Epithelial Fluid Secretion Research Articles

Control of intracellular chloride signaling by IRBIT‐mediated recruitment of multiple kinase and phosphatase pathways

Cl− is the major anion and important osmolyte in most living cells and has a prominent role in cellular homeostasis and intracellular chloride (Cl−in) affect the activity of several transporters. IRBIT is a multifunctional protein that controls the activity of IP3 receptors and multiple Cl− and HCO3− transporters by unknown mechanism. In the case of the ubiquitous NBCe1‐B, IRBIT interacts with the NBCe1‐B N terminus autoinhibitory domain (AID), exposing two cryptic Cl‐interacting sites to confer regulation of NBCe1‐B by Cl−in. Here, we report that IRBIT do so by recruiting a panel of phosphatases and kinases that both regulate signaling by Cl−in and modulate NBCe1‐B conformation to open the transport pathway. Thus, IRBIT recruits the phosphatase PP1 and the kinase SPAK to control phosphorylation of S65 to control regulation of NBCe1‐B by Cl−in at a 32GXXXP36 site. IRBIT recruits the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of S12 to control regulation of NBCe1‐B by Cl−in at a 195GXXXP199 site. IRBIT then acts on S232, S233, S235 to generate multiple conformations with either dephosphorylated S232/S233, S232/S235 or S233/S235. Each conformation has different activation by IRBIT and regulation by Cl−in. S232/S233/S235 are conserved in several members of the NBC transporters. Mutation of serine to alanine resulted in a fully activated conformation, while mutation of serine to phosphomimetic aspartate inhibited transport and eliminated IRBIT mediated activation, suggesting that S232/S233/S235 determine whether NBC transporter is in an opened or closed conformation. These findings together with structural modeling of NBCe1‐B uncover an intricate mechanism by Cl−in signals through IRBIT‐mediated recruitment of kinases and phosphatases to regulate key transporters involved in epithelial fluid and electrolyte secretion.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mouse models of SLC4-linked disorders of HCO3--transporter dysfunction.

The SLC4 family Cl-/[Formula: see text] cotransporters (NBCe1, NBCe2, NBCn1, and NBCn2) contribute to a variety of vital physiological processes including pH regulation and epithelial fluid secretion. Accordingly, their dysfunction can have devastating effects. Disorders such as epilepsy, hemolytic anemia, glaucoma, hearing loss, osteopetrosis, and renal tubular acidosis are all genetically linked to SLC4-family gene loci. This review summarizes how studies of Slc4-modified mice have enhanced our understanding of the etiology of SLC4-linked pathologies and the interpretation of genetic linkage studies. The review also surveys the novel disease signs exhibited by Slc4-modified mice which could either be considered to presage their description in humans, or to highlight interspecific differences. Finally, novel Slc4-modified mouse models are proposed, the study of which may further our understanding of the basis and treatment of SLC4-linked disorders of [Formula: see text]-transporter dysfunction.

Steviol stabilizes polycystin 1 expression and promotes lysosomal degradation of CFTR and β-catenin proteins in renal epithelial cells

Malfunction of polycystin 1 (PC1) is linked to abnormally high epithelial cell proliferation and fluid secretion, eventually leading to renal cyst development and declined renal function as found in autosomal dominant polycystic kidney disease (ADPKD). Currently, there is no effective therapy for ADPKD. Recent studies report PC1 regulates CFTR chloride channels and β-catenin levels in normal renal epithelial cells. Concurrently, our previous study found steviol retarded renal cyst enlargement in an in vitro and in an in vivo models by reducing CFTR expression and activity. Therefore, a potential relationship between steviol and PC1 is worthy of exploration. The present study was aimed to determine the effect of steviol on PC1, CFTR, and β-catenin levels in renal epithelial cells with defective PC1 biogenesis and expression (Prkcsh−/− cell) and postnatal Pkd1 homozygous cell (Pkd1−/− cells). Using western blot analysis, it was found that steviol treatment at 100μM for 24–48h substantially enhanced and stabilized PC1 C-terminal expression, while decreasing CFTR and β-catenin protein expression in both Prkcsh−/− and Pkd1−/− cells. In addition, steviol promoted LAMP2 expression, a lysosomal enzyme marker. Interestingly, hydroxychloroquine (a lysosome inhibitor) treatment abolished steviol’s effect in reducing CFTR and β-catenin protein expression. Taken together, these findings suggest steviol slows cyst progression in cells and animal models of PKD, in part, by enhancing and stabilizing PC1 protein expression as well as by promoting lysosomal degradation of CFTR and β-catenin. Therefore, steviol may represent a promising compound for treatment of polycystic kidney disease.

Stem cell-derived organoids to model gastrointestinal facets of cystic fibrosis.

Cystic fibrosis (CF) is one of the most frequently occurring inherited human diseases caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) which lead to ample defects in anion transport and epithelial fluid secretion. Existing models lack both access to early stages of CF development and a coeval focus on the gastrointestinal CF phenotypes, which become increasingly important due increased life span of the affected individuals. Here, we provide a comprehensive overview of gastrointestinal facets of CF and the opportunity to model these in various systems in an attempt to understand and treat CF. A particular focus is given on forward-leading organoid cultures, which may circumvent current limitations of existing models and thereby provide a platform for drug testing and understanding of disease pathophysiology in gastrointestinal organs.

Polycystic Kidney Disease.

Renal cysts, which arise from renal tubules, can be seen in a variety of hereditary and nonhereditary entities. Common mechanisms associated with renal cyst formation include increased cell proliferation, epithelial fluid secretion, and extracellular matrix remodeling. Hereditary polycystic kidney disease (PKD) is seen as a component of numerous diseases. Autosomal dominant (AD) PKD is the most common potentially fatal hereditary disease in humans, causes renal failure in approximately 50% of affected individuals, and accounts for approximately 5% of end stage renal disease cases in the United States. ADPKD is caused by mutation in one of two genes-85% of cases are caused by mutation in PKD1 on chromosome 16 and 15% of cases are caused by mutation in PKD2 on chromosome 4. Polycystin-1, encoded by PKD1, is a large protein, has multiple transmembrane spanning domains, has extracellular regions suggesting a role in cell-cell or cell-matrix interactions, has intracellular domains suggesting a role in signal transduction, and can physically interact with Polycystin-2. Polycystin-2 is smaller, has transmembrane domains, can act as a cation channel with calcium permeability, and may be regulated by Polycystin-1. These proteins, and many others associated with cystic kidney disease, localize to primary cilia, which may act as flow sensors in the kidney; cystic kidney diseases have also been termed ciliopathies. An increasing number of intracellular mechanisms, which are abnormally regulated in PKD, have been described and are potential targets for therapy, which is lacking in this common hereditary disease. © 2017 American Physiological Society. Compr Physiol 7:945-975, 2017.

Substituted 2-Acylaminocycloalkylthiophene-3-carboxylic Acid Arylamides as Inhibitors of the Calcium-Activated Chloride Channel Transmembrane Protein 16A (TMEM16A).

Transmembrane protein 16A (TMEM16A), also called anoctamin 1 (ANO1), is a calcium-activated chloride channel expressed widely mammalian cells, including epithelia, vascular smooth muscle tissue, electrically excitable cells, and some tumors. TMEM16A inhibitors have been proposed for treatment of disorders of epithelial fluid and mucus secretion, hypertension, asthma, and possibly cancer. Herein we report, by screening, the discovery of 2-acylaminocycloalkylthiophene-3-carboxylic acid arylamides (AACTs) as inhibitors of TMEM16A and analysis of 48 synthesized analogs (10ab-10bw) of the original AACT compound (10aa). Structure-activity studies indicated the importance of benzene substituted as 2- or 4-methyl, or 4-fluoro, and defined the significance of thiophene substituents and size of the cycloalkylthiophene core. The most potent compound (10bm), which contains an unusual bromodifluoroacetamide at the thiophene 2-position, had IC50 of ∼30 nM, ∼3.6-fold more potent than the most potent previously reported TMEM16A inhibitor 4 (Ani9), and >10-fold improved metabolic stability. Direct and reversible inhibition of TMEM16A by 10bm was demonstrated by patch-clamp analysis. AACTs may be useful as pharmacological tools to study TMEM16A function and as potential drug development candidates.

Marked increases in mucociliary clearance produced by synergistic secretory agonists or inhibition of the epithelial sodium channel.

Mucociliary clearance (MCC) is a critical host innate defense mechanism in airways, and it is impaired in cystic fibrosis (CF) and other obstructive lung diseases. Epithelial fluid secretion and absorption modify MCC velocity (MCCV). We tested the hypotheses that inhibiting fluid absorption accelerates MCCV, whereas inhibiting fluid secretion decelerates it. In airways, ENaC is mainly responsible for fluid absorption, while anion channels, including CFTR and Ca2+-activated chloride channels mediate anion/fluid secretion. MCCV was increased by the cAMP-elevating agonists, forskolin or isoproterenol (10 μM) and by the Ca2+-elevating agonist, carbachol (0.3 μM). The CFTR-selective inhibitor, CFTRinh-172, modestly reduced MCCV-increases induced by forskolin or isoproterenol but not increases induced by carbachol. The ENaC inhibitor benzamil increased basal MCCV as well as MCCV increases produced by forskolin or carbachol. MCC velocity was most dramatically accelerated by the synergistic combination of forskolin and carbachol, which produced near-maximal clearance rates regardless of prior treatment with CFTR or ENaC inhibitors. In CF airways, where CFTR-mediated secretion (and possibly synergistic MCC) is lost, ENaC inhibition via exogenous agents may provide therapeutic benefit, as has long been proposed.

The Farnesoid X Receptor: Good for BAD

Diarrhea is a feature of several chronic intestinal disorders that are associated with increased delivery of bile acids into the colon. Although the prevalence of bile acid diarrhea is high, affecting approximately 1% of the adult population, current therapies often are unsatisfactory. By virtue of its capacity to inhibit colonic epithelial fluid secretion and to down-regulate hepatic bile acid synthesis through induction of the ileal fibroblast growth factor 19 release, the nuclear bile acid receptor, farnesoid X receptor, represents a promising target for the development of new therapeutic approaches. Here, we review our current understanding of the pathophysiology of bile acid diarrhea and the current evidence supporting a role for farnesoid X receptor agonists in treatment of the disease.

Hydrogen Sulfide Facilitates Vaginal Lubrication by Activation of Epithelial ATP-Sensitive K(+) Channels and Cystic Fibrosis Transmembrane Conductance Regulator.

Hydrogen sulfide (H2S) plays a large role in female and male sexual responses characterized by a smooth muscle relaxant effect. Moreover, H2S is a novel pro-secretory neuromodulator that modulates epithelial ion transport. However, whether H2S has a role in regulating vaginal epithelial ion transport and fluid secretion has not been extensively studied. To identify the effects of H2S on vaginal epithelial ion transport and lubrication in an exploratory investigation. The mRNA, protein expression, and localization of cystathionine γ-lyase (CSE) and H2S production in vaginal epithelium were examined by reverse transcriptase polymerase chain reaction, Western blot, H2S synthesizing activity assay, and immunohistochemistry, respectively. The effect of H2S on vaginal epithelial ion transport, vaginal fluid secretion, and ionic concentration was investigated using a short-circuit current (ISC), a measurement of vaginal lubrication, and ion chromatography, respectively. The mRNA, protein expression, and localization of CSE, H2S formation, changes of ISC responses, vaginal lubrication, and K(+) and Cl(-) concentrations were studied. CSE mRNA and protein were predominantly expressed in vaginal epithelium. Sodium hydrosulfide hydrate (NaHS) caused concentration-dependent changes in ISC across isolated rat vaginal epithelium, which consisted of an initial decrease phase and then an increase phase. The increase phase in ISC was mainly Cl(-) dependent and abolished by cystic fibrosis transmembrane conductance regulator inhibitor, whereas the decrease phase was sensitive to the adenosine triphosphate-sensitive K(+) (KATP) channel blocker. Furthermore, intravaginal treatment of NaHS significantly enhanced vaginal lubrication invivo, and this effect was prevented by cystic fibrosis transmembrane conductance regulator and KATP channel inhibitors. In addition, the ionic concentrations of K(+) and Cl(-) in rat vaginal fluid were significantly increased by NaHS treatment. The CSE-H2S pathway participates in the regulation of vaginal epithelial K(+) and Cl(-) ion transport to modulate lumen fluid secretion.

Increased TMEM16A Involved in Alveolar Fluid Clearance After Lipopolysaccharide Stimulation.

Transmembrane protein 16A (TMEM16A) regulates a wide variety of cellular activities, including epithelial fluid secretion and maintenance of ion homeostasis. Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, is one of the major causes of acute lung injury (ALI). In this study, we investigated the effects of LPS on the expression of TMEM16A in LA795 cells and mouse lung tissue and the potential mechanism. We detected the expression of TMEM16A in LA795 cells and mouse lung tissue by RT-PCR, Western blot, and RNA interference techniques. TMEM16A expression was significantly increased by LPS stimulation in LA795 cells and in mouse lung tissue. Moreover, the LPS-induced TMEM16A expression enhancement in lung tissue was much more prominent in the alveolar epithelial region than in bigger airway epithelial cells. The typical TMEM16A current was recorded, and LPS treatment significantly enhances the current amplitude in LA795 cells. TMEM16A shRNA or TMEM16A inhibitor (T16Ainh-A01) did not affect alveolar fluid clearance (AFC), while co-application of T16Ainh-A01 induced a stronger AFC inhibition than LPS alone. LPS notably and synchronously enhanced Akt phosphorylation (p-Akt) and TMEM16A expression in a time-dependent manner in LA795 cells. Taken together, our results suggest that TMEM16A maybe plays an important role in pathological conditions of LPS-induced ALI as a protective protein.

Synthesis and evaluation of 5,6-disubstituted thiopyrimidine aryl aminothiazoles as inhibitors of the calcium-activated chloride channel TMEM16A/Ano1

Transmembrane protein 16A (TMEM16A), also called Ano1, is a Ca2+ activated Cl− channel expressed widely in mammalian epithelia, as well as in vascular smooth muscle and some tumors and electrically excitable cells. TMEM16A inhibitors have potential utility for treatment of disorders of epithelial fluid and mucus secretion, hypertension, some cancers and other diseases. 4-Aryl-2-amino thiazole T16Ainh-01 was previously identified by high-throughput screening. Here, a library of 47 compounds were prepared that explored the 5,6-disubstituted pyrimidine scaffold found in T16Ainh-01. TMEM16A inhibition activity was measured using fluorescence plate reader and short-circuit current assays. We found that very little structural variation of T16Ainh-01 was tolerated, with most compounds showing no activity at 10 μM. The most potent compound in the series, 9bo, which substitutes 4-methoxyphenyl in T16Ainh-01 with 2-thiophene, had IC50 ∼1 μM for inhibition of TMEM16A chloride conductance.

Evidence for a "Pathogenic Triumvirate" in Congenital Hepatic Fibrosis in Autosomal Recessive Polycystic Kidney Disease.

Autosomal recessive polycystic kidney disease (ARPKD) is a severe monogenic disorder that occurs due to mutations in the PKHD1 gene. Congenital hepatic fibrosis (CHF) associated with ARPKD is characterized by the presence of hepatic cysts derived from dilated bile ducts and a robust, pericystic fibrosis. Cyst growth, due to cyst wall epithelial cell hyperproliferation and fluid secretion, is thought to be the driving force behind disease progression. Liver fibrosis is a wound healing response in which collagen accumulates in the liver due to an imbalance between extracellular matrix synthesis and degradation. Whereas both hyperproliferation and pericystic fibrosis are hallmarks of CHF/ARPKD, whether or not these two processes influence one another remains unclear. Additionally, recent studies demonstrate that inflammation is a common feature of CHF/ARPKD. Therefore, we propose a “pathogenic triumvirate” consisting of hyperproliferation of cyst wall growth, pericystic fibrosis, and inflammation which drives CHF/ARPKD progression. This review will summarize what is known regarding the mechanisms of cyst growth, fibrosis, and inflammation in CHF/ARPKD. Further, we will discuss the potential advantage of identifying a core pathogenic feature in CHF/ARPKD to aid in the development of novel therapeutic approaches. If a core pathogenic feature does not exist, then developing multimodality therapeutic approaches to target each member of the “pathogenic triumvirate” individually may be a better strategy to manage this debilitating disease.

KCa3.1 K+ Channel Expression and Function in Human Bronchial Epithelial Cells.

The KCa3.1 K+ channel has been proposed as a novel target for pulmonary diseases such as asthma and pulmonary fibrosis. It is expressed in epithelia but its expression and function in primary human bronchial epithelial cells (HBECs) has not been described. Due to its proposed roles in the regulation of cell proliferation, migration, and epithelial fluid secretion, inhibiting this channel might have either beneficial or adverse effects on HBEC function. The aim of this study was to assess whether primary HBECs express the KCa3.1 channel and its role in HBEC function. Primary HBECs from the airways of healthy and asthmatic subjects, SV-transformed BEAS-2B cells and the neoplastic H292 epithelial cell line were studied. Primary HBECs, BEAS-2B and H292 cells expressed KCa3.1 mRNA and protein, and robust KCa3.1 ion currents. KCa3.1 protein expression was increased in asthmatic compared to healthy airway epithelium in situ, and KCa3.1 currents were larger in asthmatic compared to healthy HBECs cultured in vitro. Selective KCa3.1 blockers (TRAM-34, ICA-17043) had no effect on epithelial cell proliferation, wound closure, ciliary beat frequency, or mucus secretion. However, several features of TGFβ1-dependent epithelial-mesenchymal transition (EMT) were inhibited by KCa3.1 blockade. Treatment with KCa3.1 blockers is likely to be safe with respect to airway epithelial biology, and may potentially inhibit airway remodelling through the inhibition of EMT.

Essential role of carbonic anhydrase XII in secretory gland fluid and HCO3 (-) secretion revealed by disease causing human mutation.

Fluid and HCO3 (-) secretion is essential for all epithelia; aberrant secretion is associated with several diseases. Carbonic anhydrase XII (CA12) is the key carbonic anhydrase in epithelial fluid and HCO3 (-) secretion and works by activating the ductal Cl(-) -HCO3 (-) exchanger AE2. Delivery of CA12 to salivary glands increases salivation in mice and of the human mutation CA12(E143K) markedly inhibits it. The human mutation CA12(E143K) causes disease due to aberrant CA12 glycosylation, and misfolding resulting in loss of AE2 activity. Aberrant epithelial fluid and HCO3 (-) secretion is associated with many diseases. The activity of HCO3 (-) transporters depends of HCO3 (-) availability that is determined by carbonic anhydrases (CAs). Which CAs are essential for epithelial function is unknown. CA12 stands out since the CA12(E143K) mutation causes salt wasting in sweat and dehydration in humans. Here, we report that expression of CA12 and of CA12(E143K) in mice salivary glands respectively increased and prominently inhibited ductal fluid secretion and salivation in vivo. CA12 markedly increases the activity and is the major HCO3 (-) supplier of ductal Cl(-) -HCO3 (-) exchanger AE2, but not of NBCe1-B. The E143K mutation alters CA12 glycosylation at N28 and N80, resulting in retention of the basolateral CA12 in the ER. Knockdown of AE2 and of CA12 inhibited pancreatic and salivary gland ductal AE2 activity and fluid secretion. Accordingly, patients homozygous for the CA12(E143K) mutation have a dry mouth, dry tongue phenotype. These findings reveal an unsuspected prominent role of CA12 in epithelial function, explain the disease and call for caution in the use of CA12 inhibitors in cancer treatment.

Pancreatic epithelial fluid and bicarbonate secretion is significantly elevated in the absence of serotonin in mice

Pancreatic epithelial fluid and bicarbonate secretion is significantly elevated in the absence of serotonin in mice

CRISPR/Cas mediated genome editing reveals new insights into the WNK‐SPAK/OSR1 network

Sodium‐coupled SLC12 cation chloride cotransporters play important roles in cell volume and chloride homeostasis, epithelial fluid secretion, and renal tubular salt reabsorption. These cotransporters are phosphorylated and activated indirectly by With‐No‐Lysine (WNK) kinases through their downstream effector kinases, SPAK and OSR1. Multiple WNKs can coexist within a single cell type, although their relative contributions to SPAK/OSR1 activation and salt transport remain incompletely understood. Here, we outline a simple method to selectively knock out WNK expression from mammalian cells using single guide RNA (sgRNA) targeted CRISPR/Cas9 endonucleases. Genetically and biochemically validated WNK1 knockout HEK293T cells exhibited low baseline WNK4 and SPAK/OSR1 activity. This was associated with increased expression of KLHL3/CUL3, suggesting that WNK1 regulates WNK4 stability through their cognate E3 ubiquitin ligase. WNK1 KO cells also failed to trigger regulatory volume increase (RVI) during hypertonic stress, proving that WNK1 is necessary for cell volume control. Currently, we are adapting this technology to generate WNK4 and WNK3 KOs. To optimize sgRNA efficiency, we used a surrogate reporter‐based assay that triggers eGFP fluorescence upon target site editing, permitting enrichment of edited cells by FACS. This technique dramatically enhanced detection of genomic modification by T7E1 mismatch screening. Collectively, our data illustrate how this new and powerful technology can be used to dissect and analyze complex signaling networks. Support: NIDDK, VA

Intracellular Cl− as a signaling ion that potently regulates Na+/HCO3− transporters

Cl(-) is a major anion in mammalian cells involved in transport processes that determines the intracellular activity of many ions and plasma membrane potential. Surprisingly, a role of intracellular Cl(-) (Cl(-) in) as a signaling ion has not been previously evaluated. Here we report that Cl(-) in functions as a regulator of cellular Na(+) and HCO3 (-) concentrations and transepithelial transport through modulating the activity of several electrogenic Na(+)-HCO3 (-) transporters. We describe the molecular mechanism(s) of this regulation by physiological Cl(-) in concentrations highlighting the role of GXXXP motifs in Cl(-) sensing. Regulation of the ubiquitous Na(+)-HCO3(-) co-transport (NBC)e1-B is mediated by two GXXXP-containing sites; regulation of NBCe2-C is dependent on a single GXXXP motif; and regulation of NBCe1-A depends on a cryptic GXXXP motif. In the basal state NBCe1-B is inhibited by high Cl(-) in interacting at a low affinity GXXXP-containing site. IP3 receptor binding protein released with IP3 (IRBIT) activation of NBCe1-B unmasks a second high affinity Cl(-) in interacting GXXXP-dependent site. By contrast, NBCe2-C, which does not interact with IRBIT, has a single high affinity N-terminal GXXP-containing Cl(-) in interacting site. NBCe1-A is unaffected by Cl(-) in between 5 and 140 mM. However, deletion of NBCe1-A residues 29-41 unmasks a cryptic GXXXP-containing site homologous with the NBCe1-B low affinity site that is involved in inhibition of NBCe1-A by Cl(-) in. These findings reveal a cellular Cl(-) in sensing mechanism that plays an important role in the regulation of Na(+) and HCO3 (-) transport, with critical implications for the role of Cl(-) in cellular ion homeostasis and epithelial fluid and electrolyte secretion.

Intracellular C1 as a Signaling Molecule that Potently Regulates Na and HCO3 Transporters

Cl- and HCO3- are the two major cellular anions. Surprisingly, a role of intracellular Cl- (Cl-in) as a signaling molecule that regulates the activity of other transporters has not been evaluated. We found that Cl-in functions as a regulator of cellular Na+ and HCO3- concentrations by regulating the activity of several NBCs. All forms of regulation by Cl-in are mediated by the Cl- interacting GXXXP motifs. In the basal state NBCe1-B is inhibited by high Cl-in, while NBCe1-A is resistant to Cl-in between 5-140 mM. By contracts, the IRBIT-activated NBCe1-B and the basal activity of NBCe2-C are inhibited by high affinity Cl-in sites, with apparent affinity of about 10 mM. The NBCe1-B high and low affinity Cl-in sites are mediated by separate GXXXP motifs. Mutations in the GXXXP motifs in the autoinhibitory site of NBCe1-B and the GXXXP motif in the N terminus of NBCe2-C eliminated inhibition by low Cl-in, while sparing inhibition of NBCe1-B by high Cl-in. Mutation of a second N terminus GXXXP motif was required to eliminate inhibition of NBCe1-B by Cl-in. Deletion of residues 29-41 of NBCe1-A uncovered inhibition by Cl-in that was mediated by a hidden GXXXP motif homologous with the NBCe1-B low affinity site. These finding reveal a novel Cl-in sensing mechanism that is transmitted by regulation of Na+ and HCO3- transporters with major implication for cellular Na+ and HCO3- homeostasis and epithelial fluid and electrolyte secretion.

Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing.

Sodium-coupled SLC12 cation chloride cotransporters play important roles in cell volume and chloride homeostasis, epithelial fluid secretion, and renal tubular salt reabsorption. These cotransporters are phosphorylated and activated indirectly by With-No-Lysine (WNK) kinases through their downstream effector kinases, Ste20- and SPS1-related proline alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1). Multiple WNK kinases can coexist within a single cell type, although their relative contributions to SPAK/OSR1 activation and salt transport remain incompletely understood. Deletion of specific WNKs from cells that natively express a functional WNK-SPAK/OSR1 network will help resolve these knowledge gaps. Here, we outline a simple method to selectively knock out full-length WNK1 expression from mammalian cells using RNA-guided clustered regularly interspaced short palindromic repeats/Cas9 endonucleases. Two clonal cell lines were generated by using a single-guide RNA (sgRNA) targeting exon 1 of the WNK1 gene, which produced indels that abolished WNK1 protein expression. Both cell lines exhibited reduced endogenous WNK4 protein abundance, indicating that WNK1 is required for WNK4 stability. Consistent with an on-target effect, the reduced WNK4 abundance was associated with increased expression of the KLHL3/cullin-3 E3 ubiquitin ligase complex and was rescued by exogenous WNK1 overexpression. Although the morphology of the knockout cells was indistinguishable from control, they exhibited low baseline SPAK/OSR1 activity and failed to trigger regulatory volume increase after hypertonic stress, confirming an essential role for WNK1 in cell volume regulation. Collectively, our data show how this new, powerful, and accessible gene-editing technology can be used to dissect and analyze WNK signaling networks.

Kidney: polycystic kidney disease

Polycystic kidney disease (PKD) is a life-threatening genetic disorder characterized by the presence of fluid-filled cysts primarily in the kidneys. PKD can be inherited as autosomal recessive (ARPKD) or autosomal dominant (ADPKD) traits. Mutations in either the PKD1 or PKD2 genes, which encode polycystin 1 and polycystin 2, are the underlying cause of ADPKD. Progressive cyst formation and renal enlargement lead to renal insufficiency in these patients, which need to be managed by lifelong dialysis or renal transplantation. While characteristic features of PKD are abnormalities in epithelial cell proliferation, fluid secretion, extracellular matrix and differentiation, the molecular mechanisms underlying these events are not understood. Here we review the progress that has been made in defining the function of the polycystins, and how disruption of these functions may be involved in cystogenesis.