WNKs on the Fly

A central role of the With No Lysine (K) (WNK) kinases in renal electrolyte balance and BP control was illuminated when mutations in WNK1 and WNK4 were identified as a cause of a genetic intolerance to sodium and potassium¹ (pseudohypoaldosteronism type 2; also known as familial hyperkalemic hypertension or Gordon syndrome). In this issue of the Journal of the American Society of Nephrology (JASN), Sun et al.² report on a new player in the WNK pathway and a surprising intricacy of the signaling mechanism.

According to current understanding, the WNK kinases orchestrate a switch response that toggles the activities of two distal nephron segments (distal convoluted tubule and aldosterone sensitive distal nephron) to maintain sodium and potassium balance over widely varied potassium intake.³ WNK kinases in the distal convoluted tubule together with a downstream kinase, Ste20-related proline/alanine-rich kinase (SPAK), form a potassium-sensitive signaling cascade that controls the activity of the thiazide-sensitive sodium-chloride cotransporter (NCC) on demand. WNK signaling is activated in response to low plasma potassium in dietary potassium deficiency, and this stimulates NCC to limit potassium loss from the aldosterone sensitive distal nephron at the expense of retaining sodium.^4–6 Conversely, when dietary potassium is plentiful, the WNK cascade is inhibited, and this suppresses NaCl absorption and enhances potassium excretion.⁷ Understanding why the WNK-SPAK signaling is so exquisitely sensitive to plasma potassium has been the subject of great interest.

In this issue of the JASN, Sun et al.² report that potassium-dependent signaling mechanism may be more complex than originally imagined. This elegant series of studies makes wonderful use of a model system, the Drosophila melanogaster Malpighian tubule, to explore the complexities of WNK signaling, building on the rich history of model organisms in renal physiology. When stimulated, the Malpighian tubule secretes a potassium-chloride–rich solution at a copious rate, equivalent to a cell volume of fluid per second. Sun et al.² already established that potassium secretion is driven by activation of WNK, which activates the SPAK ortholog Fray; this, in turn, phosphoactivates NKCC1.⁸ In this study, Sun et al.² exploited the genetic tractability of the D. melanogaster Malpighian tubule model. Together with a marvelous combination of physiologic and biochemical tools, they were able to probe deeper into the intracellular signaling mechanism.

Like the mammalian counterparts,^4,9 Sun et al.² found that the D. melanogaster WNK is an intracellular chloride (Cl_i⁻)–sensing kinase. In vitro kinase measurements revealed that chloride stabilizes the inactive conformation of WNK, preventing kinase activation until Cl_i⁻ is physiologically decreased. As a consequence, WNK activation can be sensitive to changes in plasma potassium and membrane potential, which have a powerful influence over [Cl_i⁻].^4,5

In the mammalian distal convoluted tubule, Kir4.1/Kir5.1 potassium channels are believed to translate changes in plasma potassium to WNK signaling through changes in membrane potential and Cl_i⁻.^5,10,11 Consistent with this idea, heterologous coexpression studies of Kir 4.1, NCC, and WNK in human embryonic kidney cells revealed that lowering extracellular potassium caused membrane potential hyperpolarization, which in turn, lowered Cl_i⁻ to stimulate WNK and increase SPAK and NCC phosphorylation.⁵ Increasing potassium had the opposite effect. Although these beautiful studies established the WNK/Cl⁻-sensing hypothesis, they left many wondering if this really happens in vivo.

Sun et al.² now show for the first time that the mechanism operates in native transporting cells but with a twist. Using a genetically encoded Cl⁻ sensor expressed in the Malpighian tubule cells, activation of ion transport and WNK signaling in the Malpighian tubule was found to coincide with a fall in Cl_i⁻, just as predicted. Surprisingly, however, mutation of residues in WNK that form the Cl⁻ binding site was not sufficient to activate WNK signaling and transport. Full activation of ion transport in the Malpighian tubule with the Cl⁻-insensitive WNK kinase required the coexpression of another protein, a kinase scaffolding protein distantly related to armadillo proteins named Drosophila MO25¹² (also known as calcium binding protein 39). Knockdown studies established that MO25 is required for physiologic activation of transepithelial ion flux with the wild-type WNK. Because in vitro phosphorylation studies revealed that Drosophila Mo25 influences chloride sensitivity of WNK, it seems likely that cooperative interactions between chloride and Mo25 directly regulate WNK signaling.

These findings likely have immediate applicability to the mammalian kidney. MO25 colocalizes with NCC and NKCC2 on the apical membrane of the mouse kidney.¹³ Furthermore, biochemical studies revealed that mammalian MO25 enhances WNK4/SPAK-mediated phosphorylation of NCC and NKCC,¹⁴ likely by facilitating structural changes in the kinases. Together with the intriguing discovery in the Drosophila Malpighian tubule reported by Sun et al.,² these observations provide compelling reason to suggest that MO25 influences WNK signaling in the mammalian kidney.

The discovery of MO25 in WNK pathway has important implications. Because low potassium consumption, common in modern diets, presses the switch pathway to conserve potassium at the expense of increasing sodium absorption, the pathway provides a mechanism to explain why the modern diet feeds the fire of salt-sensitive hypertension. Given its potential role in determining the sensitivity of the pathway to potassium, MO25 should be considered as a potential antihypertensive drug target.

Disclosures

None.