Deciphering the mechanism of calcineurin inhibitor‐induced hypertension

In the current issue of Acta Physiologica, Gao et al. explore the possible role of the inward rectifying potassium channel (Kir) 4.1 in cyclosporin A-induced hyperkalemic hypertension.1 Calcineurin inhibitors, such as cyclosporin A and tacrolimus, are immunosuppressive drugs, which are widely used to prevent transplant rejection and treat autoimmune diseases. Side effects of calcineurin inhibitors include hypertension, hyperkalemia, hypercalciuria, and acidosis.2 These effects of calcineurin inhibitors resemble familial hyperkalemic hypertension, a genetic disease characterized by overactivity of the thiazide-sensitive sodium chloride cotransporter (NCC) expressed in the renal distal convoluted tubule.2 Previous work has established the involvement of NCC underlying the hypertensive effect of calcineurin inhibitors. In fact, in contrast to the effects in wildtype mice, calcineurin inhibition does not cause hypertension, hypercalciuria, acidosis, or increased potassium levels in NCC-deficient mice.2 Furthermore, thiazide-dependent inhibition of NCC reverses the hypertensive effect observed with calcineurin inhibition, whereas an exaggerated hypertensive response was seen in mice over-expressing NCC.2 In agreement with this, treatment with calcineurin inhibitors has been shown to increase NCC phosphorylation, an indirect measure of NCC activity, in murine kidney, tubule suspensions, and cell models.2, 3 Studies of kidney biopsies or urinary extracellular vesicles of kidney transplant recipients treated with calcineurin inhibitors showed higher total and phosphorylated levels of NCC compared to controls. Furthermore, these patients had a greater fractional chloride excretion in response to NCC blockade, indicative of an increased NCC activity.2, 3 So overall, the effect of calcineurin inhibitors on NCC is well established. However, the mechanism linking calcineurin inhibition to altered NCC activity is unclear. A potential candidate linking calcineurin with NCC is the Kir4.1/Kir5.1 channel. In the kidney, Kir4.1 interacts with Kir5.1 to form a basolateral potassium channel in the late cortical thick ascending limb, distal convoluted tubule, connecting tubule, and cortical collecting duct. Kir4.1 confers permeability to potassium, whereas Kir5.1 is thought to serve as a regulatory subunit.4 The Kir4.1/Kir5.1 channel determines the basolateral potassium conductance in the distal convoluted tubule and plays a key role in setting the membrane potential. Interestingly, in this issue of Acta Physiologica, Gao et al. show by patch-clamp measurements that cyclosporin A stimulates Kir4.1/Kir5.1 activity in the distal convoluted tubule and makes the basolateral membrane potential more negative.1 Studies in recent years have demonstrated the importance of the Kir4.1/Kir5.1 channel in the regulation of NCC. Loss-of-function mutations of KCNJ10, encoding the human Kir4.1 gene, cause EAST syndrome, which symptoms include sodium wasting and hypokalemic alkalosis, symptoms mirroring calcineurin inhibition.4 Deletion of Kcnj10 in the mouse kidney leads to depolarization of the membrane potential and a reduced NCC expression and activity in the distal convoluted tubule, along with a reduced blood pressure, sodium and potassium wasting.4 In addition, changes in Kir4.1/Kir5.1 activity have been shown to be indispensable for the regulation of NCC by dietary changes in sodium and potassium, bradykinin, activation of the type 2 angiotensin II receptor, and the β-adrenergic receptor.4 Collectively, this shows the central role of Kir4.1/5.1 in the regulation of NCC. The mechanism linking Kir4.1/Kir5.1 channels to NCC is not completely understood but is thought to depend on modulation of the with no lysine (WNK) kinases by intracellular chloride (Figure 1). Increased activity of the Kir4.1/Kir5.1 channel hyperpolarizes the membrane thereby increasing the driving force for chloride exit. In vitro studies in HEK cells have delineated that transfection with loss-of-function mutants of Kir4.1 results in an increased intracellular chloride concentration and reduced phosphorylated NCC levels. Conversely, low extracellular potassium, known to increase Kir4.1/Kir5.1 activity, led to decreased intracellular chloride concentrations and increased phosphorylated NCC levels, confirming the relation between Kir4.1/Kir5.1 activity and intracellular chloride levels.5 High intracellular chloride will inhibit WNK activity, by binding to the catalytic domain of WNK1 and WNK4. NCC activity is known to be regulated by WNK kinases, which exert their effect on NCC phosphorylation via the Ste-20-related proline alanine-rich protein kinase (SPAK) and the oxidative stress-responsive kinase 1 (OSR1).4 Therefore, decreased conductance by Kir4.1/Kir5.1 is thought to decrease NCC activity via a WNK-SPAK signaling cascade. On the other hand, the cyclosporin-induced activation of Kir4.1/Kir5.1 and consequent hyperpolarization of the membrane demonstrated by Gao et al. is expected to decrease intracellular chloride levels, leading to stimulation of the WNK-SPAK-NCC pathway.1 To explore a possible involvement of the Kir4.1/Kir5.1 channel in the effect of cyclosporin A on NCC, Gao et al. used a kidney-specific Kcnj10 knockout model. Cyclosporin A did not affect phosphorylated NCC levels in Kcnj10 knockout mice, in contrast to wildtype mice. In addition, cyclosporin A treatment reduced urinary sodium excretion in wildtype mice and NCC blockade by thiazide led to a greater sodium excretion in mice treated with cyclosporin A, indicative of an increased NCC activity.1 These effects were not seen in the Kcnj10-deficient mice, indicating that Kir4.1 is required for the effect of calcineurin inhibitors on NCC activity. Cyclosporin A treatment for 2 weeks increased blood pressure and raised plasma potassium levels in wildtype mice, but these changes were abolished by deletion of Kcnj10. Furthermore, cyclosporin A increased phosphorylated and total SPAK levels, which was not seen in the Kcnj10 knockout mice, suggesting the intracellular pathway leading from cyclosporin A to NCC is via the WNK-SPAK-pathway. In agreement with this, previous studies in mice showed that the effects of calcineurin inhibitors on NCC were accompanied by changes in the expression or phosphorylation of proteins in WNK-SPAK pathway. For example, calcineurin inhibitors have been shown to increase the abundance of WNK1, WNK3, WNK4, and SPAK as well as the phosphorylation of SPAK.2, 6 The connection between calcineurin inhibitors and NCC activity has previously been investigated, demonstrating that Kelch-like 3 (KLHL3) is dephosphorylated by calcineurin.6 KLHL3 dephosphorylation stimulates binding to WNK kinases, which would target the WNK kinases for degradation. The calcineurin inhibitor tacrolimus has been shown to increase KLHL3 phosphorylation, thereby preventing the binding of KLHL3 to WNKs. This is expected to lead to increased WNK levels, which was proposed to be the mechanism of the tacrolimus-mediated NCC activation.6 Another study suggested that calcineurin directly dephosphorylates NCC, independent of the WNK-SPAK pathway, as it was shown that tacrolimus changed phosphorylated NCC levels in mice without a detectable change in WNK4 or SPAK levels, at least not in total kidney lysates.7 The study of Gao et al. did not investigate whether KLHL3 phosphorylation was still affected by cyclosporin A in the Kcnj10 knockout mice or whether NCC was directly dephosphorylated by NCC. However, as no effect of cyclosporin A on phosphorylated NCC was seen in the Kcnj10 knockout mice in the study of Gao et al., this indicates that the effect of calcineurin on Kir4.1 is essential for mediating the effect of calcineurin on NCC.1 A question remaining is how calcineurin affects Kir4.1/Kir5.1 activity. Calcineurin is a serine/threonine protein phosphatase and the intracellular localization, activity, and ubiquitination of Kir4.1/Kir5.1 have been shown to be regulated by phosphorylation.4 If the channel is a direct substrate of calcineurin or if this regulation is indirect would be interesting to investigate in future studies. Altogether, the study by Gao et al. comprehensively shows that Kir4.1/Kir5.1 channel is critical for the cyclosporin A-induced NCC activation and hypertension.1 Their study further establishes the crucial role of Kir4.1/Kir5.1 in the regulation of NCC and blood pressure, which holds promising implications for future research on the therapeutic targeting of this pathway in hypertension treatment. None.