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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.

Clinically, potassium supplementation has been shown to lower blood pressure and reduce the risk of stroke through modulation of potassium excretion and sodium reabsorption. Hypokalemia activates the renal sodium chloride cotransporter (NCC) along the distal convoluted tubule (DCT), at least in part, through with-no-lysine 4 (WNK4) kinase and STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) signaling. The DCT also expresses a kinase-deficient, kidney-specific form of WNK1 (KS-WNK1), but its role in NCC activation is unclear. In this issue of the JCI, Boyd-Shiwarski and colleagues found that KS-WNK1 enhanced the effects of potassium on NCC activation in vivo. Specifically, they showed that mice lacking KS-WNK1 did not respond as robustly to dietary challenge. Additionally, in vivo expression of a mutated KS-WNK1 disrupted WNK body, or biomolecular condensate, formation and renal function. These findings, along with those of previous studies, indicate that KS-WNK1 may regulate potassium homeostasis by increasing the kidney's sensitivity to salt-dependent stress.

Epidemiological and clinical observations have shown that potassium ingestion is inversely correlated with arterial hypertension prevalence and cardiovascular mortality. The higher the dietary potassium, the lower the blood pressure and mortality. This phenomenon is explained, at least in part, by the interaction between salt reabsorption in the distal convoluted tubule (DCT) and potassium secretion in the connecting tubule/collecting duct of the mammalian nephron: In order to achieve adequate K + secretion levels under certain conditions, salt reabsorption in the DCT must be reduced. Because salt handling by the kidney constitutes the basis for the long‐term regulation of blood pressure, losing salt prevents hypertension. Here, we discuss how the study of inherited diseases in which salt reabsorption in the DCT is affected has revealed the molecular players, including membrane transporters and channels, kinases, and ubiquitin ligases that form the potassium sensing mechanism of the DCT and the processes through which the consequent adjustments in salt reabsorption are achieved. Graphical Abstract In this review, G. Gamba et al present the current knowledge on the regulation of sodium and potassium homeostasis in the distal convoluted tubule, and the related control of blood pressure.

Pseudohypoaldosteronism type II is a salt-sensitive form of hypertension with hyperkalemia in humans caused by mutations in the with-no-lysine kinase 4 (WNK4). Several studies have shown that WNK4 modulates the activity of the renal Na'Cl cotransporter, NCC. Because the renal consequences of WNK4 carrying pseudoaldosteronism type II mutations resemble the response to intravascular volume depletion (promotion of salt reabsorption without K⁺ secretion), a condition that is associated with high angiotensin II (AngII) levels, it has been proposed that AngII signaling might affect WNK4 modulation of the NCC. In Xenopus laevis oocytes, WNK4 is required for modulation of NCC activity by AngII. To demonstrate that WNK4 is required in the AngII-mediated regulation of NCC in vivo, we used a total WNK4-knockout mouse strain (WNK4-/-). WNK4 mRNA and protein expression were absent in WNK4-/- mice, which exhibited a mild Gitelman-like syndrome, with normal blood pressure, increased plasma renin activity, and reduced NCC expression and phosphorylation at T-58. Immunohistochemistry revealed normal morphology of the distal convoluted tubule with reduced NCC expression. Low-salt diet or infusion of AngII for 4 d induced phosphorylation of STE20/SPS1 -related proline/alanine-rich kinase (SPAK) and of NCC at S-383 and T-58, respectively, in WNK4 +/+ but not WNK4\" 7 \"mice. Thus, the absence of WNK4 in vivo precludes NCC and SPAK phosphorylation promoted by a low-salt diet or AngII infusion, suggesting that AngII action on the NCC occurs via a WNK4-SPAK-dependent signaling pathway. Additionally, stimulation of aldosterone secretion by AngII, but not by a high-K⁺ diet, was impaired in WNK4-/- mice.

With-no-lysine kinase 4 (WNK4) regulates electrolyte homeostasis and blood pressure. WNK4 phosphorylates the kinases SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress responsive kinase), which then phosphorylate and activate the renal Na-Cl cotransporter (NCC). WNK4 levels are regulated by binding to Kelch-like 3, targeting WNK4 for ubiquitylation and degradation. Phosphorylation of Kelch-like 3 by PKC or PKA downstream of AngII or vasopressin signaling, respectively, abrogates binding. We tested whether these pathways also affect WNK4 phosphorylation and activity. By tandem mass spectrometry and use of phosphosite-specific antibodies, we identified five WNK4 sites (S47, S64, S1169, S1180, S1196) that are phosphorylated downstream of AngII signaling in cultured cells and in vitro by PKC and PKA. Phosphorylation at S64 and S1196 promoted phosphorylation of the WNK4 kinase T-loop at S332, which is required for kinase activation, and increased phosphorylation of SPAK. Volume depletion induced phosphorylation of these sites in vivo, predominantly in the distal convoluted tubule. Thus, AngII, in addition to increasing WNK4 levels, also modulates WNK4 kinase activity via phosphorylation of sites outside the kinase domain.

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.

Severe cases of COVID-19 are characterized by development of acute respiratory distress syndrome (ARDS). Water accumulation in the lungs is thought to occur as consequence of an exaggerated inflammatory response. A possible mechanism could involve decreased activity of the epithelial Na + channel, ENaC, expressed in type II pneumocytes. Reduced transepithelial Na + reabsorption could contribute to lung edema due to reduced alveolar fluid clearance. This hypothesis is based on the observation of the presence of a novel furin cleavage site in the S protein of SARS-CoV-2 that is identical to the furin cleavage site present in the alpha subunit of ENaC. Proteolytic processing of αENaC by furin-like proteases is essential for channel activity. Thus, competition between S protein and αENaC for furin-mediated cleavage in SARS-CoV-2-infected cells may negatively affect channel activity. Here we present experimental evidence showing that coexpression of the S protein with ENaC in a cellular model reduces channel activity. In addition, we show that bidirectional competition for cleavage by furin-like proteases occurs between 〈ENaC and S protein. In transgenic mice sensitive to lethal SARS-CoV-2, however, a significant decrease in gamma ENaC expression was not observed by immunostaining of lungs infected as shown by SARS-CoV2 nucleoprotein staining.

Acute kidney injury (AKI) has been recognized as a risk factor for the development of chronic kidney disease (CKD). Aldosterone has a critical role in promoting renal injury induced by ischemia. Here, we evaluated whether spironolactone administered before or after AKI caused by ischemia protects against CKD. In the first set of experiments, Wistar rats underwent a sham operation without or with prior spironolactone treatment, or underwent 45minutes of bilateral renal ischemia without or with spironolactone treatment before ischemia and assessed over 270 days. The second set of rats received low (20mg/kg) or high (80mg/kg) doses of spironolactone at three different times after the sham operation or bilateral renal ischemia and were assessed after 90 days. Untreated animals developed CKD following ischemia-induced AKI as characterized by a progressive increase in proteinuria, renal dysfunction, podocyte injury, glomerular hypertrophy, and focal sclerosis. This was associated with increased oxidative stress, an upregulation of tumor growth factor (TGF)-β, followed by upregulation of the TGF-β downstream effectors phospho-Smad3, collagen I, fibronectin, and proinflammatory cytokines. Treatment with spironolactone either before or after ischemia prevented subsequent CKD by avoiding the activation of fibrotic and inflammatory pathways. Thus, spironolactone may be a promising treatment for the prevention of AKI-induced CKD.

Chronic hypoxia is a major contributor to Chronic Kidney Disease (CKD) after Acute Kidney Injury (AKI). However, the temporal relation between the acute insult and maladaptive renal response to hypoxia remains unclear. In this study, we analyzed the time-course of renal hemodynamics, oxidative stress, inflammation, and fibrosis, as well as epigenetic modifications, with focus on HIF1α/VEGF signaling, in the AKI to CKD transition. Sham-operated, right nephrectomy (UNx), and UNx plus renal ischemia (IR + UNx) groups of rats were included and studied at 1, 2, 3, or 4 months. The IR + UNx group developed CKD characterized by progressive proteinuria, renal dysfunction, tubular proliferation, and fibrosis. At first month post-ischemia, there was a twofold significant increase in oxidative stress and reduction in global DNA methylation that was maintained throughout the study. Hif1α and Vegfa expression were depressed in the first and second-months post-ischemia, and then Hif1α but not Vegfa expression was recovered. Interestingly, hypermethylation of the Vegfa promoter gene at the HIF1α binding site was found, since early stages of the CKD progression. Our findings suggest that renal hypoperfusion, inefficient hypoxic response, increased oxidative stress, DNA hypomethylation, and, Vegfa promoter gene hypermethylation at HIF1α binding site, are early determinants of AKI-to-CKD transition.

ObjectiveObesity is associated with metabolic abnormalities, including insulin resistance and dyslipidemias. Previous studies demonstrated that genistein intake modifies the gut microbiota in mice by selectively increasing Akkermansia muciniphila, leading to reduction of metabolic endotoxemia and insulin sensitivity. However, it is not known whether the consumption of genistein in humans with obesity could modify the gut microbiota reducing the metabolic endotoxemia and insulin sensitivity.Research design and methods45 participants with a Homeostatic Model Assessment (HOMA) index greater than 2.5 and body mass indices of ≥30 and≤40 kg/m2 were studied. Patients were randomly distributed to consume (1) placebo treatment or (2) genistein capsules (50 mg/day) for 2 months. Blood samples were taken to evaluate glucose concentration, lipid profile and serum insulin. Insulin resistance was determined by means of the HOMA for insulin resistance (HOMA-IR) index and by an oral glucose tolerance test. After 2 months, the same variables were assessed including a serum metabolomic analysis, gut microbiota, and a skeletal muscle biopsy was obtained to study the gene expression of fatty acid oxidation.ResultsIn the present study, we show that the consumption of genistein for 2 months reduced insulin resistance in subjects with obesity, accompanied by a modification of the gut microbiota taxonomy, particularly by an increase in the Verrucomicrobia phylum. In addition, subjects showed a reduction in metabolic endotoxemia and an increase in 5′-adenosine monophosphate-activated protein kinase phosphorylation and expression of genes involved in fatty acid oxidation in skeletal muscle. As a result, there was an increase in circulating metabolites of β-oxidation and ω-oxidation, acyl-carnitines and ketone bodies.ConclusionsChange in the gut microbiota was accompanied by an improvement in insulin resistance and an increase in skeletal muscle fatty acid oxidation. Therefore, genistein could be used as a part of dietary strategies to control the abnormalities associated with obesity, particularly insulin resistance; however, long-term studies are needed.