MicroRNA-19 is regulated by aldosterone in a sex-specific manner to alter kidney sodium transport.

A key regulator of blood pressure homeostasis is the steroid hormone aldosterone, which is released as the final signaling hormone of the renin-angiotensin-aldosterone-signaling (RAAS) system. Aldosterone increases sodium (Na+) reabsorption in the kidney distal nephron to regulate blood volume. Unregulated RAAS signaling can lead to hypertension and cardiovascular disease. The serum and glucocorticoid kinase (SGK1) coordinates much of the Na+ reabsorption in the cortical collecting duct (CCD) tubular epithelial cells. We previously demonstrated that aldosterone alters the expression of microRNAs (miRs) in CCD principal cells. The aldosterone-regulated miRs can modulate Na+ transport and the cellular response to aldosterone signaling. However, the sex-specific regulation of miRs by aldosterone in the kidney distal nephron has not been explored. In this study, we report that miR-19, part of the miR-17-92 cluster, is upregulated in female mouse CCD cells in response to aldosterone activation. Mir-19 binding to the 3'-untranslated region of SGK1 was confirmed using a dual-luciferase reporter assay. Increasing miR-19 expression in CCD cells decreased SGK1 message and protein expression. Removal of this cluster using a nephron-specific, inducible knockout mouse model increased SGK1 expression in female mouse CCD cells. The miR-19-induced decrease in SGK1 protein expression reduced the response to aldosterone stimulation and may account for sex-specific differences in aldosterone signaling. By examining evolution of the miR-17-92 cluster, phylogenetic sequence analysis indicated that this cluster arose at the same time that other Na+-sparing and salt regulatory proteins, specifically SGK1, first emerged, indicating a conserved role for these miRs in kidney function of salt and water homeostasis.NEW & NOTEWORTHY Expression of the microRNA-17-92 cluster is upregulated by aldosterone in mouse cortical collecting duct principal cells, exclusively in female mice. MiR-19 in this cluster targets the serum and glucocorticoid kinase (SGK1) to downregulate both mRNA and protein expression, resulting in a decrease in sodium transport across epithelial cells of the collecting duct. The miR-17-92 cluster is evolutionarily conserved and may act as a novel feedback regulator for aldosterone signaling in females.

Histone deacetylase inhibitors (HDACi) increase expression of KCa2.3 (SK3) in primary microvascular endothelial cells.

The small conductance calcium-activated potassium channel (KCa2.3) has long been recognized for its role in mediating vasorelaxation through the endothelium-derived hyperpolarization (EDH) response. Histone deacetylases (HDACs) have been implicated as potential modulators of blood pressure and histone deacetylase inhibitors (HDACi) are being explored as therapeutics for hypertension. Herein, we show that HDACi increase KCa2.3 expression when heterologously expressed in HEK cells and endogenously expressed in primary cultures of human umbilical vein endothelial cells (HUVECs) and human intestinal microvascular endothelial cells (HIMECs). When primary endothelial cells were exposed to HDACi, KCa2.3 transcripts, subunits, and functional current are increased. Quantitative RT-PCR (qPCR) demonstrated increased KCa2.3 mRNA following HDACi, confirming transcriptional regulation of KCa2.3 by HDACs. By using pharmacological agents selective for different classes of HDACs, we discriminated between cytoplasmic and epigenetic modulation of KCa2.3. Biochemical analysis revealed an association between the cytoplasmic HDAC6 and KCa2.3 in immunoprecipitation studies. Specifically inhibiting HDAC6 increases expression of KCa2.3. In addition to increasing the expression of KCa2.3, we show that nonspecific inhibition of HDACs causes an increase in the expression of the molecular chaperone Hsp70 in endothelial cells. When Hsp70 is inhibited in the presence of HDACi, the magnitude of the increase in KCa2.3 expression is diminished. Finally, we show a slower rate of endocytosis of KCa2.3 as a result of exposure of primary endothelial cells to HDACi. These data provide the first demonstrated approach to increase KCa2.3 channel number in endothelial cells and may partially account for the mechanism by which HDACi induce vasorelaxation.

Aldosterone-induced microRNAs act as feedback regulators of mineralocorticoid receptor signaling in kidney epithelia.

The final steps in the Renin-Angiotensin-Aldosterone signaling System (RAAS) involve binding of the corticosteroid hormone, aldosterone to its mineralocorticoid receptor (MR). The bound MR interacts with response elements to induce or repress the transcription of aldosterone-regulated genes. A well characterized aldosterone-induced gene is the serum and glucocorticoid-induced kinase (SGK1), which acts downstream to increase sodium transport in distal kidney nephron epithelial cells. The role of microRNAs (miRs) induced by extended aldosterone stimulation in regulating MR and SGK1 has not been reported. In these studies, miRs predicted to bind to the 3'-UTR of mouse MR were profiled by qRT-PCR after aldosterone stimulation. The miR-466a/b/c/e family was upregulated in mouse kidney cortical collecting duct epithelial cells. A luciferase reporter assay confirmed miR-466 binding to both MR and SGK1 3'-UTRs. Inhibition of miR-466 increased MR and SGK1 mRNA and protein levels. Inhibiting miR-466b and preventing its upregulation after aldosterone stimulation increased amiloride-sensitive sodium transport and sensitivity to aldosterone stimulation. In vivo upregulation of miR-466 was confirmed in distal nephrons of mice on low Na+ diets. Repression of MR and SGK1 by aldosterone-induced miRs may represent a negative feedback loop that contributes to a form of aldosterone escape in vivo.

Loss of miR-17~92 results in dysregulation of Cftr in nephron progenitors.

We have previously demonstrated that loss of miR-17~92 in nephron progenitors in a mouse model results in renal hypodysplasia and chronic kidney disease. Clinically, decreased congenital nephron endowment because of renal hypodysplasia is associated with an increased risk of hypertension and chronic kidney disease, and this is at least partly dependent on the self-renewal of nephron progenitors. Here, we present evidence for a novel molecular mechanism regulating the self-renewal of nephron progenitors and congenital nephron endowment by the highly conserved miR-17~92 cluster. Whole transcriptome sequencing revealed that nephron progenitors lacking this cluster demonstrated increased Cftr expression. We showed that one member of the cluster, miR-19b, is sufficient to repress Cftr expression in vitro and that perturbation of Cftr activity in nephron progenitors results in impaired proliferation. Together, these data suggest that miR-19b regulates Cftr expression in nephron progenitors, with this interaction playing a role in appropriate nephron progenitor self-renewal during kidney development to generate normal nephron endowment.

Ankyrin G Expression Regulates Apical Delivery of the Epithelial Sodium Channel (ENaC).

The epithelial sodium channel (ENaC) is the limiting entry point for Na+ reabsorption in the distal kidney nephron and is regulated by numerous hormones, including the mineralocorticoid hormone aldosterone. Previously we identified ankyrin G (AnkG), a cytoskeletal protein involved in vesicular transport, as a novel aldosterone-induced protein that can alter Na+ transport in mouse cortical collecting duct cells. However, the mechanisms underlying AnkG regulation of Na+ transport were unknown. Here we report that AnkG expression directly regulates Na+ transport by altering ENaC activity in the apical membrane. Increasing AnkG expression increased ENaC activity while depleting AnkG reduced ENaC-mediated Na+ transport. These changes were due to a change in ENaC directly rather than through alterations to the Na+ driving force created by Na+/K+-ATPase. Using a constitutively open mutant of ENaC, we demonstrate that the augmentation of Na+ transport is caused predominantly by increasing the number of ENaCs at the surface. To determine the mechanism of AnkG action on ENaC surface number, changes in rates of internalization, recycling, and membrane delivery were investigated. AnkG did not alter ENaC delivery to the membrane from biosynthetic pathways or removal by endocytosis. However, AnkG did alter ENaC insertion from constitutive recycling pathways. These findings provide a mechanism to account for the role of AnkG in the regulation of Na+ transport in the distal kidney nephron.

Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel.

The epithelial sodium channel (ENaC) has an important role in regulating extracellular fluid volume and blood pressure, as well as airway surface liquid volume and mucociliary clearance. ENaC is a trimer of three homologous subunits (α, ß, and γ). We previously reported that cytoplasmic residues on the ß (ßCys-43 and ßCys-557) and γ (γCys-33 and γCys-41) subunits are palmitoylated. Mutation of Cys that blocked ENaC palmitoylation also reduced channel open probability. Furthermore, γ subunit palmitoylation had a dominant role over ß subunit palmitoylation in regulating ENaC. To determine which palmitoyltransferases (termed DHHCs) regulate the channel, mouse ENaCs were co-expressed in Xenopus oocytes with each of the 23 mouse DHHCs. ENaC activity was significantly increased by DHHCs 1, 2, 3, 7, and 14. ENaC activation by DHHCs was lost when γ subunit palmitoylation sites were mutated, whereas DHHCs 1, 2, and 14 still activated ENaC lacking ß subunit palmitoylation sites. ß subunit palmitoylation was increased by ENaC co-expression with DHHC 7. Both wild type ENaC and channels lacking ß and γ palmitoylation sites co-immunoprecipitated with the five activating DHHCs, suggesting that ENaC forms a complex with multiple DHHCs. RT-PCR revealed that transcripts for the five activating DHHCs were present in cultured mCCDcl1 cells, and DHHC 3 was expressed in aquaporin 2-positive principal cells of mouse aldosterone-sensitive distal nephron where ENaC is localized. Treatment of polarized mCCDcl1 cells with a general inhibitor of palmitoylation reduced ENaC-mediated Na+ currents within minutes. Our results indicate that specific DHHCs have a role in regulating ENaC.

Role of microRNAs in aldosterone signaling.

PURPOSE OF REVIEW: The review describes studies investigating the role of microRNAs in the signaling pathway of the mineralocorticoid hormone, aldosterone. RECENT FINDINGS: Emerging evidence indicates that aldosterone alters the expression of microRNAs in target tissues thereby modulating the expression of key regulatory proteins. SUMMARY: The mineralocorticoid hormone aldosterone is released by the adrenal glands in a homeostatic mechanism to regulate blood volume. The long-term renal action of aldosterone is to increase the retrieval of sodium from filtered plasma to restore blood pressure. Emerging evidence indicates aldosterone may alter noncoding RNAs (ncRNAs) to integrate this hormonal response in target tissue. Expression of the best characterized small ncRNAs, microRNAs, is regulated by aldosterone stimulation. MicroRNAs modulate protein expression at all steps in the renin-angiotensin-aldosterone-signaling (RAAS) system. In addition to acting as a rheostat to fine-tune protein levels in aldosterone-responsive cells, there is evidence that microRNAs down-regulate components of the signaling cascade as a feedback mechanism. The role of microRNAs is, therefore, as signal integrator, and damper in aldosterone signaling, which has implications in understating the RAAS system from both a physiological and pathophysiological perspective. Recent evidence for microRNA's role in RAAS signaling will be discussed.

A MicroRNA Cluster miR-23-24-27 Is Upregulated by Aldosterone in the Distal Kidney Nephron Where it Alters Sodium Transport.

The epithelial sodium channel (ENaC) is expressed in the epithelial cells of the distal convoluted tubules, connecting tubules, and cortical collecting duct (CCD) in the kidney nephron. Under the regulation of the steroid hormone aldosterone, ENaC is a major determinant of sodium (Na+ ) and water balance. The ability of aldosterone to regulate microRNAs (miRs) in the kidney has recently been realized, but the role of miRs in Na+ regulation has not been well established. Here we demonstrate that expression of a miR cluster mmu-miR-23-24-27, is upregulated in the CCD by aldosterone stimulation both in vitro and in vivo. Increasing the expression of these miRs increased Na+ transport in the absence of aldosterone stimulation. Potential miR targets were evaluated and miR-27a/b was verified to bind to the 3'-untranslated region of intersectin-2, a multi-domain protein expressed in the distal kidney nephron and involved in the regulation of membrane trafficking. Expression of Itsn2 mRNA and protein was decreased after aldosterone stimulation. Depletion of Itsn2 expression, mimicking aldosterone regulation, increased ENaC-mediated Na+ transport, while Itsn2 overexpression reduced ENaC's function. These findings reinforce a role for miRs in aldosterone regulation of Na+ transport, and implicate miR-27 in aldosterone's action via a novel target. J. Cell. Physiol. 232: 1306-1317, 2017. © 2016 Wiley Periodicals, Inc.

Reconstitution of the death-inducing signaling complex reveals a substrate switch that determines CD95-mediated death or survival.

The death-inducing signaling complex (DISC) is critical for initiation of death-receptor-mediated apoptosis; however, paradoxically, CD95 also signals for cell survival. Here, we reconstitute a functional DISC using only purified CD95, FADD, and procaspase-8 and unveil a two-step activation mechanism involving both dimerization and proteolytic cleavage of procaspase-8 that is obligatory for death-receptor-induced apoptosis. Initially, dimerization yields active procaspase-8 with a very restricted substrate repertoire, limited to itself or c-FLIP. Proteolytic cleavage is then required to fully activate caspase-8, thereby permitting DISC-mediated cleavage of the critical exogenous apoptotic substrates, caspase-3 and Bid. This switch in catalytic activity and substrate range is a key determinant of DISC signaling, as cellular expression of noncleavable procaspase-8 mutants, which undergo DISC-mediated oligomerization, but not cleavage, fails to initiate CD95-induced apoptosis. Thus, using the reconstituted DISC, we have delineated a crucial two-step activation mechanism whereby activated death receptor complexes can trigger death or survival.

BH3 profiling and a toolkit of BH3-mimetic drugs predict anti-apoptotic dependence of cancer cells.

BACKGROUND: Anti-apoptotic BCL-2 family members antagonise apoptosis by sequestering their pro-apoptotic counterparts. The balance between the different BCL-2 family members forms the basis of BH3 profiling, a peptide-based technique used to predict chemosensitivity of cancer cells. Recent identification of cell-permeable, selective inhibitors of BCL-2, BCL-XL and MCL-1, further facilitates the determination of the BCL-2 family dependency of cancer cells. METHODS: We use BH3 profiling in combination with cell death analyses using a chemical inhibitor toolkit to assess chemosensitivity of cancer cells. RESULTS: Both BH3 profiling and the inhibitor toolkit effectively predict chemosensitivity of cells addicted to a single anti-apoptotic protein but a combination of both techniques is more instructive when cell survival depends on more than one anti-apoptotic protein. CONCLUSIONS: The inhibitor toolkit provides a rapid, inexpensive and simple means to assess the chemosensitivity of tumour cells and in conjunction with BH3 profiling offers much potential in personalising cancer therapy.

MicroRNAs and the regulation of aldosterone signaling in the kidney.

The role of small noncoding RNAs, termed microRNAs (miRs), in development and disease has been recognized for many years. The number of miRs and regulated targets that reinforce a role for miRs in human disease and disease progression is ever-increasing. However, less is known about the involvement of miRs in steady-state, nondisease homeostatic pathways. In the kidney, much of the regulated ion transport is under the control of hormonal signaling. Evidence is emerging that miRs are involved in the hormonal regulation of kidney function and, particularly, in ion transport. In this short review, the production and intra- and extracellular signaling of miRs and the involvement of miRs in kidney disease are discussed. The discussion also focuses on the role of these small biological molecules in the homeostatic control of ion transport in the kidney. MiR regulation of and by corticosteroid hormones, in particular the mineralocorticoid hormone aldosterone, is considered. While information about the role of aldosterone-regulated miRs in the kidney is limited, an increase in the research in this area will undoubtedly highlight the involvement of miRs as central mediators of hormonal signaling in normal physiology.

Aldosterone regulates microRNAs in the cortical collecting duct to alter sodium transport.

A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells. Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport. Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein. A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3' untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.

Potentiation of BKCa channels by cystic fibrosis transmembrane conductance regulator correctors VX-445 and VX-121.

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, ultimately leading to diminished transepithelial anion secretion and mucociliary clearance. CFTR correctors are therapeutics that restore the folding/trafficking of mutated CFTR to the plasma membrane. The large-conductance calcium-activated potassium channel (BKCa, KCa1.1) is also critical for maintaining lung airway surface liquid (ASL) volume. Here, we show that the class 2 (C2) CFTR corrector VX-445 (elexacaftor) induces K+ secretion across WT and F508del CFTR primary human bronchial epithelial cells (HBEs), which was entirely inhibited by the BKCa antagonist paxilline. Similar results were observed with VX-121, a corrector under clinical evaluation. Whole-cell patch-clamp recordings verified that CFTR correctors potentiated BKCa activity from both primary HBEs and HEK cells stably expressing the α subunit (HEK-BK cells). Furthermore, excised patch-clamp recordings from HEK-BK cells verified direct action on the channel and demonstrated a significant increase in open probability. In mouse mesenteric artery, VX-445 induced a paxilline-sensitive vasorelaxation of preconstricted arteries. VX-445 also reduced firing frequency in primary rat hippocampal and cortical neurons. We raise the possibilities that C2 CFTR correctors gain additional clinical benefit by activation of BKCa in the lung yet may lead to adverse events through BKCa activation elsewhere.

An optimised patient-derived explant platform for breast cancer reflects clinical responses to chemotherapy and antibody-directed therapy.

Breast Cancer is the most common cancer among women globally. Despite significant improvements in overall survival, many tumours are refractory to therapy and so novel approaches are required to improve patient outcomes. We have evaluated patient-derived explants (PDEs) as a novel preclinical platform for breast cancer (BC) and implemented cutting-edge digital pathology and multi-immunofluorescent approaches for investigating biomarker changes in both tumour and stromal areas at endpoint. Short-term culture of intact fragments of BCs as PDEs retained an intact immune microenvironment, and tumour architecture was augmented by the inclusion of autologous serum in the culture media. Cell death/proliferation responses to FET chemotherapy in BC-PDEs correlated significantly with BC patient progression-free survival (p = 0.012 and p = 0.0041, respectively) and cell death responses to the HER2 antibody therapy trastuzumab correlated significantly with HER2 status (p = 0.018). These studies show that the PDE platform combined with digital pathology is a robust preclinical approach for informing clinical responses to chemotherapy and antibody-directed therapies in breast cancer. Furthermore, since BC-PDEs retain an intact tumour architecture over the short-term, they facilitate the preclinical testing of anti-cancer agents targeting the tumour microenvironment.

Activation of the epithelial sodium channel (ENaC) by the alkaline protease from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that significantly contributes to the mortality of patients with cystic fibrosis. Chronic infection by Pseudomonas induces sustained immune and inflammatory responses and damage to the airway. The ability of Pseudomonas to resist host defenses is aided, in part, by secreted proteases, which act as virulence factors in multiple modes of infection. Recent studies suggest that misregulation of protease activity in the cystic fibrosis lung may alter fluid secretion and pathogen clearance by proteolytic activation of the epithelial sodium channel (ENaC). To evaluate the possibility that proteolytic activation of ENaC may contribute to the virulence of Pseudomonas, primary human bronchial epithelial cells were exposed to P. aeruginosa and ENaC function was assessed by short circuit current measurements. Apical treatment with a strain known to express high levels of alkaline protease (AP) resulted in an increase in basal ENaC current and a loss of trypsin-inducible ENaC current, consistent with sustained activation of ENaC. To further characterize this AP-induced ENaC activation, AP was purified, and its folding, activity, and ability to activate ENaC were assessed. AP folding was efficient under pH and calcium conditions thought to exist in the airway surface liquid of normal and cystic fibrosis (CF) lungs. Short circuit measurements of ENaC in polarized monolayers indicated that AP activated ENaC in immortalized cell lines as well as post-transplant, primary human bronchial epithelial cells from both CF and non-CF patients. This activation was mapped to the γ-subunit of ENaC. Based on these data, patho-mechanisms associated with AP in the CF lung are proposed wherein secretion of AP leads to decreased airway surface liquid volume and a corresponding decrease in mucocilliary clearance of pulmonary pathogens.

Novel modeling of combinatorial miRNA targeting identifies SNP with potential role in bone density.

MicroRNAs (miRNAs) are post-transcriptional regulators that bind to their target mRNAs through base complementarity. Predicting miRNA targets is a challenging task and various studies showed that existing algorithms suffer from high number of false predictions and low to moderate overlap in their predictions. Until recently, very few algorithms considered the dynamic nature of the interactions, including the effect of less specific interactions, the miRNA expression level, and the effect of combinatorial miRNA binding. Addressing these issues can result in a more accurate miRNA:mRNA modeling with many applications, including efficient miRNA-related SNP evaluation. We present a novel thermodynamic model based on the Fermi-Dirac equation that incorporates miRNA expression in the prediction of target occupancy and we show that it improves the performance of two popular single miRNA target finders. Modeling combinatorial miRNA targeting is a natural extension of this model. Two other algorithms show improved prediction efficiency when combinatorial binding models were considered. ComiR (Combinatorial miRNA targeting), a novel algorithm we developed, incorporates the improved predictions of the four target finders into a single probabilistic score using ensemble learning. Combining target scores of multiple miRNAs using ComiR improves predictions over the naïve method for target combination. ComiR scoring scheme can be used for identification of SNPs affecting miRNA binding. As proof of principle, ComiR identified rs17737058 as disruptive to the miR-488-5p:NCOA1 interaction, which we confirmed in vitro. We also found rs17737058 to be significantly associated with decreased bone mineral density (BMD) in two independent cohorts indicating that the miR-488-5p/NCOA1 regulatory axis is likely critical in maintaining BMD in women. With increasing availability of comprehensive high-throughput datasets from patients ComiR is expected to become an essential tool for miRNA-related studies.

Rab11b regulates the trafficking and recycling of the epithelial sodium channel (ENaC).

Expression of the epithelial sodium channel (ENaC) at the apical membrane of cortical collecting duct (CCD) principal cells is modulated by regulated trafficking mediated by vesicle insertion and retrieval. Small GTPases are known to facilitate vesicle trafficking, recycling, and membrane fusion events; however, little is known about the specific Rab family members that modify ENaC surface density. Using a mouse CCD cell line that endogenously expresses ENaC (mpkCCD), the channel was localized to both Rab11a- and Rab11b-positive endosomes by immunoisolation and confocal fluorescent microscopy. Expression of a dominant negative (DN) form of Rab11a or Rab11b significantly reduced the basal and cAMP-stimulated ENaC-dependent sodium (Na(+)) transport. The greatest reduction in Na(+) transport was observed with the expression of DN-Rab11b. Furthermore, small interfering RNA-mediated knockdown of each Rab11 isoform demonstrated the requirement for Rab11b in ENaC surface expression. These data indicate that Rab11b, and to a lesser extent Rab11a, is involved in establishing the constitutive and cAMP-stimulated Na(+) transport in mpkCCD cells.

Regulation of the epithelial sodium channel (ENaC) by membrane trafficking.

The epithelial Na(+) channel (ENaC) is a major regulator of salt and water reabsorption in a number of epithelial tissues. Abnormalities in ENaC function have been directly linked to several human disease states including Liddle syndrome, psuedohypoaldosteronism, and cystic fibrosis and may be implicated in salt-sensitive hypertension. ENaC activity in epithelial cells is regulated both by open probability and channel number. This review focuses on the regulation of ENaC in the cells of the kidney cortical collecting duct by trafficking and recycling. The trafficking of ENaC is discussed in the broader context of epithelial cell vesicle trafficking. Well-characterized pathways and protein interactions elucidated using epithelial model cells are discussed, and the known overlap with ENaC regulation is highlighted. In following the life of ENaC in CCD epithelial cells the apical delivery, internalization, recycling, and destruction of the channel will be discussed. While a number of pathways presented still need to be linked to ENaC regulation and many details of the regulation of ENaC trafficking remain to be elucidated, knowledge of these mechanisms may provide further insights into ENaC activity in normal and disease states.

Concurrent up-regulation of BCL-XL and BCL2A1 induces approximately 1000-fold resistance to ABT-737 in chronic lymphocytic leukemia.

ABT-737 and its orally active analog, ABT-263, are rationally designed inhibitors of BCL2 and BCL-X(L). ABT-263 shows promising activity in early phase 1 clinical trials in B-cell malignancies, particularly chronic lymphocytic leukemia (CLL). In vitro, peripheral blood CLL cells are extremely sensitive to ABT-737 (EC(50) approximately 7 nM), with rapid induction of apoptosis in all 60 patients tested, independent of parameters associated with disease progression and chemotherapy resistance. In contrast to data from cell lines, ABT-737-induced apoptosis in CLL cells was largely MCL1-independent. Because CLL cells within lymph nodes are more resistant to apoptosis than those in peripheral blood, CLL cells were cultured on CD154-expressing fibroblasts in the presence of interleukin-4 (IL-4) to mimic the lymph node microenvironment. CLL cells thus cultured developed an approximately 1000-fold resistance to ABT-737 within 24 hours. Investigations of the underlying mechanism revealed that this resistance occurred upstream of mitochondrial perturbation and involved de novo synthesis of the antiapoptotic proteins BCL-X(L) and BCL2A1, which were responsible for resistance to low and high ABT-737 concentrations, respectively. Our data indicate that after therapy with ABT-737-related inhibitors, resistant CLL cells might develop in lymph nodes in vivo and that treatment strategies targeting multiple BCL2 antiapoptotic members simultaneously may have synergistic activity.