Arterial Blood Pressure, Neuronal Excitability, Mineral Metabolism and Cell Volume Regulation Mechanisms Revealed by Xenopus laevis oocytes.

Xenopus laevis oocytes have been an invaluable tool to discover and explore the molecular mechanisms and characteristics of many proteins, in particular integral membrane proteins. The oocytes were fundamental in many projects designed to identify the cDNA encoding a diversity of membrane proteins including receptors, transporters, channels and pores. In addition to being a powerful tool for cloning, oocytes were later used to experiment with the functional characterization of many of the identified proteins. In this review I present an overview of my personal 30-year experience using Xenopus laevis oocytes and the impact this had on a variety of fields such as arterial blood pressure, neuronal excitability, mineral metabolism and cell volume regulation.

Role of KLHL3 and dietary K+ in regulating KS-WNK1 expression.

The physiological role of the shorter isoform of with no lysine kinase (WNK)1 that is exclusively expressed in the kidney (KS-WNK1), with particular abundance in the distal convoluted tubule, remains elusive. KS-WNK1, despite lacking the kinase domain, is nevertheless capable of stimulating the NaCl cotransporter, apparently through activation of WNK4. It has recently been shown that a less severe form of familial hyperkalemic hypertension featuring only hyperkalemia is caused by missense mutations in the WNK1 acidic domain that preferentially affect cullin 3 (CUL3)-Kelch-like protein 3 (KLHL3) E3-induced degradation of KS-WNK1 rather than that of full-length WNK1. Here, we show that full-length WNK1 is indeed less impacted by the CUL3-KLHL3 E3 ligase complex compared with KS-WNK1. We demonstrated that the unique 30-amino acid NH2-terminal fragment of KS-WNK1 is essential for its activating effect on the NaCl cotransporter and recognition by KLHL3. We identified specific amino acid residues in this region critical for the functional effect of KS-WNK1 and KLHL3 sensitivity. To further explore this, we generated KLHL3-R528H knockin mice that mimic human mutations causing familial hyperkalemic hypertension. These mice revealed that the KLHL3 mutation specifically increased expression of KS-WNK1 in the kidney. We also observed that in wild-type mice, the expression of KS-WNK1 was only detectable after exposure to a low-K+ diet. These findings provide new insights into the regulation and function of KS-WNK1 by the CUL3-KLHL3 complex in the distal convoluted tubule and indicate that this pathway is regulated by dietary K+ levels.NEW & NOTEWORTHY In this work, we demonstrated that the kidney-specific isoform of with no lysine kinase 1 (KS-WNK1) in the kidney is modulated by dietary K+ and activity of the ubiquitin ligase protein Kelch-like protein 3. We analyzed the role of different amino acid residues of KS-WNK1 in its activity against the NaCl cotransporter and sensitivity to Kelch-like protein 3.

Regulation of the renal NaCl cotransporter by the WNK/SPAK pathway: lessons learned from genetically altered animals.

The renal thiazide-sensitive NaCl cotransporter (NCC) is the major salt transport pathway in the distal convoluted tubule of the mammalian nephron. NCC activity is critical for modulation of arterial blood pressure and serum potassium levels. Reduced activity of NCC in genetic diseases results in arterial hypotension and hypokalemia, while increased activity results in genetic diseases featuring hypertension and hyperkalemia. Several hormones and physiological conditions modulate NCC activity through a final intracellular complex pathway involving kinases and ubiquitin ligases. A substantial amount of work has been conducted to understand this pathway in the last 15 yr, but advances over the last 3 yr have helped to begin to understand how these regulatory proteins interact with each other and modulate the activity of this important cotransporter. In this review, we present the current model of NCC regulation by the Cullin 3 protein/Kelch-like 3 protein/with no lysine kinase/STE20-serine-proline alanine-rich kinase (CUL3/KELCH3-WNK-SPAK) pathway. We present a review of all genetically altered mice that have been used to translate most of the proposals made from in vitro experiments into in vivo observations that have helped to elucidate the model at the physiological level. Many questions have been resolved, but some others will require further models to be constructed. In addition, unexpected observations in mice have raised new questions and identified regulatory pathways that were previously unknown.

Kidney-specific WNK1 isoform (KS-WNK1) is a potent activator of WNK4 and NCC.

Familial hyperkalemic hypertension (FHHt) can be mainly attributed to increased activity of the renal Na+:Cl- cotransporter (NCC), which is caused by altered expression and regulation of the with-no-lysine (K) 1 (WNK1) or WNK4 kinases. The WNK1 gene gives rise to a kidney-specific isoform that lacks the kinase domain (KS-WNK1), the expression of which occurs primarily in the distal convoluted tubule. The role played by KS-WNK1 in the modulation of the WNK/STE20-proline-alanine rich kinase (SPAK)/NCC pathway remains elusive. In the present study, we assessed the effect of human KS-WNK1 on NCC activity and on the WNK4-SPAK pathway. Microinjection of oocytes with human KS-WNK1 cRNA induces remarkable activation and phosphorylation of SPAK and NCC. The effect of KS-WNK1 was abrogated by eliminating a WNK-WNK-interacting domain and by a specific WNK inhibitor, WNK463, indicating that the activation of SPAK/NCC by KS-WNK1 is due to interaction with another WNK kinase. Under control conditions in oocytes, the activating serine 335 of the WNK4 T loop is not phosphorylated. In contrast, this serine becomes phosphorylated when the intracellular chloride concentration ([Cl-]i) is reduced or when KS-WNK1 is coexpressed with WNK4. KS-WNK1-mediated activation of WNK4 is not due to a decrease of the [Cl-]i. Coimmunoprecipitation analysis revealed that KS-WNK1 and WNK4 interact with each other and that WNK4 becomes autophosphorylated at serine 335 when it is associated with KS-WNK1. Together, these observations suggest that WNK4 becomes active in the presence of KS-WNK1, despite a constant [Cl-]i.

Physiological role of SLC12 family members in the kidney.

The solute carrier family 12, as numbered according to Human Genome Organisation (HUGO) nomenclature, encodes the electroneutral cation-coupled chloride cotransporters that are expressed in many cells and tissues; they play key roles in important physiological events, such as cell volume regulation, modulation of the intracellular chloride concentration, and transepithelial ion transport. Most of these family members are expressed in specific regions of the nephron. The Na-K-2Cl cotransporter NKCC2, which is located in the thick ascending limb, and the Na-Cl cotransporter, which is located in the distal convoluted tubule, play important roles in salt reabsorption and serve as the receptors for loop and thiazide diuretics, respectively (Thiazide diuretics are among the most commonly prescribed drugs in the world.). The activity of these transporters correlates with blood pressure levels; thus, their regulation has been a subject of intense research for more than a decade. The K-Cl cotransporters KCC1, KCC3, and KCC4 are expressed in several nephron segments, and their role in renal physiology is less understood but nevertheless important. Evidence suggests that they are involved in modulating proximal tubule glucose reabsorption, thick ascending limb salt reabsorption and collecting duct proton secretion. In this work, we present an overview of the physiological roles of these transporters in the kidney, with particular emphasis on the knowledge gained in the past few years.

Increased phosphorylation of the renal Na+-Cl- cotransporter in male kidney transplant recipient patients with hypertension: a prospective cohort.

Evidence in rodents suggests that tacrolimus-induced posttransplant hypertension is due to upregulation of the thiazide-sensitive Na+-Cl- cotransporter NCC. Here, we analyzed whether a similar mechanism is involved in posttransplant hypertension in humans. From January 2013 to June 2014, all adult kidney transplant recipients receiving a kidney allograft were enrolled in a prospective cohort study. All patients received tacrolimus as part of the immunosuppressive therapy. Six months after surgery, we assessed general clinical and laboratory variables, tacrolimus trough blood levels, and ambulatory 24-h blood pressure monitoring. Urinary exosomes were extracted to perform Western blot analysis using total and phospho-NCC antibodies. A total of 52 patients, including 17 women and 35 men, were followed. At 6 mo after transplantation, of the 35 men, 17 developed hypertension and 18 remained normotensive, while high blood pressure was observed in only 3 of 17 women. The hypertensive patients were significantly older than the normotensive group; however, there were no significant differences in body weight, history of acute rejection, renal function, and tacrolimus trough levels. In urinary exosomes, hypertensive patients showed higher NCC expression (1.7±0.19) than normotensive (1±0.13) (P=0.0096). Also, NCC phosphorylation levels were significantly higher in the hypertensive patients (1.57±0.16 vs. 1±0.07; P=0.0049). Our data show that there is a positive correlation between NCC expression/phosphorylation in urinary exosomes and the development of hypertension in posttransplant male patients treated with tacrolimus. Our results are consistent with the hypothesis that NCC activation plays a major role in tacrolimus-induced hypertension.