The evolving field of salt transport regulation in the Steve Hebert Lecture

IN A RECENT ISSUE of the American Journal of Physiology-Renal Physiology, Melanie Cobb’s group presents an interesting review about the renal and vascular effects of the With No Lysine kinases, WNKs (5). This work is showing an overview of what was presented by Dr. Cobb as the Steven Hebert Lecturer of the American Physiological Society during the Experimental Biology meeting in Boston, 2015. This yearly lecture was inaugurated in 2010 by the Epithelial Transport Group to honor the memory of Steven Charles Hebert, an innovative and visionary physiologist who made very important contributions to the renal and endocrinology field, including the molecular identification of the cDNA encoding for the furosemide-sensitive Na -K -2Cl cotransporter (NKCC2) (7), the thiazide-sensitive Na -Cl cotransporter (NCC) (7, 8), the renal outer medullary potassium channel (ROMK) (9), and the calcium-sensing receptor (CaSR), from the bovine parathyroid gland (3) and rat kidney (17). The WNKs were discovered in 2000 by Cobb’s group in an attempt to identify new members of the MAP kinase cascades in the central nervous system (29). In that seminal paper, WNKs were described as serine/threonine kinases with the unique property of lacking the catalytic lysine located in subdomain II that is characteristic of kinases. Hence the name WNK, for With No Lysine kinases. It was later shown that in WNKs the catalytic lysine resides in subdomain I (13), and, as discussed by Dbouk et al. (5), more than a decade later came the realization that the absence of the lysine in subdomain II confers the WNKs with the unique property of being sensitive to intracellular chloride concentration ([Cl ]i). When [Cl ]i rises, this ion binds to key leucine residues in the kinase domain, preventing the autophosphorylation of the WNK and thus maintaining the inactive state, while a decrease in [Cl ]i allows the autophosphorylation and thus activation of the kinase (15). The WNKs attracted the interest of the cardiovascular and renal community when it was demonstrated by Lifton’s group (28) that mutations in two members of the family, WNK1 and WNK4, were the cause of the familial hyperkalemic hypertension (FHHt), also known as pseudohypoaldosteronism type II (PHAII) (Table 1). This is an inherited dominant disease featuring arterial hypertension with hyperkalemia, metabolic acidosis, and hypercalciuria. Because this phenotype is the mirror image of the Gitelman syndrome (hypotensive hypokalemic metabolic alkalosis) that is due to inactivating mutations of the NCC (Table 1), it was soon proposed that these kinases were regulators of the NCC and that mutations affected regulation of the cotransporter, increasing its activity and producing the disease. As discussed by Dbouk et al. (5), other regulatory players were added in the following years, unmasking a complicated set of interactive proteins involved in the regulation of renal electrolyte homeostasis (Table 1). On the one hand, the downstream serine/threonine kinases SPAK and OSR1 were discovered to be essential for WNKs’ action to occur in certain target proteins (16, 26). WNKs interact with and phosphorylate SPAK/OSR1, which are then directly responsible for the phosphorylation of the target cotransporters, such as NCC, NKCC2, and KCCs (6, 18, 19). Thus the effects of WNKs that are dependent on their catalytic activity require the presence of SPAK/OSR1. On the other hand, the fact that mutations in WNKs did not explain FHHt in all studied families allowed the identification of upstream WNKs regulators, known as Kelch-like 3 and cullin 3 proteins, which form a ubiquitylation-ligase complex that targets WNKs. Mutations in genes encoding the Kelch-like 3 or the cullin 3 proteins explain the remaining cases of FHHt, which present a more severe phenotype, consistent with the upstream position of these proteins in the cascade. Thus the Kelch3-Cul3 complex modulates the ubiquitylation and thus half-life of WNKs, which in turn phosphorylate SPAK/OSR1 that ultimately modulate the activity of target Address for reprint requests and other correspondence: G. Gamba, Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México and Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga No. 15, Tlalpan 14080, Mexico City, Mexico (e-mail: gamba@biomedicas.unam.mx).

IN A RECENT ISSUE of the American Journal of Physiology-Renal Physiology, Melanie Cobb's group presents an interesting review about the renal and vascular effects of the With No Lysine kinases, WNKs (5).This work is showing an overview of what was presented by Dr. Cobb as the Steven Hebert Lecturer of the American Physiological Society during the Experimental Biology meeting in Boston, 2015.This yearly lecture was inaugurated in 2010 by the Epithelial Transport Group to honor the memory of Steven Charles Hebert, an innovative and visionary physiologist who made very important contributions to the renal and endocrinology field, including the molecular identification of the cDNA encoding for the furosemide-sensitive Na ϩ -K ϩ -2Cl Ϫ cotransporter (NKCC2) (7), the thiazide-sensitive Na ϩ -Cl Ϫ cotransporter (NCC) (7,8), the renal outer medullary potassium channel (ROMK) (9), and the calcium-sensing receptor (CaSR), from the bovine parathyroid gland (3) and rat kidney (17).
The WNKs were discovered in 2000 by Cobb's group in an attempt to identify new members of the MAP kinase cascades in the central nervous system (29).In that seminal paper, WNKs were described as serine/threonine kinases with the unique property of lacking the catalytic lysine located in subdomain II that is characteristic of kinases.Hence the name WNK, for With No Lysine kinases.It was later shown that in WNKs the catalytic lysine resides in subdomain I (13), and, as discussed by Dbouk et al. (5), more than a decade later came the realization that the absence of the lysine in subdomain II confers the WNKs with the unique property of being sensitive to intracellular chloride concentration ([Cl Ϫ ] i ).When [Cl Ϫ ] i rises, this ion binds to key leucine residues in the kinase domain, preventing the autophosphorylation of the WNK and thus maintaining the inactive state, while a decrease in [Cl Ϫ ] i allows the autophosphorylation and thus activation of the kinase (15).
The WNKs attracted the interest of the cardiovascular and renal community when it was demonstrated by Lifton's group (28) that mutations in two members of the family, WNK1 and WNK4, were the cause of the familial hyperkalemic hypertension (FHHt), also known as pseudohypoaldosteronism type II (PHAII) (Table 1).This is an inherited dominant disease featuring arterial hypertension with hyperkalemia, metabolic acidosis, and hypercalciuria.Because this phenotype is the mirror image of the Gitelman syndrome (hypotensive hypokalemic metabolic alkalosis) that is due to inactivating mutations of the NCC (Table 1), it was soon proposed that these kinases were regulators of the NCC and that mutations affected regulation of the cotransporter, increasing its activity and producing the disease.As discussed by Dbouk et al. ( 5), other regulatory players were added in the following years, unmasking a complicated set of interactive proteins involved in the regulation of renal electrolyte homeostasis (Table 1).On the one hand, the downstream serine/threonine kinases SPAK and OSR1 were discovered to be essential for WNKs' action to occur in certain target proteins (16,26).WNKs interact with and phosphorylate SPAK/OSR1, which are then directly responsible for the phosphorylation of the target cotransporters, such as NCC, NKCC2, and KCCs (6,18,19).Thus the effects of WNKs that are dependent on their catalytic activity require the presence of SPAK/OSR1.On the other hand, the fact that mutations in WNKs did not explain FHHt in all studied families allowed the identification of upstream WNKs regulators, known as Kelch-like 3 and cullin 3 proteins, which form a ubiquitylation-ligase complex that targets WNKs.Mutations in genes encoding the Kelch-like 3 or the cullin 3 proteins explain the remaining cases of FHHt, which present a more severe phenotype, consistent with the upstream position of these proteins in the cascade.Thus the Kelch3-Cul3 complex modulates the ubiquitylation and thus half-life of WNKs, which in turn phosphorylate SPAK/OSR1 that ultimately modulate the activity of target

Editorial Focus
proteins such as NCC by phosphorylating key residues in the amino terminal domain (14,18).WNKs are very complex kinases, as discussed by Dbouk et al. (5).In addition to the unique kinase domain lacking the catalytic lysine in subdomain II, these kinases contains a low-complexity C-terminal domain that make them belong to the intrinsically disordered proteins (IDPs) that can undergo numerous posttranslational modifications and may act as interaction hubs with an impact on many cellular processes.Thus many WNKs' effects are known to be independent of their catalytic activity (11).This characteristic, together with the unique sensitivity of WNKs for [Cl Ϫ ] i , produced a series of contradictory observations through the years, complicating the understanding of the role of WNK kinases in modulating ion transport in the distal nephron.Today, however, some key points have been clarified, allowing the field to move toward a better understanding of the molecular physiology of the kinases and the disease associated with them, opening the next questions that requires research to be resolved.
First, it is clear that FHHt-type mutations in genes encoding WNKs, Kelch-like 3, or cullin 3 result in increased expression of the kinases.What is not yet understood is why the increased WNK or WNKs escape the modulation by [Cl Ϫ ] i , maintaining the increased activity of NCC, even though the FHHt phenotype, salt-sensitive hypertension and hyperkalemia, would be expected to inhibit them.Second, it is now clear that WNKs can have inhibitory or excitatory effects in their target proteins, depending on the activity mode of the kinases.What is not known yet is how and under what conditions this modulation takes place in vivo.Third, apparently the affinity of WNKs for [Cl Ϫ ] i is different, with WNK4 being more sensitive than WNK1 and WNK3.What is not yet known is the structural reason for such a different sensitivity.Fourth, WNKs modulate transport proteins involved in the cellular response to cell volume changes and are activated or inhibited by osmotic stress.However, changes in cell volume often go with opposite changes in the [Cl Ϫ ] i .For instance, cell shrinkage is associated with activation of WNKs and their target protein, NKCC1.However, during cell shrinkage [Cl Ϫ ] i increases, and that will be expected to reduce the activity of the WNKs-SPAK-NKCC1 complex.

GRANTS
This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT) México Grants No. 165815 and 188712 (to G. Gamba).DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors.AUTHOR CONTRIBUTIONS G.G. provided conception and design of research; G.G. performed experiments; G.G. analyzed data; G.G. interpreted results of experiments; G.G. prepared figures; G.G. drafted manuscript; G.G. edited and revised manuscript; G.G. approved final version of manuscript.

Table 1 .
Address for reprint requests and other correspondence: G. Gamba, Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México and Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga No. 15, Tlalpan 14080, Mexico City, Mexico (e-mail: gamba@biomedicas.unam.mx).Genes causing inherited disease due to altered activity of the renal Na ϩ -Cl Ϫ cotransporter (NCC) Thus the modulation of WNKs by osmotic stress occurs by different mechanisms not yet understood.Fifth, much of what we know about WNKs has been studied in kidneys, but a growing number of studies revealed the presence of WNKs in blood vessels.In this regards, Dbouk et al. (5) present a review of what is known about WNKs in blood vessels and propose what could happen if what is known for WNKs in epithelial cells also occurs in endothelial cells and vascular smooth muscle cells.