Aldosterone mediates activation of the thiazide-sensitive Na-Cl cotransporter through an SGK1 and WNK4 signaling pathway

Aldosterone regulates volume homeostasis and blood pressure by enhancing sodium reabsorption in the kidney’s distal nephron (DN). On the apical surface of these renal epithelia, aldosterone increases expression and activity of the thiazide-sensitive Na-Cl cotransporter (NCC) and the epithelial sodium channel (ENaC). While the cellular mechanisms by which aldosterone regulates ENaC have been well characterized, the molecular mechanisms that link aldosterone to NCC-mediated Na⁺/Cl^– reabsorption remain elusive. The serine/threonine kinase with-no-lysine 4 (WNK4) has previously been shown to reduce cell surface expression of NCC. Here we measured sodium uptake in a Xenopus oocyte expression system and found that serum and glucocorticoid–induced kinase 1 (SGK1), an aldosterone-responsive gene expressed in the DN, attenuated the inhibitory effect of WNK4 on NCC activity. In addition, we showed — both in vitro and in a human kidney cell line — that SGK1 bound and phosphorylated WNK4. We found one serine located within an established SGK1 consensus target sequence, and the other within a motif that was, to our knowledge, previously uncharacterized. Mutation of these target serines to aspartate, in order to mimic phosphorylation, attenuated the effect of WNK4 on NCC activity in the Xenopus oocyte system. These data thus delineate what we believe to be a novel mechanism for aldosterone activation of NCC through SGK1 signaling of WNK4 kinase.     

Suppression of WNK1-SPAK/OSR1 Attenuates Bone Cancer Pain by Regulating NKCC1 and KCC2

Our preliminary experiment indicated the activation of with-nolysine kinases 1 (WNK1) in bone cancer pain (BCP) rats. This study aimed to investigate the underlying mechanisms via which WNK1 contributed to BCP. A rat model of BCP was induced by Walker-256 tumor cell implantation. WNK1 expression and distribution in the lumbar spinal cord dorsal horn and dorsal root ganglion were examined. SPS1-related proline/alanine-rich kinase (SPAK), oxidative stress-responsive kinase 1 (OSR1), sodium-potassium-chloride cotransporter 1 (NKCC1), and potassium-chloride cotransporter 2 (KCC2) expression were assessed. Pain behaviors including mechanical allodynia and movement-evoked pain were measured. BCP rats exhibited significant mechanical allodynia, with increased WNK1 expression in the dorsal horn and dorsal root ganglion neurons, elevated SPAK/OSR1 and NKCC1 expression in the dorsal root ganglion, and decreased KCC2 expression in the dorsal horn. WNK1 knock-down by small interfering alleviated mechanical allodynia and movement-evoked pain, inhibited WNK1-SPAK/OSR1-NKCC1 activities, and restored KCC2 expression. In addition, closantel (a WNK1-SPAK/OSR1 inhibitor) improved pain behaviors, downregulated SPAK/OSR1 and NKCC1 expression, and upregulated KCC2 expression in BCP rats. Activation of WNK1-SPAK/OSR1 signaling contributed to BCP in rats by modulating NKCC1 and KCC2 expression. Therefore, suppression of WNK1-SPAK/OSR1 may serve as a potential target for BCP therapy.PerspectiveOur findings demonstrated that the WNK1-SPAK/OSR1 signaling contributed to BCP in rats via regulating NKCC1 and KCC2. Suppressing this pathway reduced pain behaviors. Based on these findings, the WNK1-SPAK/OSR1 signaling may be a potential target for BCP therapy.     

Enhanced phosphorylation of Na+-Cl- co-transporter in experimental metabolic syndrome: role of insulin

In the present study, we investigated the activity of the thiazide-sensitive NCC (Na+-Cl- co-transporter) in experimental metabolic syndrome and the role of insulin in NCC activation. Renal responses to the NCC inhibitor HCTZ (hydrochlorothiazide), as a measure of NCC activity in vivo, were studied in 12-week-old ZO (Zucker obese) rats, a model of the metabolic syndrome, and in ZL (Zucker lean) control animals, together with renal NCC expression and molecular markers of NCC activity, such as localization and phosphorylation. Effects of insulin were studied further in mammalian cell lines with inducible and endogenous expression of this molecule. ZO rats displayed marked hyperinsulinaemia, but no differences in plasma aldosterone, compared with ZL rats. In ZO rats, natriuretic and diuretic responses to NCC inhibition with HCTZ were enhanced compared with ZL rats, and were associated with a decrease in BP (blood pressure). ZO rats displayed enhanced Thr53 NCC phosphorylation and predominant membrane localization of both total and phosphorylated NCC, together with a different profile in expression of SPAK (Ste20-related proline/alanine-rich kinase) isoforms, and lower expression of WNK4. In vitro, insulin induced NCC phosphorylation, which was blocked by a PI3K (phosphoinositide 3-kinase) inhibitor. Insulin-induced reduction in WNK4 expression was also observed, but delayed compared with the time course of NCC phosphorylation. In summary, we report increased NCC activity in hyperinsulinaemic rodents in conjunction with the SPAK expression profile consistent with NCC activation and reduced WNK4, as well as an ability of insulin to induce NCC stimulatory phosphorylation in vitro. Together, these findings indicate that hyperinsulinaemia is an important driving force of NCC activity in the metabolic syndrome with possible consequences for BP regulation.     

Ubiquitylation and the pathogenesis of hypertension

Liddle syndrome is monogenic hypertension caused by mutations in the epithelial Na⁺ channel (ENaC) that interfere with its ubiquitylation by Nedd4-2. In this issue, Ronzaud and colleagues found that deleting Nedd4-2 from kidney tubules in adult mice led to ENaC accumulation, but not at the plasma membrane, as predicted from current models. Instead, abundance of the sodium chloride transporter NCC increased at the plasma membrane, and the mice have some features of increased NCC activity. Together, the results suggest that defective ubiquitylation of ENaC by Nedd4-2 may not fully explain Liddle syndrome and that Nedd4-2 modulates NCC more strongly. Because something is happening here But you don’t know what it is Do you, Mister Jones? —Bob Dylan,“Ballad of A Thin Man” (1965)Liddle syndrome is a monogenic hypertensive disease that results from excess activity of the epithelial sodium channel ENaC (1). In 1996, Staub, Rotin, and colleagues found that the ubiquitin ligase Nedd 4-2 interacts with ENaC via PY motifs within the carboxyl terminal domains of ENaC subunits (1). The disease-causing mutations in the β- or γENAC subunits result in deletion of the PY motifs (2), which abrogates ubiquitylation of the channel in vitro (3). As ubiquitylation of proteins appears to accelerate their removal from the plasma membrane and to increase degradation (or recycling), these deletions are expected to result in an increase in ENaC at the plasma membrane, an effect that has been observed both in vitro (3) and in vivo (4); mice with a mutated βENaC also exhibit many of the phenotypic features of Liddle syndrome, supporting the fidelity of this model (4). Although attention has focused on the ability of Nedd4-2 to modulate ENaC, it may also interact with another sodium transport protein expressed by the distal nephron, the thiazide-sensitive Na⁺Cl^– cotransporter (NCC, SLC12A3). Ko and colleagues reported that NCC is ubiquitylated, and suggested that this process might be involved in its endocytosis (5). Arroyo et al. reported that NCC coimmunoprecipitates with Nedd4-2 when transfected into HEK293 cells and that coexpression of Nedd4-2 with NCC inhibited NCC activity in oocytes (6). Surprisingly, however, the interaction of Nedd4-2 with NCC did not occur through classical PY motifs, suggesting that the interaction occurs via alternative domains. Thus, emerging evidence posits a role for Nedd4-2 in mediating NCC ubiquitylation and endocytosis. Nedd4-2 ubiquitin ligase activity is not constitutive, but instead is believed to be important in modulating aldosterone effects on ENaC and perhaps NCC. According to consensus models (Figure ​(Figure1),1), aldosterone increases SGK1 expression, which phosphorylates Nedd4-2 on key serine residues, thereby promoting interaction with 14-3-3 proteins. This interaction reduces Nedd4-2’s ubiquitin ligase activity with respect to ENaC. The model suggests that ENaC that is not highly ubiquitylated will remain in the plasma membrane longer (7). This hypothesis is supported by abundant data, but like all models, it must be tested in vivo to confirm its essential features. The paper by Ronzaud and colleagues (8) in this issue of the JCI takes this model out for a test drive, with some very surprising results. Open in a separate windowFigure 1Models of ENaC and NCC regulation by Nedd4-2.(A) Aldosterone (Aldo) interaction with the mineralocorticoid receptor (MR) induces SGK1 activity, which suppresses the ubiquitin ligase activity of Nedd4-2. Nedd4-2 normally catalyzes the addition of ubiquitin moieties (Ub) to ENaC (step i). Ubiquitylated ENaC is then removed from the plasma membrane (step ii) and undergoes degradation (or recycling) inside the cell (step iii). Poorly defined alternate pathways for SGK1 to stimulate ENaC independently of Nedd4-2 also exist. Ronzaud and colleagues (8) found that Nedd4-2 deletion increased in intracellular ENaC, suggesting that Nedd4-2 may affect step iii (dotted blue arrow) more than step ii. (B) Similar signaling pathway to NCC. An alternative pathway for SGK1 activation of NCC may involve WNK4, as described in text. In contrast with effects on ENaC, NCC abundance was increased at the plasma membrane by Nedd4-2 deletion, suggesting a defect at step ii, as shown by the dotted blue arrow. Note that sites of ubiquitylation on NCC have not been defined.     

The thiazide-sensitive Na-Cl cotransporter is regulated by a WNK kinase signaling complex

The pathogenesis of essential hypertension remains unknown, but thiazide diuretics are frequently recommended as first-line treatment. Recently, familial hyperkalemic hypertension (FHHt) was shown to result from activation of the thiazide-sensitive Na-Cl cotransporter (NCC) by mutations in WNK4, although the mechanism for this effect remains unknown. WNK kinases are unique members of the human kinome, intimately involved in maintaining electrolyte balance across cell membranes and epithelia. Previous work showed that WNK1, WNK4, and a kidney-specific isoform of WNK1 interact to regulate NCC activity, suggesting that WNK kinases form a signaling complex. Here, we report that WNK3, another member of the WNK kinase family expressed by distal tubule cells, interacts with WNK4 and WNK1 to regulate NCC in both human kidney cells and Xenopus oocytes, further supporting the WNK signaling complex hypothesis. We demonstrate that physiological regulation of NCC in oocytes results from antagonism between WNK3 and WNK4 and that FHHt-causing WNK4 mutations exert a dominant-negative effect on wild-type (WT) WNK4 to mimic a state of WNK3 excess. The results provide a mechanistic explanation for the divergent effects of WT and FHHt-mutant WNK4 on NCC activity, and for the dominant nature of FHHt in humans and genetically modified mice.     

WNK kinases regulate thiazide-sensitive Na-Cl cotransport

Pseudohypoaldosteronism type II (PHAII) is an autosomal dominant disorder of hyperkalemia and hypertension. Mutations in two members of the WNK kinase family, WNK1 and WNK4, cause the disease. WNK1 mutations are believed to increase WNK1 expression; the effect of WNK4 mutations remains unknown. The clinical phenotype of PHAII is opposite to Gitelman syndrome, a disease caused by dysfunction of the thiazide-sensitive Na-Cl cotransporter. We tested the hypothesis that WNK kinases regulate the mammalian thiazide-sensitive Na-Cl cotransporter (NCC). Mouse WNK4 was cloned and expressed in Xenopus oocytes with or without NCC. Coexpression with WNK4 suppressed NCC activity by more than 85%. This effect did not result from defects in NCC synthesis or processing, but was associated with an 85% reduction in NCC abundance at the plasma membrane. Unlike WNK4, WNK1 did not affect NCC activity directly. WNK1, however, completely prevented WNK4 inhibition of NCC. Some WNK4 mutations that cause PHAII retained NCC-inhibiting activity, but the Q562E WNK4 demonstrated diminished activity, suggesting that some PHAII mutations lead to loss of NCC inhibition. Gain-of-function WNK1 mutations would be expected to inhibit WNK4 activity, thereby activating NCC, contributing to the PHAII phenotype. Together, these results identify WNK kinases as a previously unrecognized sodium regulatory pathway of the distal nephron. This pathway likely contributes to normal and pathological blood pressure homeostasis.     

IRBIT governs epithelial secretion in mice by antagonizing the WNK/SPAK kinase pathway

Fluid and HCO(3)(-) secretion are fundamental functions of epithelia and determine bodily fluid volume and ionic composition, among other things. Secretion of ductal fluid and HCO(3)(-) in secretory glands is fueled by Na(+)/HCO(3)(-) cotransport mediated by basolateral solute carrier family 4 member 4 (NBCe1-B) and by Cl(-)/HCO(3)(-) exchange mediated by luminal solute carrier family 26, member 6 (Slc26a6) and CFTR. However, the mechanisms governing ductal secretion are not known. Here, we have shown that pancreatic ductal secretion in mice is suppressed by silencing of the NBCe1-B/CFTR activator inositol-1,4,5-trisphosphate (IP(3)) receptor-binding protein released with IP(3) (IRBIT) and by inhibition of protein phosphatase 1 (PP1). In contrast, silencing the with-no-lysine (WNK) kinases and Ste20-related proline/alanine-rich kinase (SPAK) increased secretion. Molecular analysis revealed that the WNK kinases acted as scaffolds to recruit SPAK, which phosphorylated CFTR and NBCe1-B, reducing their cell surface expression. IRBIT opposed the effects of WNKs and SPAK by recruiting PP1 to the complex to dephosphorylate CFTR and NBCe1-B, restoring their cell surface expression, in addition to stimulating their activities. Silencing of SPAK and IRBIT in the same ducts rescued ductal secretion due to silencing of IRBIT alone. These findings stress the pivotal role of IRBIT in epithelial fluid and HCO(3)(-) secretion and provide a molecular mechanism by which IRBIT coordinates these processes. They also have implications for WNK/SPAK kinase-regulated processes involved in systemic fluid homeostasis, hypertension, and cystic fibrosis.     

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.     

Mechanisms of WNK1 and WNK4 interaction in the regulation of thiazide-sensitive NaCl cotransport

With-no-lysine (WNK) kinases are highly expressed along the mammalian distal nephron. Mutations in either WNK1 or WNK4 cause familial hyperkalemic hypertension (FHHt), suggesting that the protein products converge on a final common pathway. We showed previously that WNK4 downregulates thiazide-sensitive NaCl cotransporter (NCC) activity, an effect suppressed by WNK1. Here we investigated the mechanisms by which WNK1 and WNK4 interact to regulate ion transport. We report that WNK1 suppresses the WNK4 effect on NCC activity and associates with WNK4 in a protein complex involving the kinase domains. Although a kinase-dead WNK1 also associates with WNK4, it fails to suppress WNK4-mediated NCC inhibition; the WNK1 kinase domain alone, however, is not sufficient to block the WNK4 effect. The carboxyterminal 222 amino acids of WNK4 are sufficient to inhibit NCC, but this fragment is not blocked by WNK1. Instead, WNK1 inhibition requires an intact WNK4 kinase domain, the region that binds to WNK1. In summary, these data show that: (a) the WNK4 carboxyl terminus mediates NCC suppression, (b) the WNK1 kinase domain interacts with the WNK4 kinase domain, and (c) WNK1 inhibition of WNK4 is dependent on WNK1 catalytic activity and an intact WNK1 protein. These findings provide insight into the complex interrelationships between WNK1 and WNK4 and provide a molecular basis for FHHt.     

Intersectin links WNK kinases to endocytosis of ROMK1

With-no-lysine (WNK) kinases are a novel family of protein kinases characterized by an atypical placement of the catalytic lysine. Mutations of 2 family members, WNK1 and WNK4, cause pseudohypoaldosteronism type 2 (PHA2), an autosomal-dominant disease characterized by hypertension and hyperkalemia. WNK1 and WNK4 stimulate clathrin-dependent endocytosis of renal outer medullar potassium 1 (ROMK1), and PHA2-causing mutations of WNK4 increase the endocytosis. How WNKs stimulate endocytosis of ROMK1 and how mutations of WNK4 increase the endocytosis are unknown. Intersectin (ITSN) is a multimodular endocytic scaffold protein. Here we show that WNK1 and WNK4 interacted with ITSN and that the interactions were crucial for stimulation of endocytosis of ROMK1 by WNKs. The stimulation of endocytosis of ROMK1 by WNK1 and WNK4 required specific proline-rich motifs of WNKs, but did not require their kinase activity. WNK4 interacted with ROMK1 as well as with ITSN. Disease-causing WNK4 mutations enhanced interactions of WNK4 with ITSN and ROMK1, leading to increased endocytosis of ROMK1. These results provide a molecular mechanism for stimulation of endocytosis of ROMK1 by WNK kinases.     

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Na_vs) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Na_vs remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Na_vs, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury–induced neuropathic pain was used, and an Na_v1.7-specific inhibitor, ProTxII, allowed the isolation of Na_v1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Na_v1.7 and Na_v1.8 currents. The redistribution of Na_v1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L^–/–). SNS-Nedd4L^–/– mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Na_v1.7 and Na_v1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Na_vs and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.     

Contribution of the WNK1 kinase to corneal wound healing using the tissue‐engineered human cornea as an in vitro model

Damage to the corneal epithelium triggers important changes in the extracellular matrix (ECM) to which basal human corneal epithelial cells (hCECs) attach. These changes are perceived by integrin receptors that activate different intracellular signalling pathways, ultimately leading to re‐epithelialization of the injured epithelium. In this study, we investigated the impact of pharmacological inhibition of specific signal transduction mediators on corneal wound healing using both monolayers of hCECs and the human tissue‐engineered cornea (hTEC) as an in vitro 3D model. RNA and proteins were isolated from the wounded and unwounded hTECs to conduct gene profiling analyses and protein kinase arrays. The impact of WNK1 inhibition was evaluated on the wounded hTECs as well as on hCECs monolayers using a scratch wound assay. Gene profiling and protein kinase arrays revealed that expression and activity of several mediators from the integrin‐dependent signaling pathways were altered in response to the ECM changes occurring during corneal wound healing. Phosphorylation of the WNK1 kinase turned out to be the most striking activation event going on during this process. The inhibition of WNK1 by WNK463 reduced the rate of corneal wound closure in both the hTEC and hCECs grown in monolayer compared with their respective negative controls. WNK463 also reduced phosphorylation of the WNK1 downstream targets SPAK/OSR1 in wounded hTECs. These in vitro results allowed for a better understanding of the cellular and molecular mechanisms involved in corneal wound healing and identified WNK1 as a kinase important to ensure proper wound healing of the cornea.     

WNK kinases and the control of blood pressure

The WNK kinases are a small group of serine/threonine kinases with unique catalytic domains that lack the lysine residue used in other kinases to co-ordinate ATP (hence, With No K [WNK]). Their closest homologues are found within the mitogen-activated protein kinase (MAPK) pathway suggesting a role in signalling. Two WNK isoforms, WNK1 and WNK4, have been identified as the disease genes for a rare monogenic hypertension syndrome (Gordon's syndrome or pseudohypoaldosteronism type 2 [PHA2]) implicating them in salt homeostasis by the kidney. This is supported by recent data showing widespread expression of WNK1 and WNK4 in mammalian transporting epithelia. Within the kidney, WNKs probably regulate the surface expression of several proteins involved in ion transport, including the sodium-chloride cotransporter (NCCT) and the potassium channel renal outer medullary potassium channel (ROMK), based on co-expression studies in Xenopus oocytes. WNKs, especially WNK4, have been suggested as candidate genes for essential hypertension itself, but evidence for this is lacking. Some of the effects of the WNKs are independent of their kinase function, suggesting that they are dependent on specific protein-protein interactions. It seems likely that the WNKs are part of much larger protein scaffolds in cells and have effects in cells beyond ion transport. However, because of their effect on expression of the NCCT they are attractive drug targets for the development of novel antihypertensive agents. These agents could potentially offer the efficacy of a thiazide diuretic, but without the metabolic side effects usually seen with this class of antihypertensive therapy.     

Aldosterone alleviates lipopolysaccharide-induced acute lung injury by regulating epithelial sodium channel through PI3K/Akt/SGK1 signaling pathway

Reduced alveolar fluid clearance (AFC) is a major pathological feature of acute lung injury (ALI). Epithelial sodium channel (ENaC) plays a key role in regulating the transport of Na⁺ and clearing alveolar edema fluid effectively. ENaC has been reported to be regulated by aldosterone in the distal collecting tube of the kidney. We hypothesized whether aldosterone regulated ENaC in alveolar epithelium and correspondingly played a role in ALI. In this study we found that the expression of aldosterone synthesis encoding gene, CYP11B2, and ENaC were decreased in the lung tissue of LPS-induced ALI mice. Furthermore, aldosterone alleviated ALI by increasing the expression of ENaC-α and relieving pulmonary edema. Besides, we found that aldosterone upregulated ENaC-α through PI3K/Akt/SGK1 pathway. In conclusion, our study demonstrated that aldosterone attenuated pulmonary edema by upregulating ENaC-α through the PI3K/Akt/SGK1 pathway in LPS-induced ALI, indicating that aldosterone might be a promising adjuvant drug for ALI treatment.     

Collecting duct-specific knockout of endothelin-1 causes hypertension and sodium retention

In vitro studies suggest that collecting duct-derived (CD-derived) endothelin-1 (ET-1) can regulate renal Na reabsorption; however, the physiologic role of CD-derived ET-1 is unknown. Consequently, the physiologic effect of selective disruption of the ET-1 gene in the CD of mice was determined. Mice heterozygous for aquaporin2 promoter Cre recombinase and homozygous for loxP-flanked exon 2 of the ET-1 gene (called CD-specific KO of ET-1 [CD ET-1 KO] mice) were generated. These animals had no CD ET-1 mRNA and had reduced urinary ET-1 excretion. CD ET-1 KO mice on a normal Na diet were hypertensive, while body weight, Na excretion, urinary aldosterone excretion, and plasma renin activity were unchanged. CD ET-1 KO mice on a high-Na diet had worsened hypertension, reduced urinary Na excretion, and excessive weight gain, but showed no differences between aldosterone excretion and plasma renin activity. Amiloride or furosemide reduced BP in CD ET-1 KO mice on a normal or high-Na diet and prevented excessive Na retention in salt-loaded CD ET-1 KO mice. These studies indicate that CD-derived ET-1 is an important physiologic regulator of renal Na excretion and systemic BP.     

Nerve injury-induced changes in Homer/glutamate receptor signaling contribute to the development and maintenance of neuropathic pain

While group 1 metabotropic glutamate receptors (mGluRs) and ionotropic N-methyl-d-aspartate (NMDA) receptors regulate nociception, the precise molecular mechanism(s) contributing to glutamate signaling in chronic pain remain unclear. Here we not only confirmed the key involvement of Homer proteins in neuropathic pain, but also distinguished between the functional roles for different Homer family members and isoforms. Chronic constriction injury (CCI) of the sciatic nerve induced long-lasting, time-dependent increases in the postsynaptic density expression of the constitutively expressed (CC) isoforms Homer1b/c and/or Homer2a/b in the spinal dorsal horn and supraspinal structures involved in nociception (prefrontal cortex, thalamus), that co-occurred with increases in their associated mGluRs, NR2 subunits of the NMDA receptor, and the activation of downstream kinases. Virus-mediated overexpression of Homer1c and Homer2b after spinal (intrathecal) virus injection exacerbated CCI-induced mechanical and cold hypersensitivity, however, Homer1 and Homer2 gene knockout (KO) mice displayed no changes in their neuropathic phenotype. In contrast, overexpression of the immediate early gene (IEG) Homer1a isoform reduced, while KO of Homer1a gene potentiated neuropathic pain hypersensitivity. Thus, nerve injury-induced increases in CC-Homers expression promote pain in pathological states, but IEG-Homer induction protects against both the development and maintenance of neuropathy. Additionally, exacerbated pain hypersensitivity in transgenic mice with reduced Homer binding to mGluR5 supports also an inhibitory role for Homer interactions with mGluR5 in mediating neuropathy. Such data indicate that nerve injury-induced changes in glutamate receptor/Homer signaling contribute in dynamic but distinct ways to neuropathic pain processing, which has relevance for the etiology of chronic pain symptoms and its treatment.     

Cotransporters, WNKs and hypertension: important leads from the study of monogenetic disorders of blood pressure regulation

Major advances are being made in identifying the structure and behaviour of regulatory cascades that control the activity of cation-Cl(-) cotransporters and certain Na(+), K(+) and Cl(-) channels. These transporters play key roles in regulating arterial blood pressure as they are not only responsible for NaCl reabsorption in the thick ascending limb and distal tubule of the kidney, but are also involved in regulating smooth muscle Ca(2+) levels. It is now apparent that defects in these transporters, and particularly in the regulatory cascades, cause some monogenetic forms of hypertension and may contribute to essential hypertension and problems with K(+) homoeostasis. Two families of kinases are prominent in these processes: the Ste-20-related kinases [OSR1 (oxidative stress-responsive kinase 1) and SPAK (Ste20/SPS1-related proline/alanine-rich kinase)] and the WNKs [with no lysine kinases]. These kinases affect the behaviour of their targets through both phosphorylation and by acting as scaffolding proteins, bringing together regulatory complexes. This review analyses how these kinases affect transport by activating or inhibiting individual transporters at the cell surface, or by changing the surface density of transporters by altering the rate of insertion or removal of transporters from the cell surface, and perhaps through controlling the rate of transporter degradation. This new knowledge should not only help us target antihypertensive therapy more appropriately, but could also provide the basis for developing new therapeutic approaches to essential hypertension.     

Monogenetische Hypertonien

Zusammenfassung. q Vier monogenetische Hypertonieformen gehören in die Gruppe der Mineralokortikoidhypertonie, deren Hauptcharakteristika eine gesteigerte renale Natrium- und Wasserretention mit daraus resultierender Suppression der Plasmareninaktivität (PRA) und eine verstärkte renale Kaliumausscheidung mit konsekutiver Hypokaliämie sind: 1. erhöhte Produktion des Hormons Aldosteron: glukokortikoidsupprimierbarer Hyperaldosteronismus (GSH), 2. Prärezeptorstörung mit Verlust der Selektivität des Mineralokortikoidrezeptors: apparenter Mineralokortikoidexzess (AME), 3. Rezeptorstörung mit konstitutiver Aktivierung des Mineralokortikoidrezeptors: "Geller-Syndrom", 4. Postrezeptorstörung mit erhöhter Funktion des epithelialen Natriumkanals (ENaC): Liddle-Syndrom. q Während bei der Aldosteronüberproduktion des GSH Plasmaaldosteron hoch und PRA niedrig sind, werden bei den primär renalen Störungen des AME, der konstitutiven Aktivierung des Mineralokortikoidrezeptors sowie dem Liddle-Syndrom niedriges Plasmaaldosteron und niedrige PRA gemessen. q In ihrer vollen Ausprägung kommen diese Hypertonieformen sehr selten vor, weisen jedoch auf wichtige Kandidatengene hin, deren Mutationen mit weniger starken Auswirkungen zu einer erhöhten Prädisposition zur Hypertonie führen können. q Eine Punktmutation der b-Untereinheit (T594M) des ENaC ist bei Personen afrikanischer Herkunft mit etwa 5% so häufig, dass als Voraussetzung für eine gezielte Behandlung mit Amilorid eine Genotypisierung bei Hypertonikern afrikanischer Herkunft empfohlen wird. Umgekehrt soll der relativ häufige (A[2139]G) Polymorphismus des Promotors der a-Untereinheit das Hypertonierisiko senken. q Mutationen der im distalen Nephron exprimierten Serin-Threonin-Kinasen WNK1 oder WNK4 sind Ursache für Hypertonie beim Pseudohypoaldosteronismus Typ II. Die Hypertonie ist durch eine verstärkte renale Satzretention und die Hyperkaliämie durch eine verminderte renale K⁺-Exkretion bedingt. Aldosteron stimuliert die Nariumrückresorption, beeinflusst aber nicht die Hyperkaliämie. q Eine nichtrenale Störung wird bei der autosomal-dominant vererbten Hypertonie mit Brachydaktylie angenommen: Die Patienten zeigen einen stark gedämpften Barorezeptorreflex und ein neurovaskuläres Kompressionssyndrom. Das mutierte Gen ist unbekannt. Abstract. q Four types of monogenic hypertension belong to the group of mineralocorticoid hypertension, which are characterized by high renal water and sodium retention and resulting suppression of plasma renin activity (PRA), high urinary potassium secretion and consecutive low plasma potassium: 1. increased production of the hormone aldosterone: glucocorticoid-remediable aldosteronism (GRH), 2. prereceptor disorder with loss of selectivity of the mineralocorticoid receptor: apparent mineralocorticoid excess (AME), 3. receptor disorder with constitutive activation of the mineralocorticoid receptor: "Geller syndrome", 4. postreceptor disorder with enhanced function of the epithelial sodium channel: Liddle's syndrome. q While in GRH high synthesis of aldosterone results in high plasma aldosterone and low PRA, in the primary renal malfunctions of the AME, constitutive activation of the mineralocorticoid receptor and the Liddle's syndrome both plasma aldosterone and PRA are low. q These forms of hypertension are rather rare in their complete expression, but they point to candidate genes whose mutations may predispose to hypertension. q A point mutation of the ENaC b-subunit (T594M) occurs rather frequent in people of African origin, with 5%. Therefore it is suggested to analyze the genotype of black hypertensive patients as a prerequisite for a rational amiloride therapy. Contrarily, the rather frequent (A[2139]G) polymorphism of the promoter of the a-subunit is supposed to mark a lower risk of hypertension. q Mutations in the serine-threonine kinases WNK1 or WNK4 cause pseudohypoaldosteronism type II. WNK1 and WNK4 are expressed in the distal part of the nephron. Stimulation of sodium reabsorption by aldosterone is normal but without influence on hyperkalemia. q An extrarenal disorder is suggested to be the cause of autosomal-dominant hypertension with brachydactyly: the patients react with a severely impaired baroreflex und show neurovascular contact. The mutation causing this syndrome is not known.     

mTORC2 critically regulates renal potassium handling

The mTOR pathway orchestrates cellular homeostasis. The rapamycin-sensitive mTOR complex (mTORC1) in the kidney has been widely studied; however, mTORC2 function in renal tubules is poorly characterized. Here, we generated mice lacking mTORC2 in the distal tubule (Rictor^fl/fl Ksp-Cre mice), which were viable and had no obvious phenotype, except for a 2.5-fold increase in plasma aldosterone. Challenged with a low-Na⁺ diet, these mice adequately reduced Na⁺ excretion; however, Rictor^fl/fl Ksp-Cre mice rapidly developed hyperkalemia on a high-K⁺ diet, despite a 10-fold increase in serum aldosterone levels, implying that mTORC2 regulates kaliuresis. Phosphorylation of serum- and glucocorticoid-inducible kinase 1 (SGK1) and PKC-α was absent in Rictor^fl/fl Ksp-Cre mice, indicating a functional block in K⁺ secretion activation via ROMK channels. Indeed, patch-clamp experiments on split-open tubular segments from the transition zone of the late connecting tubule and early cortical collecting duct demonstrated that Ba²⁺-sensitive apical K⁺ currents were barely detectable in the majority of Rictor^fl/fl Ksp-Cre mice. Conversely, epithelial sodium channel (ENaC) activity was largely preserved, suggesting that the reduced ability to maintain K⁺ homeostasis is the result of impaired apical K⁺ conductance and not a reduced electrical driving force for K⁺ secretion. Thus, these data unravel a vital and nonredundant role of mTORC2 for distal tubular K⁺ handling.