Effect of SARS-CoV-2 S protein on the proteolytic cleavage of the epithelial Na+ channel ENaC.

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.

Arginine vasopressin regulates the renal Na+-Cl- and Na+-K+-Cl- cotransporters through with-no-lysine kinase 4 and inhibitor 1 phosphorylation.

Vasopressin regulates water homeostasis via the V2 receptor in the kidney at least in part through protein kinase A (PKA) activation. Vasopressin, through an unknown pathway, upregulates the activity and phosphorylation of Na+-Cl- cotransporter (NCC) and Na+-K+-2Cl- cotransporter 2 (NKCC2) by Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1), which are regulated by the with-no-lysine kinase (WNK) family. Phosphorylation of WNK4 at PKA consensus motifs may be involved. Inhibitor 1 (I1), a protein phosphatase 1 (PP1) inhibitor, may also play a role. In human embryonic kidney (HEK)-293 cells, we assessed the phosphorylation of WNK4, SPAK, NCC, or NKCC2 in response to forskolin or desmopressin. WNK4 and cotransporter phosphorylation were studied in desmopressin-infused WNK4-/- mice and in tubule suspensions. In HEK-293 cells, only wild-type WNK4 but not WNK1, WNK3, or a WNK4 mutant lacking PKA phosphorylation motifs could upregulate SPAK or cotransporter phosphorylation in response to forskolin or desmopressin. I1 transfection maximized SPAK phosphorylation in response to forskolin in the presence of WNK4 but not of mutant WNK4 lacking PP1 regulation. We observed direct PP1 regulation of NKCC2 dephosphorylation but not of NCC or SPAK in the absence of WNK4. WNK4-/- mice with desmopressin treatment did not increase SPAK/OSR1, NCC, or NKCC2 phosphorylation. In stimulated tubule suspensions from WNK4-/- mice, upregulation of pNKCC2 was reduced, whereas upregulation of SPAK phosphorylation was absent. These findings suggest that WNK4 is a central node in which kinase and phosphatase signaling converge to connect cAMP signaling to the SPAK/OSR1-NCC/NKCC2 pathway.NEW & NOTEWORTHY With-no-lysine kinases regulate the phosphorylation and activity of the Na+-Cl- and Na+-K+-2Cl- cotransporters. This pathway is modulated by arginine vasopressin (AVP). However, the link between AVP and WNK signaling remains unknown. Here, we show that AVP activates WNK4 through increased phosphorylation at putative protein kinase A-regulated sites and decreases its dephosphorylation by protein phosphatase 1. This work increases our understanding of the signaling pathways mediating AVP actions in the kidney.

Transient response of serpinA3 during cellular stress.

We demonstrated that serpinA3c/k relocates from the cytoplasm to the apical tubular membrane (ATM) in chronic kidney disease (CKD), suggesting its secretion in luminal space in pathophysiological contexts. Here, we studied serpinA3c/k expression and secretion under different stressful conditions in vitro and in vivo. HEK-293 cells were transfected with a FLAG-tagged serpinA3c/k clone and exposed to H2 O2 or starvation. Both stressors induced serpinA3c/k secretion but with a higher molecular weight. Glycanase treatment established that serpinA3c/k is glycosylated. Site-directed mutagenesis for each of the four glycosylation sites was performed. During cellular stress, serpinA3c/k secretion increased with each mutant except in the quadruple mutant. In rats and patients suffering acute kidney injury (AKI), an atypical urinary serpinA3c/k excretion (uSerpinA3c/k) was observed. In rats with AKI, the greater the induced kidney damage, the greater the uSerpinA3 c/k, together with relocation toward ATM. Our findings show that: (1) serpinA3c/k is glycosylated and secreted, (2) serpinA3c/k secretion increases during cellular stress, (3) its appearance in urine reveals a pathophysiological state, and (4) urinary serpinA3 excretion could become a potential biomarker for AKI.

Vegfa promoter gene hypermethylation at HIF1α binding site is an early contributor to CKD progression after renal ischemia.

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.

WNK4 kinase: from structure to physiology.

With no lysine kinase-4 (WNK4) belongs to a serine-threonine kinase family characterized by the atypical positioning of its catalytic lysine. Despite the fact that WNK4 has been found in many tissues, the majority of its study has revolved around its function in the kidney, specifically as a positive regulator of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule of the nephron. This is explained by the description of gain-of-function mutations in the gene encoding WNK4 that causes familial hyperkalemic hypertension. This disease is mainly driven by increased downstream activation of the Ste20/SPS1-related proline-alanine-rich kinase/oxidative stress responsive kinase-1-NCC pathway, which increases salt reabsorption in the distal convoluted tubule and indirectly impairs renal K+ secretion. Here, we review the large volume of information that has accumulated about different aspects of WNK4 function. We first review the knowledge on WNK4 structure and enumerate the functional domains and motifs that have been characterized. Then, we discuss WNK4 physiological functions based on the information obtained from in vitro studies and from a diverse set of genetically modified mouse models with altered WNK4 function. We then review in vitro and in vivo evidence on the different levels of regulation of WNK4. Finally, we go through the evidence that has suggested how different physiological conditions act through WNK4 to modulate NCC activity.

Physiological Processes Modulated by the Chloride-Sensitive WNK-SPAK/OSR1 Kinase Signaling Pathway and the Cation-Coupled Chloride Cotransporters.

The role of Cl- as an intracellular signaling ion has been increasingly recognized in recent years. One of the currently best described roles of Cl- in signaling is the modulation of the With-No-Lysine (K) (WNK) - STE20-Proline Alanine rich Kinase (SPAK)/Oxidative Stress Responsive Kinase 1 (OSR1) - Cation-Coupled Cl- Cotransporters (CCCs) cascade. Binding of a Cl- anion to the active site of WNK kinases directly modulates their activity, promoting their inhibition. WNK activation due to Cl- release from the binding site leads to phosphorylation and activation of SPAK/OSR1, which in turn phosphorylate the CCCs. Phosphorylation by WNKs-SPAK/OSR1 of the Na+-driven CCCs (mediating ions influx) promote their activation, whereas that of the K+-driven CCCs (mediating ions efflux) promote their inhibition. This results in net Cl- influx and feedback inhibition of WNK kinases. A wide variety of alterations to this pathway have been recognized as the cause of several human diseases, with manifestations in different systems. The understanding of WNK kinases as Cl- sensitive proteins has allowed us to better understand the mechanistic details of regulatory processes involved in diverse physiological phenomena that are reviewed here. These include cell volume regulation, potassium sensing and intracellular signaling in the renal distal convoluted tubule, and regulation of the neuronal response to the neurotransmitter GABA.

Molecular mechanisms for the regulation of blood pressure by potassium.

It has been well documented that the amount of potassium in the diet is associated with blood pressure levels in the population: the higher the potassium consumption, the lower the blood pressure and the cardiovascular mortality. In the last few years certain mechanisms for potassium regulation of salt reabsorption in the kidney have been elucidated at the molecular level. In this work we discuss the evidence demonstrating the relationship between potassium intake and blood pressure levels in human populations and in animal models, as well as the experimental data that reveal the effects of potassium on transepithelial Na+ reabsorption in different nephron segments. We also discuss the physiological relevance of K+-induced natriuresis, and finally, we focus on the molecular mechanisms by which extracellular potassium modulates the activity of the renal NaCl cotransporter, which is the mechanism that has been best dissected so far.

C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity.

WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overactivation of the renal Na+/Cl- cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4-/- mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4's C terminus at several positions increased its kinase activity toward Ste20-related proline/alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-binding site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2+-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.

Epigenetic Effects of an Adenosine Derivative in a Wistar Rat Model of Liver Cirrhosis.

The pathological characteristic of cirrhosis is scarring which results in a structurally distorted and dysfunctional liver. Previously, we demonstrated that Col1a1 and Pparg genes are deregulated in CCl4 -induced cirrhosis but their normal expression levels are recovered upon treatment with IFC-305, an adenosine derivative. We observed that adenosine was able to modulate S-adenosylmethionine-dependent trans-methylation reactions, and recently, we found that IFC-305 modulates HDAC3 expression. Here, we investigated whether epigenetic mechanisms, involving DNA methylation processes and histone acetylation, could explain the re-establishment of gene expression mediated by IFC-305 in cirrhosis. Therefore, Wistar rats were CCl4 treated and a sub-group received IFC-305 to reverse fibrosis. Global changes in DNA methylation, 5-hydroxymethylation, and histone H4 acetylation were observed after treatment with IFC-305. In particular, during cirrhosis, the Pparg gene promoter is depleted of histone H4 acetylation, whereas IFC-305 administration restores normal histone acetylation levels which correlates with an increase of Pparg transcript and protein levels. In contrast, the promoter of Col1a1 gene is hypomethylated during cirrhosis but gains DNA methylation upon treatment with IFC-305 which correlates with a reduction of Col1a1 transcript and protein levels. Our results suggest a model in which cirrhosis results in a general loss of permissive chromatin histone marks which triggers the repression of the Pparg gene and the upregulation of the Col1a1 gene. Treatment with IFC-305 restores epigenetic modifications globally and specifically at the promoters of Pparg and Col1a1 genes. These results reveal one of the mechanisms of action of IFC-305 and suggest a possible therapeutic function in cirrhosis. J. Cell. Biochem. 119: 401-413, 2018. © 2017 Wiley Periodicals, Inc.