Kenneth R. Hallows

Essential Regulation of Cell Bioenergetics by Constitutive InsP3 Receptor Ca2+ Transfer to Mitochondria

Mechanisms that regulate cellular metabolism are a fundamental requirement of all cells. Most eukaryotic cells rely on aerobic mitochondrial metabolism to generate ATP. Nevertheless, regulation of mitochondrial activity is incompletely understood. Here we identified an unexpected and essential role for constitutive InsP(3)R-mediated Ca(2+) release in maintaining cellular bioenergetics. Macroautophagy provides eukaryotes with an adaptive response to nutrient deprivation that prolongs survival. Constitutive InsP(3)R Ca(2+) signaling is required for macroautophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to diminished mitochondrial Ca(2+) uptake. Mitochondrial uptake of InsP(3)R-released Ca(2+) is fundamentally required to provide optimal bioenergetics by providing sufficient reducing equivalents to support oxidative phosphorylation. Absence of this Ca(2+) transfer results in enhanced phosphorylation of pyruvate dehydrogenase and activation of AMPK, which activates prosurvival macroautophagy. Thus, constitutive InsP(3)R Ca(2+) release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration and maintenance of normal cell bioenergetics.

Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated Cl(-) channel that regulates other epithelial transport proteins by uncharacterized mechanisms. We employed a yeast two-hybrid screen using the COOH-terminal 70 residues of CFTR to identify proteins that might be involved in such interactions. The alpha1 (catalytic) subunit of AMP-activated protein kinase (AMPK) was identified as a dominant and novel interacting protein. The interaction is mediated by residues 1420-1457 in CFTR and by the COOH-terminal regulatory domain of alpha1-AMPK. Mutations of two protein trafficking motifs within the 38-amino acid region in CFTR each disrupted the interaction. GST-fusion protein pull-down assays in vitro and in transfected cells confirmed the CFTR-alpha1-AMPK interaction and also identified alpha2-AMPK as an interactor with CFTR. AMPK is coexpressed in CFTR-expressing cell lines and shares an apical distribution with CFTR in rat nasal epithelium. AMPK phosphorylated full-length CFTR in vitro, and AMPK coexpression with CFTR in Xenopus oocytes inhibited cAMP-activated CFTR whole-cell Cl(-) conductance by approximately 35-50%. Because AMPK is a metabolic sensor in cells and responds to changes in cellular ATP, regulation of CFTR by AMPK may be important in inhibiting CFTR under conditions of metabolic stress, thereby linking transepithelial transport to cell metabolic state.

Mechanisms of ENaC Regulation and Clinical Implications

The epithelial Na+ channel (ENaC) transports Na+ across tight epithelia, including the distal nephron. Different paradigms of ENaC regulation include extrinsic and intrinsic factors that affect the expression, single-channel properties, and intracellular trafficking of the channel. In particular, recent discoveries highlight new findings regarding proteolytic processing, ubiquitination, and recycling of the channel. Understanding the regulation of this channel is critical to the understanding of various clinical phenomena, including normal physiology and several diseases of kidney and lung epithelia, such as blood pressure (BP) control, edema, and airway fluid clearance. Significant progress has been achieved in this active field of research. Although ENaC is classically thought to be a mediator of BP and volume status through Na+ reabsorption in the distal nephron, several studies in animal models highlight important roles for ENaC in lung pathophysiology, including in cystic fibrosis. The purpose of this review is to highlight the various modes and mechanisms of ENaC regulation, with a focus on more recent studies and their clinical implications.

Activating AMP-activated protein kinase (AMPK) slows renal cystogenesis

Renal cyst development and expansion in autosomal dominant polycystic kidney disease (ADPKD) involves both fluid secretion and abnormal proliferation of cyst-lining epithelial cells. The chloride channel of the cystic fibrosis transmembrane conductance regulator (CFTR) participates in secretion of cyst fluid, and the mammalian target of rapamycin (mTOR) pathway may drive proliferation of cyst epithelial cells. CFTR and mTOR are both negatively regulated by AMP-activated protein kinase (AMPK). Metformin, a drug in wide clinical use, is a pharmacological activator of AMPK. We find that metformin stimulates AMPK, resulting in inhibition of both CFTR and the mTOR pathways. Metformin induces significant arrest of cystic growth in both in vitro and ex vivo models of renal cystogenesis. In addition, metformin administration produces a significant decrease in the cystic index in two mouse models of ADPKD. Our results suggest a possible role for AMPK activation in slowing renal cystogenesis as well as the potential for therapeutic application of metformin in the context of ADPKD.

AMP-activated kinase inhibits the epithelial Na- channel through functional regulation of the ubiquitin ligase Nedd4-2

We recently found that the metabolic sensor AMP-activated kinase (AMPK) inhibits the epithelial Na+ channel (ENaC) through decreased plasma membrane ENaC expression, an effect requiring the presence of a binding motif in the cytoplasmic tail of the β-ENaC subunit for the ubiquitin ligase Nedd4-2. To further examine the role of Nedd4-2 in the regulation of ENaC by AMPK, we studied the effects of AMPK activation on ENaC currents in Xenopus oocytes co-expressing ENaC and wild-type (WT) or mutant forms of Nedd4-2. ENaC inhibition by AMPK was preserved in oocytes expressing WT Nedd4-2 but blocked in oocytes expressing either a dominant-negative (DN) or constitutively active (CA) Nedd4-2 mutant, suggesting that AMPK-dependent modulation of Nedd4-2 function is involved. Similar experiments utilizing WT or mutant forms of the serum- and glucocorticoid-regulated kinase (SGK1), modulators of protein kinase A (PKA), or extracellular-regulated kinase (ERK) did not affect ENaC inhibition by AMPK, suggesting that these pathways known to modulate the Nedd4-2-ENaC interaction are not responsible. AMPK-dependent phosphorylation of Nedd4-2 expressed in HEK-293 cells occurred both in vitro and in vivo, suggesting a potential mechanism for modulation of Nedd4-2 and thus cellular ENaC activity. Moreover, cellular AMPK activation significantly enhanced the interaction of the β-ENaC subunit with Nedd4-2, as measured by co-immunoprecipitation assays in HEK-293 cells. In summary, these results suggest a novel mechanism for ENaC regulation in which AMPK promotes ENaC-Nedd4-2 interaction, thereby inhibiting ENaC by increasing Nedd4-2-dependent ENaC retrieval from the plasma membrane. AMPK-dependent ENaC inhibition may limit cellular Na+ loading under conditions of metabolic stress when AMPK becomes activated.

Role of the energy sensor AMP-activated protein kinase in renal physiology and disease

The ultrasensitive energy sensor AMP-activated protein kinase (AMPK) orchestrates the regulation of energy-generating and energy-consuming pathways. AMPK is highly expressed in the kidney where it is reported to be involved in a variety of physiological and pathological processes including ion transport, podocyte function, and diabetic renal hypertrophy. Sodium transport is the major energy-consuming process in the kidney, and AMPK has been proposed to contribute to the coupling of ion transport with cellular energy metabolism. Specifically, AMPK has been identified as a regulator of several ion transporters of significance in renal physiology, including the cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial sodium channel (ENaC), the Na(+)-K(+)-2Cl(-) cotransporter (NKCC), and the vacuolar H(+)-ATPase (V-ATPase). Identified regulators of AMPK in the kidney include dietary salt, diabetes, adiponectin, and ischemia. Activation of AMPK in response to adiponectin is described in podocytes, where it reduces albuminuria, and in tubular cells, where it reduces glycogen accumulation. Reduced AMPK activity in the diabetic kidney is associated with renal accumulation of triglyceride and glycogen and the pathogenesis of diabetic renal hypertrophy. Acute renal ischemia causes a rapid and powerful activation of AMPK, but the functional significance of this observation remains unclear. Despite the recent advances, there remain significant gaps in the present understanding of both the upstream regulating pathways and the downstream substrates for AMPK in the kidney. A more complete understanding of the AMPK pathway in the kidney offers potential for improved therapies for several renal diseases including diabetic nephropathy, polycystic kidney disease, and ischemia-reperfusion injury.

The Urine/Plasma Electrolyte Ratio: A Predictive Guide to Water Restriction

Patients with hypotonic hyponatremia are encountered commonly in the general practice of medicine. Nearly all strategies for the management of subacute or chronic hyponatremia call for some amount of water restriction. The considerations for such a prescription have not been addressed in the literature. We describe therefore a simple approach grounded in the physiology of electrolyte-free water clearance that can be used at the bedside.

Noncanonical control of vasopressin receptor type 2 signaling by retromer and arrestin

Background: It remains unclear why vasopressin induces greater antidiuresis through V2R than does oxytocin. Results: Vasopressin sustains cAMP signaling during V2R internalization, a process promoted by β-arrestins, and is halted by the retromer complex. Conclusion: This new noncanonical model of GPCR signaling differentiates the actions of vasopressin and oxytocin. Significance: This emerging model may explain the physiological bias between ligands. The vasopressin type 2 receptor (V2R) is a critical G protein-coupled receptor (GPCR) for vertebrate physiology, including the balance of water and sodium ions. It is unclear how its two native hormones, vasopressin (VP) and oxytocin (OT), both stimulate the same cAMP/PKA pathway yet produce divergent antinatriuretic and antidiuretic effects that are either strong (VP) or weak (OT). Here, we present a new mechanism that differentiates the action of VP and OT on V2R signaling. We found that vasopressin, as opposed to OT, continued to generate cAMP and promote PKA activation for prolonged periods after ligand washout and receptor internalization in endosomes. Contrary to the classical model of arrestin-mediated GPCR desensitization, arrestins bind the VP-V2R complex yet extend rather than shorten the generation of cAMP. Signaling is instead turned off by the endosomal retromer complex. We propose that this mechanism explains how VP sustains water and Na+ transport in renal collecting duct cells. Together with recent work on the parathyroid hormone receptor, these data support the existence of a novel “noncanonical” regulatory pathway for GPCR activation and response termination, via the sequential action of β-arrestin and the retromer complex.

Extracorporeal Therapy for Dabigatran Removal in the Treatment of Acute Bleeding: A Single Center Experience

BACKGROUND AND OBJECTIVES Dabigatran is an oral direct thrombin inhibitor that is Food and Drug Administration-approved for prevention of stroke in patients with atrial fibrillation. No antidote is available for reversal of dabigatrans anticoagulant effect. Despite limited clinical data, hemodialysis has been suggested as a strategy to remove dabigatran during acute bleeding. This work presents five cases, in which extracorporeal therapy was performed for dabigatran removal in acutely bleeding patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS The series is comprised of five consecutive cases of patients receiving dabigatran 150 mg per os two times daily who were admitted with life-threatening bleeding between March of 2012 and January of 2013. Dabigatran plasma concentrations ranged from 149 to 1200 ng/ml. Treatment included administration of blood products to all patients and then, high-flux intermittent hemodialysis alone or followed by continuous renal replacement therapy. RESULTS Dabigatran concentrations decreased by 52%-77% during intermittent hemodialysis but rebounded up to 87% within 2 hours after completion of dialysis. Initiation of continuous renal replacement therapy after intermittent hemodialysis attenuated the rebound effect in one patient and contributed to a reduction in dabigatran concentrations of 81% over 30 hours. CONCLUSIONS Extracorporeal therapy lowered dabigatran concentrations, suggesting that it removed the drug and may effectively accelerate total clearance, especially in patients with impaired kidney function. The use of prolonged intermittent hemodialysis or intermittent hemodialysis followed by continuous renal replacement therapy is recommended for the management of life-threatening bleeding in patients receiving dabigatran. The advantage of extracorporeal therapy should be weighed against the risk of bleeding with catheter insertion.

Vacuolar H+-ATPase apical accumulation in kidney intercalated cells is regulated by PKA and AMP-activated protein kinase

The vacuolar H(+)-ATPase (V-ATPase) in type A kidney intercalated cells is a major contributor to acid excretion in the collecting duct. The mechanisms of V-ATPase-trafficking regulation in kidney intercalated cells have not been well-characterized. In developmentally related epididymal clear cells, we showed previously that PKA, acting downstream of soluble adenylyl cyclase (sAC), induces V-ATPase apical membrane accumulation. These PKA-mediated effects were inhibited by activators of the metabolic sensor AMP-activated kinase (AMPK) in clear cells. Here, we examined the regulation of V-ATPase subcellular localization in intercalated cells by PKA and AMPK in rat kidney tissue slices ex vivo. Immunofluorescence labeling of kidney slices revealed that the PKA activator N(6)-monobutyryl cAMP (6-MB-cAMP) induced V-ATPase apical membrane accumulation in collecting duct intercalated cells, whereas the V-ATPase had a more cytosolic distribution when incubated in Ringer buffer alone for 30 min. V-ATPase accumulated at the apical membrane in intercalated cells in kidney slices incubated in Ringer buffer for 75 min, an effect that was prevented by treatment with PKA inhibitor (mPKI). The V-ATPase distribution was cytosolic in intercalated cells treated with the carbonic anhydrase inhibitor acetazolamide or the sAC inhibitor KH7, effects that were overridden by 6-MB-cAMP. Preincubation of kidney slices with an AMPK activator blocked V-ATPase apical membrane accumulation induced by 6-MB-cAMP, suggesting that AMPK antagonizes cAMP/PKA effects on V-ATPase distribution. Taken together, our results suggest that in intercalated cells V-ATPase subcellular localization and therefore its activity may be coupled to acid-base status via PKA, and metabolic status via AMPK.