James A. McCormick

The WNKs: Atypical Protein Kinases With Pleiotropic Actions

WNKs are serine/threonine kinases that comprise a unique branch of the kinome. They are so-named owing to the unusual placement of an essential catalytic lysine. WNKs have now been identified in diverse organisms. In humans and other mammals, four genes encode WNKs. WNKs are widely expressed at the message level, although data on protein expression is more limited. Soon after the WNKs were identified, mutations in genes encoding WNK1 and -4 were determined to cause the human disease familial hyperkalemic hypertension (also known as pseudohypoaldosteronism II, or Gordons Syndrome). For this reason, a major focus of investigation has been to dissect the role of WNK kinases in renal regulation of ion transport. More recently, a different mutation in WNK1 was identified as the cause of hereditary sensory and autonomic neuropathy type II, an early-onset autosomal disease of peripheral sensory nerves. Thus the WNKs represent an important family of potential targets for the treatment of human disease, and further elucidation of their physiological actions outside of the kidney and brain is necessary. In this review, we describe the gene structure and mechanisms regulating expression and activity of the WNKs. Subsequently, we outline substrates and targets of WNKs as well as effects of WNKs on cellular physiology, both in the kidney and elsewhere. Next, consequences of these effects on integrated physiological function are outlined. Finally, we discuss the known and putative pathophysiological relevance of the WNKs.

The calcineurin inhibitor tacrolimus activates the renal sodium chloride cotransporter to cause hypertension

Calcineurin inhibitors (CNIs) are immunosuppressive drugs that are used widely to prevent rejection of transplanted organs and to treat autoimmune disease. Hypertension and renal tubule dysfunction, including hyperkalemia, hypercalciuria and acidosis, often complicate their use. These side effects resemble familial hyperkalemic hypertension, a genetic disease characterized by overactivity of the renal sodium chloride cotransporter (NCC) and caused by mutations in genes encoding WNK kinases. We hypothesized that CNIs induce hypertension by stimulating NCC. In wild-type mice, the CNI tacrolimus caused salt-sensitive hypertension and increased the abundance of phosphorylated NCC and the NCC-regulatory kinases WNK3, WNK4 and SPAK. We demonstrated the functional importance of NCC in this response by showing that tacrolimus did not affect blood pressure in NCC-knockout mice, whereas the hypertensive response to tacrolimus was exaggerated in mice overexpressing NCC. Moreover, hydrochlorothiazide, an NCC-blocking drug, reversed tacrolimus-induced hypertension. These observations were extended to humans by showing that kidney transplant recipients treated with tacrolimus had a greater fractional chloride excretion in response to bendroflumethiazide, another NCC-blocking drug, than individuals not treated with tacrolimus; renal NCC abundance was also greater. Together, these findings indicate that tacrolimus-induced chronic hypertension is mediated largely by NCC activation, and suggest that inexpensive and well-tolerated thiazide diuretics may be especially effective in preventing the complications of CNI treatment.

Potassium modulates electrolyte balance and blood pressure through effects on distal cell voltage and chloride

Dietary potassium deficiency, common in modern diets, raises blood pressure and enhances salt sensitivity. Potassium homeostasis requires a molecular switch in the distal convoluted tubule (DCT), which fails in familial hyperkalemic hypertension (pseudohypoaldosteronism type 2), activating the thiazide-sensitive NaCl cotransporter, NCC. Here, we show that dietary potassium deficiency activates NCC, even in the setting of high salt intake, thereby causing sodium retention and a rise in blood pressure. The effect is dependent on plasma potassium, which modulates DCT cell membrane voltage and, in turn, intracellular chloride. Low intracellular chloride stimulates WNK kinases to activate NCC, limiting potassium losses, even at the expense of increased blood pressure. These data show that DCT cells, like adrenal cells, sense potassium via membrane voltage. In the DCT, hyperpolarization activates NCC via WNK kinases, whereas in the adrenal gland, it inhibits aldosterone secretion. These effects work in concert to maintain potassium homeostasis.

The WNK Kinase Network Regulating Sodium, Potassium, and Blood Pressure

The relationship between renal salt handling and hypertension is intertwined historically. The discovery of WNK kinases (With No lysine = K) now offers new insight to this relationship because WNKs are a crucial molecular pathway connecting hormones such as angiotensin II and aldosterone to renal sodium and potassium transport. To fulfill this task, the WNKs also interact with other important kinases, including serum and glucocorticoid-regulated kinase 1, STE20/SPS1-related, proline alanine-rich kinase, and oxidative stress responsive protein type 1. Collectively, this kinase network regulates the activity of the major sodium and potassium transporters in the distal nephron, including thiazide-sensitive Na-Cl cotransporters and ROMK channels. Here we show how the WNKs modulate ion transport through two distinct regulatory pathways, trafficking and phosphorylation, and discuss the physiologic and clinical relevance of the WNKs in the kidney. This ranges from rare mutations in WNKs causing familial hyperkalemic hypertension to acquired forms of hypertension caused by salt sensitivity or diabetes mellitus. Although many questions remain unanswered, the WNKs hold promise for unraveling the link between salt and hypertension, potentially leading to more effective interventions to prevent cardiorenal damage.

Pathogenesis of calcineurin inhibitor-induced hypertension.

This article reviews the current understanding of the mechanisms of calcineurin inhibitor-induced hypertension. Already early after the introduction of cyclosporine in the 1980s, vasoconstriction, sympathetic excitation and sodium retention by the kidney had been shown to play a role in this form of hypertension. The vasoconstrictive effects of calcineurin inhibitors are related to interference with the balance of vasoactive substances, including endothelin and nitric oxide. Until recently, the renal site of the sodium-retaining effect of calcineurin inhibitors was unknown. We and others have shown that calcineurin inhibitors increase the activity of the thiazide-sensitive sodium chloride cotransporter through an effect on the kinases WNK and SPAK. Here, we review the pertinent literature on the hypertensinogenic effects of calcineurin inhibitors, including neural, vascular and renal effects, and we propose an integrated model of calcineurin inhibitor-induced hypertension.

WNK Kinases and Renal Sodium Transport in Health and Disease An Integrated View

Hypertension affects 25% of the adult population in the developed world and is a major independent risk factor for stroke, myocardial infarction, and heart and kidney failure. Although many genetic and environmental contributors are involved, the kidney plays a dominant role, both in animal models,1 and in human essential hypertension.2 Most monogenic hypertensive syndromes result from increased Na+ transport along the aldosterone-sensitive distal nephron.3 The majority of these, however, are associated with hypokalemia, indicating that activation of the epithelial Na+ channel, ENaC, is a primary pathophysiologic process. In contrast, familial hyperkalemic hypertension (FHHt; also known as Gordon’s syndrome or type II pseudohypoaldosteronism) is characterized by hypertension with hyperkalemia, indicating that stimulated ENaC cannot be the primary event. FHHt was first described in 19644 and later shown to be inherited in an autosomal dominant manner.5,6 Patients with FHHt all exhibit hyperkalemia, which seems to be the most consistent feature of the disease. Hypertension, although commonly present and sometimes severe, often appears later in the natural history. Other characteristic features include mild metabolic acidosis, suppressed plasma renin activity, and aldosterone levels that are lower than would be expected, considering the hyperkalemia. Infusing the chloride salt of Na+ (NaCl) does not increase urinary potassium excretion in patients with FHHt, as it does in the normal individual, whereas infusing nonchloride salts of Na+ does increase K+ excretion in FHHt patients to normal levels.7,8 Patients are often remarkably sensitive to thiazide diuretics, which can correct both the hyperkalemia and hypertension, in many cases.9 In 2001, some cases of FHHt were shown to result from mutations in WNK1 and WNK4,10 identifying WNK kinases as previously undiscovered components of a novel electrolyte homeostasis pathway. Since that time, information about the physiological role of WNK …

SPAK Differentially Mediates Vasopressin Effects on Sodium Cotransporters

Activation of the Na(+)-K(+)-2Cl(-)-cotransporter (NKCC2) and the Na(+)-Cl(-)-cotransporter (NCC) by vasopressin includes their phosphorylation at defined, conserved N-terminal threonine and serine residues, but the kinase pathways that mediate this action of vasopressin are not well understood. Two homologous Ste20-like kinases, SPS-related proline/alanine-rich kinase (SPAK) and oxidative stress responsive kinase (OSR1), can phosphorylate the cotransporters directly. In this process, a full-length SPAK variant and OSR1 interact with a truncated SPAK variant, which has inhibitory effects. Here, we tested whether SPAK is an essential component of the vasopressin stimulatory pathway. We administered desmopressin, a V2 receptor-specific agonist, to wild-type mice, SPAK-deficient mice, and vasopressin-deficient rats. Desmopressin induced regulatory changes in SPAK variants, but not in OSR1 to the same degree, and activated NKCC2 and NCC. Furthermore, desmopressin modulated both the full-length and truncated SPAK variants to interact with and phosphorylate NKCC2, whereas only full-length SPAK promoted the activation of NCC. In summary, these results suggest that SPAK mediates the effect of vasopressin on sodium reabsorption along the distal nephron.

Hyperkalemic hypertension–associated cullin 3 promotes WNK signaling by degrading KLHL3

Familial hyperkalemic hypertension (FHHt) is a monogenic disease resulting from mutations in genes encoding WNK kinases, the ubiquitin scaffold protein cullin 3 (CUL3), or the substrate adaptor kelch-like 3 (KLHL3). Disease-associated CUL3 mutations abrogate WNK kinase degradation in cells, but it is not clear how mutant forms of CUL3 promote WNK stability. Here, we demonstrated that an FHHt-causing CUL3 mutant (CUL3 Δ403-459) not only retains the ability to bind and ubiquitylate WNK kinases and KLHL3 in cells, but is also more heavily neddylated and activated than WT CUL3. In cells, activated CUL3 Δ403-459 depleted KLHL3, preventing WNK degradation, despite increased CUL3-mediated WNK ubiquitylation; therefore, CUL3 loss in kidney should phenocopy FHHt in murine models. As predicted, nephron-specific deletion of Cul3 in mice did increase WNK kinase levels and the abundance of phosphorylated Na-Cl cotransporter (NCC). Over time, however, Cul3 deletion caused renal dysfunction, including hypochloremic alkalosis, diabetes insipidus, and salt-sensitive hypotension, with depletion of sodium potassium chloride cotransporter 2 and aquaporin 2. Moreover, these animals exhibited renal inflammation, fibrosis, and increased cyclin E. These results indicate that FHHt-associated CUL3 Δ403-459 targets KLHL3 for degradation, thereby preventing WNK degradation, whereas general loss of CUL3 activity - while also impairing WNK degradation - has widespread toxic effects in the kidney.

Free radical scavenging inhibits STAT phosphorylation following in vivo ischemia/reperfusion injury

The signal transducer and activator of transcription (STAT) family are latent transcription factors involved in a variety of signal transduction pathways, including cell death cascades. STAT1 has been shown to have a crucial role in regulating cardiac cell apoptosis in the myocardium exposed to ischemia/ reperfusion (I/R) injury. The free radical scavenger, tempol, is known to have cardioprotective properties, although little is known about the molecular mechanism(s) by which it acts. In the present study, we assessed the levels of phosphorylated STAT1 and STAT3 and examined whether tempol was able to affect STAT activation after in vivo cardiac I/R injury. We observed a reperfusion time‐dependent increase in the tyrosine phosphorylation of STAT1 and STAT3 at residues 701 and 705, respectively. Here we show for the first time that tempol dramatically reduced STAT1 and 3 phosphorylation. The reduction in STAT1 and 3 phosphorylation was accompanied by a concomitant decrease in cellular malondialdehyde (MDA) levels. To verify the role of STAT1 in modulating the cardioprotective effect of tempol, rats were injected with the STAT1 activator, IFN‐γ, and tempol during I/R injury. We found that the presence of IFN‐γ abrogated the protective effects of tempol, suggesting that the protective effects of tempol may partly operate by decreasing the phosphorylation of STAT1. This study demonstrates that careful dissection of the molecular mechanisms that underpin I/R injury may reveal cardioprotective targets for future therapy.—McCormick, J,. Barry, S. P., Sivarajah, A., Stefanutti, G., Townsend, P. A., Lawrence, K. M., Eaton, S., Knight, R. A., Thiemermann, C., Latchman, D. S., Stephanou, A. Free radical scavenging inhibits stat phosphorylation following in vivo ischemia/reperfusion injury. FASEB J. 20, E1404 –E1410 (2006)

Altered Renal FGF23-Mediated Activity Involving MAPK and Wnt: Effects of the Hyp Mutation

Fibroblast growth factor-23 (FGF23), a hormone central to renal phosphate handling, is elevated in multiple hypophosphatemic disorders. Initial FGF23-dependent Erk1/2 activity in the kidney localizes to the distal convoluted tubule (DCT) with the co-receptor α-Klotho (KL), distinct from Npt2a in proximal tubules (PT). The Hyp mouse model of X-linked hypophosphatemic rickets (XLH) is characterized by hypophosphatemia with increased Fgf23, and patients with XLH elevate FGF23 following combination therapy of phosphate and calcitriol. The molecular signaling underlying renal FGF23 activity, and whether these pathways are altered in hypophosphatemic disorders, is unknown. To examine Npt2a in vivo, mice were injected with FGF23. Initial p-Erk1/2 activity in the DCT occurred within 10 min; however, Npt2a protein was latently reduced in the PT at 30-60  min, and was independent of Npt2a mRNA changes. KL-null mice had no DCT p-Erk1/2 staining following FGF23 delivery. Under basal conditions in Hyp mice, c-Fos and Egr1, markers of renal Fgf23 activity, were increased; however, KL mRNA was reduced 60% (P<0.05). Despite the prevailing hypophosphatemia and elevated Fgf23, FGF23 injections into Hyp mice activated p-Erk1/2 in the DCT. FGF23 injection also resulted in phospho-β-catenin (p-β-cat) co-localization with KL in wild-type mice, and Hyp mice demonstrated strong p-β-cat staining under basal conditions, indicating potential crosstalk between mitogen-activated protein kinase and Wnt signaling. Collectively, these studies refine the mechanisms for FGF23 bioactivity, and demonstrate novel suppression of Wnt signaling in a KL-dependent DCT-PT axis, which is likely altered in XLH. Finally, the current treatment of phosphate and calcitriol for hypophosphatemic disorders may increase FGF23 activity.