Expression of claudin-8 is induced by aldosterone in renal collecting duct principal cells.

Fine tuning of Na+ reabsorption takes place along the aldosterone-sensitive distal nephron, which includes the collecting duct (CD), where it is mainly regulated by aldosterone. In the CD, Na+ reabsorption is mediated by the epithelial Na+ channel and Na+ pump (Na+-K+-ATPase). Paracellular ion permeability is mainly dependent on tight junction permeability. Claudin-8 is one of the main tight junction proteins expressed along the aldosterone-sensitive distal nephron. We have previously shown a coupling between transcellular Na+ reabsorption and paracellular Na+ barrier. We hypothesized that aldosterone controls the expression levels of both transcellular Na+ transporters and paracellular claudin-8 in a coordinated manner. Here, we show that aldosterone increased mRNA and protein levels as well as lateral membrane localization of claudin-8 in cultured CD principal cells. The increase in claudin-8 mRNA levels in response to aldosterone was prevented by preincubation with 17-hydroxyprogesterone, a mineralocorticoid receptor antagonist, and by inhibition of transcription with actinomycin D. We also showed that a low-salt diet, which stimulated aldosterone secretion, was associated with increased claudin-8 abundance in the mouse kidney. Reciprocally, mice subjected to a high-salt diet, which inhibits aldosterone secretion, or treated with spironolactone, a mineralocorticoid receptor antagonist, displayed decreased claudin-8 expression. Inhibition of glycogen synthase kinase-3, Lyn, and Abl signaling pathways prevented the effect of aldosterone on claudin-8 mRNA and protein abundance, suggesting that signaling of protein kinases plays a permissive role on the transcriptional activity of the mineralocorticoid receptor. This study shows that signaling via multiple protein kinases working in concert mediates aldosterone-induced claudin-8 expression in the CD.NEW & NOTEWORTHY In this study, we showed that aldosterone modulates claudin-8 expression in cultured collecting duct principal cells and in the mouse kidney. The upregulation of claudin-8 expression in response to aldosterone is dependent on at least glycogen synthase kinase-3, Lyn, and Abl signaling pathways, indicating the participation of multiple protein kinases to the effect of aldosterone.

Function and Regulation of the Epithelial Na+ Channel ENaC.

The Epithelial Na+ Channel, ENaC, comprised of 3 subunits (αßγ, or sometimes δßγENaC), plays a critical role in regulating salt and fluid homeostasis in the body. It regulates fluid reabsorption into the blood stream from the kidney to control blood volume and pressure, fluid absorption in the lung to control alveolar fluid clearance at birth and maintenance of normal airway surface liquid throughout life, and fluid absorption in the distal colon and other epithelial tissues. Moreover, recent studies have also revealed a role for sodium movement via ENaC in nonepithelial cells/tissues, such as endothelial cells in blood vessels and neurons. Over the past 25 years, major advances have been made in our understanding of ENaC structure, function, regulation, and role in human disease. These include the recently solved three-dimensional structure of ENaC, ENaC function in various tissues, and mutations in ENaC that cause a hereditary form of hypertension (Liddle syndrome), salt-wasting hypotension (PHA1), or polymorphism in ENaC that contributes to other diseases (such as cystic fibrosis). Moreover, great strides have been made in deciphering the regulation of ENaC by hormones (e.g., the mineralocorticoid aldosterone, glucocorticoids, vasopressin), ions (e.g., Na+ ), proteins (e.g., the ubiquitin-protein ligase NEDD4-2, the kinases SGK1, AKT, AMPK, WNKs & mTORC2, and proteases), and posttranslational modifications [e.g., (de)ubiquitylation, glycosylation, phosphorylation, acetylation, palmitoylation]. Characterization of ENaC structure, function, regulation, and role in human disease, including using animal models, are described in this article, with a special emphasis on recent advances in the field. © 2021 American Physiological Society. Compr Physiol 11:1-29, 2021.

SIRT7 modulates the stability and activity of the renal K-Cl cotransporter KCC4 through deacetylation.

SIRT7 is a NAD+ -dependent deacetylase that controls important aspects of metabolism, cancer, and bone formation. However, the molecular targets and functions of SIRT7 in the kidney are currently unknown. In silico analysis of kidney transcripts of the BXD murine genetic reference population revealed a positive correlation between Sirt7 and Slc12a7 mRNA expression, suggesting a link between the corresponding proteins that these transcripts encode, SIRT7, and the K-Cl cotransporter KCC4, respectively. Here, we find that protein levels and activity of heterologously expressed KCC4 are significantly modulated depending on its acetylation status in Xenopus laevis oocytes. Moreover, SIRT7 interacts with KCC4 in a NAD+ -dependent manner and increases its stability and activity in HEK293 cells. Interestingly, metabolic acidosis increases SIRT7 expression in kidney, as occurs with KCC4. In contrast, total SIRT7-deficient mice present lower KCC4 expression and an exacerbated metabolic acidosis than wild-type mice during an ammonium chloride challenge. Altogether, our data suggest that SIRT7 interacts with, stabilizes and modulates KCC4 activity through deacetylation, and reveals a novel role for SIRT7 in renal physiology.

The serine-threonine kinase PIM3 is an aldosterone-regulated protein in the distal nephron.

The mineralocorticoid hormone aldosterone plays a crucial role in the control of Na+ and K+ balance, blood volume, and arterial blood pressure, by acting in the aldosterone-sensitive distal nephron (ASDN) and stimulating a complex transcriptional, translational, and cellular program. Because the complexity of the aldosterone response is still not fully appreciated, we aimed at identifying new elements in this pathway. Here, we demonstrate that the expression of the proto-oncogene PIM3 (Proviral Integration Site of Moloney Murine Leukemia Virus 3), a serine/threonine kinase belonging to the calcium/calmodulin-regulated group of kinases, is stimulated by aldosterone in vitro (mCCDcl1 cells), ex vivo (mouse kidney slices), and in vivo in mice. Characterizing a germline Pim3-/- mouse model, we found that these mice have an upregulated Renin-Angiotensin-Aldosterone System (RAAS), with high circulating aldosterone and plasma renin activity levels on both standard or Na+ -deficient diet. Surprisingly, we did not observe any obvious salt-losing phenotype in Pim3 KO mice as shown by normal blood pressure, plasma and urinary electrolytes, as well as unchanged expression levels of the major Na+ transport proteins. These observations suggest that the potential effects of the loss of the Pim3 gene are physiologically compensated. Indeed, the 2 other family members of the PIM kinase family, PIM1 and PIM2 are upregulated in the kidney of Pim3-/- mice, and may therefore be involved in such compensation. In conclusion, our data demonstrate that the PIM3 kinase is a novel aldosterone-induced protein, but its precise role in aldosterone-dependent renal homeostasis remains to be determined.

The Role of Intercalated Cell Nedd4-2 in BP Regulation, Ion Transport, and Transporter Expression.

BackgroundNedd4-2 is an E3 ubiquitin-protein ligase that associates with transport proteins, causing their ubiquitylation, and then internalization and degradation. Previous research has suggested a correlation between Nedd4-2 and BP. In this study, we explored the effect of intercalated cell (IC) Nedd4-2 gene ablation on IC transporter abundance and function and on BP.Methods We generated IC Nedd4-2 knockout mice using Cre-lox technology and produced global pendrin/Nedd4-2 null mice by breeding global Nedd4-2 null (Nedd4-2-/- ) mice with global pendrin null (Slc26a4-/- ) mice. Mice ate a diet with 1%-4% NaCl; BP was measured by tail cuff and radiotelemetry. We measured transepithelial transport of Cl- and total CO2 and transepithelial voltage in cortical collecting ducts perfused in vitro Transporter abundance was detected with immunoblots, immunohistochemistry, and immunogold cytochemistry.Results IC Nedd4-2 gene ablation markedly increased electroneutral Cl-/HCO3- exchange in the cortical collecting duct, although benzamil-, thiazide-, and bafilomycin-sensitive ion flux changed very little. IC Nedd4-2 gene ablation did not increase the abundance of type B IC transporters, such as AE4 (Slc4a9), H+-ATPase, barttin, or the Na+-dependent Cl-/HCO3- exchanger (Slc4a8). However, IC Nedd4-2 gene ablation increased CIC-5 total protein abundance, apical plasma membrane pendrin abundance, and the ratio of pendrin expression on the apical membrane to the cytoplasm. IC Nedd4-2 gene ablation increased BP by approximately 10 mm Hg. Moreover, pendrin gene ablation eliminated the increase in BP observed in global Nedd4-2 knockout mice.Conclusions IC Nedd4-2 regulates Cl-/HCO3- exchange in ICs., Nedd4-2 gene ablation increases BP in part through its action in these cells.

AQP2 Abundance is Regulated by the E3-Ligase CHIP Via HSP70.

BACKGROUND/AIMS: AQP2 expression is mainly controlled by vasopressin-dependent changes in protein abundance which is in turn regulated by AQP2 ubiquitylation and degradation, however the proteins involved in these processes are largely unknown. Here, we investigated the potential role of the CHIP E3 ligase in AQP2 regulation. METHODS: MCD4 cells and kidney slices were used to study the involvement of the E3 ligase CHIP on AQP2 protein abundance by cell homogenization and immunoprecipitation followed by immunoblotting. RESULTS: We found that AQP2 complexes with CHIP in renal tissue. Expression of CHIP increased proteasomal degradation of AQP2 and HSP70 abundance, a molecular signature of HSP90 inhibition. Increased HSP70 level, secondary to CHIP expression, promoted ERK signaling resulting in increased AQP2 phosphorylation at S261. Phosphorylation of AQP2 at S256 and T269 were instead downregulated. Next, we investigated HSP70 interaction with AQP2, which is important for endocytosis. Compared with AQP2-wt, HSP70 binding decreased in AQP2-S256D and AQP2-S256D-S261D, while increased in AQP2-S256D-S261A. Surprisingly, expression of CHIP-delUbox, displaying a loss of E3 ligase activity, still induced AQP2 degradation, indicating that CHIP does not ubiquitylate and degrade AQP2 itself. Conversely, the AQP2 half-life was increased upon the expression of CHIP-delTPR a domain which binds Hsc70/HSP70 and HSP90. HSP70 has been reported to bind other E3 ligases such as MDM2. Notably, we found that co-expression of CHIP and MDM2 increased AQP2 degradation, whereas co-expression of CHIP with MDM2-delRING, an inactive form of MDM2, impaired AQP2 degradation. CONCLUSION: Our findings indicate CHIP as a master regulator of AQP2 degradation via HSP70 that has dual functions: (1) as chaperone for AQP2 and (2) as an anchoring protein for MDM2 E3 ligase, which is likely to be involved in AQP2 degradation.

Functional assessment of sodium chloride cotransporter NCC mutants in polarized mammalian epithelial cells.

The thiazide-sensitive sodium chloride cotransporter NCC is important for maintaining serum sodium (Na+) and, indirectly, serum potassium (K+) levels. Functional studies on NCC have used cell lines with native NCC expression, transiently transfected nonpolarized cell lines, or Xenopus laevis oocytes. Here, we developed the use of polarized Madin-Darby canine kidney type I (MDCKI) mammalian epithelial cell lines with tetracycline-inducible human NCC expression to study NCC activity and membrane abundance in the same system. In radiotracer assays, induced cells grown on filters had robust thiazide-sensitive and chloride dependent sodium-22 (22Na) uptake from the apical side. To minimize cost and maximize throughput, assays were modified to use cells grown on plastic. On plastic, cells had similar thiazide-sensitive 22Na uptakes that increased following preincubation of cells in chloride-free solutions. NCC was detected in the plasma membrane, and both membrane abundance and phosphorylation of NCC were increased by incubation in chloride-free solutions. Furthermore, in cells exposed for 15 min to low or high extracellular K+, the levels of phosphorylated NCC increased and decreased, respectively. To demonstrate that the system allows rapid and systematic assessment of mutated NCC, three phosphorylation sites in NCC were mutated, and NCC activity was examined. 22Na fluxes in phosphorylation-deficient mutants were reduced to baseline levels, whereas phosphorylation-mimicking mutants were constitutively active, even without chloride-free stimulation. In conclusion, this system allows the activity, cellular localization, and abundance of wild-type or mutant NCC to be examined in the same polarized mammalian expression system in a rapid, easy, and low-cost fashion.

Differential ubiquitylation of the mineralocorticoid receptor is regulated by phosphorylation.

Aldosterone stimulation of the mineralocorticoid receptor (MR) is involved in numerous physiological responses, including Na+ homeostasis, blood pressure control, and heart failure. Aldosterone binding to MR promotes different post-translational modifications that regulate MR nuclear translocation, gene expression, and finally receptor degradation. Here, we show that aldosterone stimulates rapid phosphorylation of MR via ERK1/2 in a dose-dependent manner (from 0.1 to 10 nM) in renal epithelial cells. This phosphorylation induces an increase of MR apparent molecular weight, with a maximal upward shift of 30 kDa. Strikingly, these modifications are critical for the regulation of the MR ubiquitylation state. Indeed, we find that MR is monoubiquitylated in its basal state, and this status is sustained by the tumor suppressor gene 101 (Tsg101). Phosphorylation leads to disruption of MR/Tsg101 association and monoubiquitin removal. These events prompt polyubiquitin-dependent destabilization of MR and degradation. Preventing MR phosphorylation by ERK1/2 inhibition or mutation of target serines affects the sequential mechanisms of MR ubiquitylation and inhibits the aldosterone-mediated degradation. Our data provide a novel model of negative feedback of aldosterone signaling, involving sequential phosphorylation, monoubiquitin removal and subsequent polyubiquitylation/degradation of MR.

Nedd4-2 and the regulation of epithelial sodium transport.

Nedd4-2 is a ubiquitin ligase previously demonstrated to regulate endocytosis and lysosomal degradation of the epithelial Na(+) channel (ENaC) and other ion channels and transporters. Recent studies using Nedd4-2 knockout mice specifically in kidney or lung epithelia has revealed a critical role for this E3 ubiquitin ligase in regulating salt and fluid transport in these tissues/organs and in maintaining homeostasis of body blood pressure. Interestingly, the primary targets for Nedd4-2 may differ in these two organs: in the lung Nedd4-2 targets ENaC, and loss of Nedd4-2 leads to excessive ENaC function and to cystic fibrosis - like lung disease, whereas in the kidney, Nedd4-2 targets the Na(+)/Cl(-) cotransporter (NCC) in addition to targeting ENaC. In accord, loss of Nedd4-2 in the distal nephron leads to increased NCC abundance and function. The aldosterone-responsive kinase, Sgk1, appears to be involved in the regulation of NCC by Nedd4-2 in the kidney, similar to its regulation of ENaC. Collectively, these new findings underscore the physiological importance of Nedd4-2 in regulating epithelial salt and fluid transport and balance.

Deubiquitylating enzyme USP2 counteracts Nedd4-2-mediated downregulation of KCNQ1 potassium channels.

BACKGROUND: KCNQ1 (Kv7.1), together with its KCNE ß subunits, plays a pivotal role both in the repolarization of cardiac tissue and in water and salt transport across epithelial membranes. Nedd4/Nedd4-like (neuronal precursor cell-expressed developmentally downregulated 4) ubiquitin-protein ligases interact with the KCNQ1 potassium channel through a PY motif located in the C terminus of KCNQ1. This interaction induces ubiquitylation of KCNQ1, resulting in a reduced surface density of the channel. It was reported recently that the epithelial sodium channel is regulated by the reverse process-deubiquitylation-mediated by USP2 (ubiquitin-specific protease 2). OBJECTIVE: In this article, we investigated whether deubiquitylation may regulate KCNQ1 channel complexes. METHODS: In this study, we used electrophysiology, biochemistry, and confocal microscopy. RESULTS: Electrophysiological investigations of KCNQ1/KCNE1 proteins coexpressed with USP2-45 or USP2-69 isoforms and Nedd4-2 in Xenopus laevis oocytes and mammalian cells revealed that both USP2 isoforms counter the Nedd4-2-specific downregulation of I(Ks). Biochemical studies showed that the total and surface-expressed KCNQ1 protein was more abundant when coexpressed with USP2 and Nedd4-2 as compared with Nedd4-2 alone. Western blotting revealed partial protection against covalent attachment of ubiquitin moieties on KCNQ1 when USP2 was coexpressed with Nedd4-2. Coimmunoprecipitation assays suggested that USP2 can bind to KCNQ1 independently of the PY motif. Immunocytochemistry confirmed that USP2 restores the membrane localization of KCNQ1. CONCLUSION: These results demonstrate that USP2 can be a potent regulator of KCNQ1 surface density. USP2, which is well expressed in many tissues, may therefore be important in controlling the KCNQ1 channel dynamics in vivo.