1, with no lysine kinase 1.

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