Search

Ischemia-reperfusion injury (IRI) due to hypotension is a common cause of human acute kidney injury (AKI). Hypoxia-inducible transcription factors (HIFs) orchestrate a protective response in renal endothelial and epithelial cells in AKI models. As human mucin 1 (MUC1) is induced by hypoxia and enhances HIF-1 activity in cultured epithelial cells, we asked whether mouse mucin 1 (Muc1) regulates HIF-1 activity in kidney tissue during IRI. Whereas Muc1 was localized on the apical surface of the thick ascending limb, distal convoluted tubule, and collecting duct in the kidneys of sham-treated mice, Muc1 appeared in the cytoplasm and nucleus of all tubular epithelia during IRI. Muc1 was induced during IRI, and Muc1 transcripts and protein were also present in recovering proximal tubule cells. Kidney damage was worse and recovery was blocked during IRI in Muc1 knockout mice compared with congenic control mice. Muc1 knockout mice had reduced levels of HIF-1a, reduced or aberrant induction of HIF-1 target genes involved in the shift of glucose metabolism to glycolysis, and prolonged activation of AMP-activated protein kinase, indicating metabolic stress. Muc1 clearly plays a significant role in enhancing the HIF protective pathway during ischemic insult and recovery in kidney epithelia, providing a new target for developing therapies to treat AKI. Moreover, our data support a role specifically for HIF-1 in epithelial protection of the kidney during IRI as Muc1 is present only in tubule epithelial cells. [ABSTRACT FROM AUTHOR]

Proximal tubule epithelial cells have a highly sophisticated endocytic machinery to retrieve the albumin in the glomerular filtrate. The megalincubilin complex and the endocytic adaptor disabled-2 (Dab2) play a pivotal role in albumin endocytosis. We previously demonstrated that protein kinase B (Akt) regulates albumin endocytosis in the proximal tubule through an interaction with Dab2. Here, we examined the nature of Akt-Dab2 interaction. The pleckstrin homology (PH) and catalytic domains (CD) of Akt interacted with the proline-rich domain (PRD) of Dab2 based on yeast-two hybrid (Y2H) experiments. Pulldown experiments utilizing the truncated constructs of Dab2 demonstrated that the initial 11 amino acids of Dab2-PRD were sufficient to mediate the interaction between Akt and Dab2. Endocytosis experiments utilizing Akt1- and Akt2-silencing RNA revealed that both Akt1 and Akt2 mediate albumin endocytosis in proximal tubule epithelial cells; therefore, Akt1 and Akt2 may play a compensatory role in albumin endocytosis. Furthermore, both Akt isoforms phosphorylated Dab2 at Ser residues 448 and 449. Ser-to-Ala mutations of these Dab2 residues inhibited albumin endocytosis and resulted in a shift in location of Dab2 from the peripheral to the perinuclear area, suggesting the physiological relevance of these phosphorylation sites in albumin endocytosis. We conclude that both Akt1 and Akt2 are involved in albumin endocytosis, and phosphorylation of Dab2 by Akt induces albumin endocytosis in proximal tubule epithelial cells. Further delineation of how Akt affects expression/phosphorylation of endocytic adaptors and receptors will enhance our understanding of the molecular network triggered by albumin overload in the proximal tubule. [ABSTRACT FROM AUTHOR]

The vacuolar H+-ATPase (V-ATPase) in intercalated cells contributes to luminal acidification in the kidney collecting duct and nonvolatile acid excretion. We previously showed that the A subunit in the cytoplasmic V1 sector of the V-ATPase (ATP6V1A) is phosphorylated by the metabolic sensor AMP-activated protein kinase (AMPK) in vitro and in kidney cells. Here, we demonstrate that treatment of rabbit isolated, perfused collecting ducts with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) inhibited V-ATPase-dependent H+ secretion from intercalated cells after an acid load. We have identified by mass spectrometry that Ser-384 is a major AMPK phosphorylation site in the V-ATPase A subunit, a result confirmed by comparing AMPK-dependent phosphate labeling of wild-type A-subunit (WT-A) with that of a Ser-384- to-Ala A subunit mutant (S384A-A) in vitro and in intact HEK-293 cells. Compared with WT-A-expressing HEK-293 cells, S384A-Aexpressing cells exhibited greater steady-state acidification of HCO3- containing media. Moreover, AICAR treatment of clone C rabbit intercalated cells expressing the WT-A subunit reduced V-ATPasedependent extracellular acidification, an effect that was blocked in cells expressing the phosphorylation-deficient S384A-A mutant. Finally, expression of the S384A-A mutant prevented cytoplasmic redistribution of the V-ATPase by AICAR in clone C cells. In summary, direct phosphorylation of the A subunit at Ser-384 by AMPK represents a novel regulatory mechanism of the V-ATPase in kidney intercalated cells. Regulation of the V-ATPase by AMPK may couple V-ATPase activity to cellular metabolic status with potential relevance to ischemic injury in the kidney and other tissues. [ABSTRACT FROM AUTHOR]

Salt reabsorption is the major energy-requiring process in the kidney, and AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism. Mice with targeted deletion of the ß1-subunit of AMPK (AMPK-ß1-/- mice) had significantly increased urinary Na+ excretion on a normal salt diet. This was associated with reduced expression of the ß-subunit of the epithelial Na+ channel (ENaC) and increased subapical tubular expression of kidney-specific Na+-K+-2Cl- cotransporter 2 (NKCC2) in the medullary thick ascending limb of Henle. AMPK-ß1-/- mice fed a salt-deficient diet were able to conserve Na+, but renin secretion increased 180% compared with control mice. Cyclooxygenase-2 mRNA also increased in the kidney cortex, indicating greater signaling through the macula densa tubular salt-sensing pathway. To determine whether the increase in renin secretion was due to a change in regulation of fatty acid metabolism by AMPK, mice with a mutation of the inhibitory AMPK phosphosite in acetyl-CoA carboxylase 1 [ACC1-knockin (KI)S79A mice] were examined. ACC1-KIS79A mice on a normal salt diet had no increase in salt loss or renin secretion, and expression of NKCC2, Na+-Cl- cotransporter, and ENaC-ß were similar to those in control mice. When mice were placed on a salt-deficient diet, however, renin secretion and cortical expression of cyclooxygenase-2 mRNA increased significantly in ACC1-KIS79A mice compared with control mice. In summary, our data suggest that renin synthesis and secretion are regulated by AMPK and coupled to metabolism by phosphorylation of ACC1. [ABSTRACT FROM AUTHOR]

Galectins (Gal) are β-galactoside-binding proteins that function in epithelial development and homeostasis. An overlapping role for Gal-3 and Gal-7 in wound repair was reported in stratified epithelia. Although Gal-7 was thought absent in simple epithelia, it was reported in a proteomic analysis of cilia isolated from cultured human airway, and we recently identified Gal-7 transcripts in Madin-Darby canine kidney (MDCK) cells (Poland PA, Rondanino C, Kinlough CL, Heimburg-Molinaro J, Arthur CM, Stowell SR, Smith DF, Hughey RP. J Biol Chem 286: 6780-6790, 2011). We now report that Gal-7 is localized exclusively on the primary cilium of MDCK, LLC-PK1 (pig kidney), and mpkCCDc14 (mouse kidney) cells as well as on cilia in the rat renal proximal tubule. Gal-7 is also present on most cilia of multiciliated cells in human airway epithelia primary cultures. Interestingly, exogenous glutathione S-transferase (GST)-Gal-7 bound the MDCK apical plasma membrane as well as the cilium, while the lectin Ulex europeaus agglutinin, with glycan preferences similar to Gal-7, bound the basolateral plasma membrane as well as the cilium. In pull-down assays, β1-integrin isolated from either the basolateral or apical/cilia membranes of MDCK cells was similarly bound by GST-Gal-7. Selective localization of Gal-7 to cilia despite the presence of binding sites on all cell surfaces suggests that intracellular Gal-7 is specifically delivered to cilia rather than simply binding to surface glycoconjugates after generalized secretion. Moreover, depletion of Gal-7 using tetracycline-induced short-hairpin RNA in mpkCCDc14 cells significantly reduced cilia length and slowed wound healing in a scratch assay. We conclude that Gal-7 is selectively targeted to cilia and plays a key role in surface stabilization of glycoconjugates responsible for integrating cilia function with epithelial repair. [ABSTRACT FROM AUTHOR]

The E3 ubiquitin ligase Nedd4-2 regulates several ion transport proteins, including the epithelial Na+ channel (ENaC). Nedd4-2 decreases apical membrane expression and activity of ENaC. Although it is subject to tight hormonal control, the mechanistic basis of Nedd4-2 regulation remains poorly understood. To characterize regulatory inputs to Nedd4-2 function, we screened for novel sites of Nedd4-2 phosphorylation using tandem mass spectrometry. Three of seven identified Xenopus Nedd4-2 Ser/Thr phosphorylation sites corresponded to previously identified target sites for SGK 1, whereas four were novel, including Ser-293, which matched the consensus for a MAPK target sequence. Further in vitro and in vivo phosphorylation experiments revealed that Nedd4-2 serves as a target of JNK1, but not of p38 MAPK or ERK1/2. Additional rounds of tandem mass spectrometry identified two other phosphorylated residues within Nedd4-2, including Thr-899, which is present within the catalytic domain. Nedd4-2 with mutations at these sites had markedly inhibited JNKl-dependent phosphorylation, virtually no ENaC inhibitory activity, and significantly reduced ubiquitin ligase activity. These data identify phosphorylatable residues that activate Nedd4-2 and may work together with residues targeted by inhibitory kinases (e.g. SGK1 and protein kinase A) to govern Nedd4-2 regulation of epithelial ion transport. [ABSTRACT FROM AUTHOR]

The metabolic sensor AMP-activated protein kinase (AMPK) regulates several transport proteins, potentially coupling transport activity to cellular stress and energy levels. The creatine transporter (CRT; SLC6A8) mediates creatine uptake into several cell types, including kidney epithelial cells, where it has been proposed that CRT is important for reclamation of filtered creatine, a process critical for total body creatine homeostasis. Creatine and phosphocreatine provide an intracellular, high-energy phosphate-buffering system essential for maintaining ATP supply in tissues with high energy demands. To test our hypothesis that CRT is regulated by AMPK in the kidney. we examined CRT and AMPK distribution in the kidney and the regulation of CRT by AMPK in cells. By immunofluorescence staining, we detected CRT at the apical pole in a polarized mouse S3 proximal tubule cell line and in native rat kidney proximal tubules, a distribution overlapping with AMPK. Two-electrode voltage-clamp (TEV) measurements of Na+-dependent creatine uptake into CRT-expressing Xenopus laevis oocytes demonstrated that AMPK inhibited CRT via a reduction in its Michaelis-Menten Vmax parameter. [14C]creatine uptake and apical surface biotinylation measurements in polarized S3 cells demonstrated parallel reductions in creatine influx and CRT apical membrane expression after AMPK activation with the AMP-mimetic compound 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside. In oocyte TEV experiments, rapamycin and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl 5'-monophosphate (ZMP) inhibited CRT currents, but there was no additive inhibition of CRT by ZMP, suggesting that AMPK may inhibit CRT indirectly via the mammalian target of rapamycin pathway. We conclude that AMPK inhibits apical membrane CRT expression in kidney proximal tubule cells, which could be important in reducing cellular energy expenditure and unnecessary creatine reabsorption under conditions of local and whole body metabolic stress. [ABSTRACT FROM AUTHOR]

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 N6-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. [ABSTRACT FROM AUTHOR]

Acidic luminal pH and low [HCO-3] maintain sperm quiescent during maturation in the epididymis. The vacuolar H+-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HCO-3, induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and non- human primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer compared with that of controls perfused without drug. Moreover, preperfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by N6-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [32P]orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNA interference- mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knock-down. In summary, our findings suggest that AMPK inhibits PKA- mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK. [ABSTRACT FROM AUTHOR]

Reduced kidney AMPK activity is associated with nutrient stress–induced chronic kidney disease (CKD) in male mice. In contrast, female mice resist nutrient stress–induced CKD. The role of kidney AMPK in sex-related organ protection against nutrient stress and metabolite changes was evaluated in diabetic kidney tubule–specific AMPKγ2KO (KTAMPKγ2ΚΟ) male and female mice. In wild-type (WT) males, diabetes increased albuminuria, urinary kidney injury molecule-1, hypertension, kidney p70S6K phosphorylation, and kidney matrix accumulation; these features were not exacerbated with KTAMPKγ2ΚΟ. Whereas WT females had protection against diabetes-induced kidney injury, KTAMPKγ2ΚΟ led to loss of female protection against kidney disease. The hormone 17β-estradiol ameliorated high glucose–induced AMPK inactivation, p70S6K phosphorylation, and matrix protein accumulation in kidney tubule cells. The mechanism for female protection against diabetes-induced kidney injury is likely via an estrogen-AMPK pathway, as inhibition of AMPK led to loss of estrogen protection to glucose-induced mTORC1 activation and matrix production. RNA sequencing and metabolomic analysis identified a decrease in the degradation pathway of phenylalanine and tyrosine resulting in increased urinary phenylalanine and tyrosine levels in females. The metabolite levels correlated with loss of female protection. The findings provide new insights to explain evolutionary advantages to females during states of nutrient challenges. Article Highlights: The basis for protection against kidney disease injury associated with obesity and diabetes in females is not known. In the present studies, genetic deletion of kidney tubule–specific gamma subunit of AMPK led to loss of female protection against diabetic kidney injury along with dysregulation of phenylalanine and tyrosine degradation. These new data indicate that AMPK in the kidney is critical for diabetic kidney disease. Importantly, differential regulation of kidney AMPK contributes to sex disparities. Our results provide a previously unrecognized mechanism for the development of precision treatments in sex dependence. [ABSTRACT FROM AUTHOR]

Nanoparticle drug delivery has many advantages over small molecule therapeutics, including reducing off-target side effects and increasing drug potency. However, many nanoparticles are administered parenterally, which is challenging for chronic diseases such as polycystic kidney disease (PKD), the most common hereditary disease worldwide in which patients need continuous treatment over decades. To address this clinical need, we present the development of nanoparticles synthesized from chitosan, a widely available polymer chosen for its ability to improve oral bioavailability. Specifically, we optimized the synthesis parameters of chitosan nanoparticles and demonstrate mucoadhesion and permeation across an intestinal barrier model in vitro. Furthermore, when administered orally to mice, ex vivo imaging of rhodamine-loaded chitosan nanoparticles showed significantly higher accumulation in the intestines compared to the free model drug, as well as 1.3 times higher serum area under the curve (AUC), demonstrating controlled release and improved serum delivery over 24 h. To test its utility for chronic diseases such as PKD, we loaded the candidate PKD drug, metformin, into chitosan nanoparticles, and upon oral administration to a PKD murine model (Pkd1fl/fl;Pax8-rtTA;Tet-O cre), a lower cyst burden was observed compared to free metformin, and was well tolerated upon repeated dosages. Blood urea nitrogen (BUN) and creatinine levels were similar to untreated mice, demonstrating kidney and biocompatibility health. Our study builds upon previous chitosan-based drug delivery approaches, and demonstrates a novel, oral nanoformulation for PKD. Unlabelled Image [ABSTRACT FROM AUTHOR]

Our previous work has established that the metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na+ channel (ENaC) by promoting its binding to neural precursor cell-expressed, developmentally down-regulated 4-2, E3 ubiquitin protein ligase (Nedd4-2). Here, using MS analysis and in vitro phosphorylation, we show that AMPK phosphorylates Nedd4-2 at the Ser-444 (Xenopus Nedd4-2) site critical for Nedd4-2 stability. We further demonstrate that the Pak-interacting exchange factor β1Pix is required for AMPK-mediated inhibition of ENaC-dependent currents in both CHO and murine kidney cortical collecting duct (CCD) cells. Short hairpin RNA-mediated knockdown of β1Pix expression in CCD cells attenuated the inhibitory effect of AMPK activators on ENaC currents. Moreover, overexpression of a β1Pix dimerization-deficient mutant unable to bind 14-3-3 proteins (Δ602-611) increased ENaC currents in CCD cells, whereas overexpression of WT β1Pix had the opposite effect. Using additional immunoblotting and co-immunoprecipitation experiments, we found that treatment with AMPK activators promoted the binding of β1Pix to 14-3-3 proteins in CCD cells. However, the association between Nedd4-2 and 14-3-3 proteins was not consistently affected by AMPK activation, β1Pix knockdown, or overexpression of WT β1Pix or the β1Pix-Δ602-611 mutant. Moreover, we found that β1Pix is important for phosphorylation of the aforementioned Nedd4-2 site critical for its stability. Overall, these findings elucidate novel molecular mechanisms by which AMPK regulates ENaC. Specifically, they indicate that AMPK promotes the assembly of β1Pix, 14-3-3 proteins, and Nedd4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and its degradation. [ABSTRACT FROM AUTHOR]

The thiazide-sensitive NaCl cotransporter (NCC) is important for renal salt handling and blood-pressure homeostasis. The canonical NCC-activating pathway consists of With-No-Lysine (WNK) kinases and their downstream effector kinases SPAK and OSR1, which phosphorylate NCC directly. The upstream mechanisms that connect physiological stimuli to this system remain obscure. Here, we have shown that aldosterone activates SPAK/OSR1 via WNK1. We identified 2 alternatively spliced exons embedded within a proline-rich region of WNK1 that contain PY motifs, which bind the E3 ubiquitin ligase NEDD4-2. PY motif-containing WNK1 isoforms were expressed in human kidney, and these isoforms were efficiently degraded by the ubiquitin proteasome system, an effect reversed by the aldosterone-induced kinase SGK1. In gene-edited cells, WNK1 deficiency negated regulatory effects of NEDD4-2 and SGK1 on NCC, suggesting that WNK1 mediates aldosterone-dependent activity of the WNK/SPAK/OSR1 pathway. Aldosterone infusion increased proline-rich WNK1 isoform abundance in WT mice but did not alter WNK1 abundance in hypertensive Nedd4-2 KO mice, which exhibit high baseline WNK1 and SPAK/ OSR1 activity toward NCC. Conversely, hypertensive Sg/d KO mice exhibited low WNK1 expression and activity. Together, our findings indicate that the proline-rich exons are modular cassettes that convert WNK1 into a NEDD4-2 substrate, thereby linking aldosterone and other NEDD4-2-suppressing antinatriuretic hormones to NCC phosphorylation status. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The vacuolar H+-ATPase (V-ATPase) is a major contributor to luminal acidification in epithelia of Wolffian duct origin. In both kidney-intercalated cells and epididymal clear cells, cAMP induces V-ATPase apical membrane accumulation, which is linked to proton secretion. We have shown previously that the A subunit in the cytoplasmic V1 sector of the V-ATPase is phosphorylated by protein kinase A (PKA). Here we have identified by mass spectrometry and mutagenesis that Ser-175 is the major PKA phosphorylation site in the A subunit. Overexpression in HEK-293T cells of either a wild-type (WT) or phosphomimic Ser-175 to Asp (S175D) A subunit mutant caused increased acidification of HCO3-containing culture medium compared with cells expressing vector alone or a PKA phosphorylationdeficient Ser-175 to Ala (S175A) mutant. Moreover, localization of the S175A A subunit mutant expressed in HEK-293T cells was more diffusely cytosolic than that of WT or S175D A subunit. Acute V-ATPase-mediated, bafilomycin-sensitive H+ secretion was up-regulated by a specific PKA activator in HEK-293T cells expressing WT A subunit in HCO-3free buffer. In cells expressing the S175D mutant, V-ATPase activity at the membrane was constitutively up-regulated and unresponsive to PKA activators, whereas cells expressing the S175A mutant had decreased V-ATPase activity that was unresponsive to PKA activation. Finally, Ser-175 was necessary for PKA-stimulated apical accumulation of the V-ATPase in a polarized rabbit cell line of collecting duct A-type intercalated cell characteristics (Clone C). In summary, these results indicate a novel mechanism for the regulation of V-ATPase localization and activity in kidney cells via direct PKA-dependent phosphorylation of the A subunit at Ser-175. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here, manipulation of p38 and YAP activity allowed for long-term clonal expansion of primary mouse and human NPCs and induced NPCs (iNPCs) from human pluripotent stem cells (hPSCs). Molecular analyses demonstrated that cultured iNPCs closely resemble primary human NPCs. iNPCs generated nephron organoids with minimal off-target cell types and enhanced maturation of podocytes relative to published human kidney organoid protocols. Surprisingly, the NPC culture medium uncovered plasticity in human podocyte programs, enabling podocyte reprogramming to an NPC-like state. Scalability and ease of genome editing facilitated genome-wide CRISPR screening in NPC culture, uncovering genes associated with kidney development and disease. Further, NPC-directed modeling of autosomal-dominant polycystic kidney disease (ADPKD) identified a small-molecule inhibitor of cystogenesis. These findings highlight a broad application for the reported iNPC platform in the study of kidney development, disease, plasticity, and regeneration., Competing Interests: Declaration of interests A.P.M. is a scientific advisor or consultant for Novartis, eGENESIS, Trestle Biotherapeutics, GentiBio, and IVIVA Medical. Zhongwei Li, B.H., Z.Z., A.P.M., K.R.H., and N.M.P.-S. have applied for intellectual property protection on the technologies discussed here., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Autosomal dominant polycystic kidney disease (ADPKD) is a complex disorder characterized by uncontrolled renal cyst growth, leading to kidney function decline. The multifaceted nature of ADPKD suggests that single-pathway interventions using individual small molecule drugs may not be optimally effective. As such, a strategy encompassing combination therapy that addresses multiple ADPKD-associated signaling pathways could offer synergistic therapeutic results. However, severe off-targeting side effects of small molecule drugs pose a major hurdle to their clinical transition. To address this, we identified four drug candidates from ADPKD clinical trials, bardoxolone methyl (Bar), octreotide (Oct), salsalate (Sal), and pravastatin (Pra), and incorporated them into peptide amphiphile micelles containing the RGD peptide (GRGDSP), which binds to the basolateral surface of renal tubules via integrin receptors on the extracellular matrix. We hypothesized that encapsulating drug combinations into RGD micelles would enable targeting to the basolateral side of renal tubules, which is the site of disease, via renal secretion, leading to superior therapeutic benefits compared to free drugs. To test this, we first evaluated the synergistic effect of drug combinations using the 20% inhibitory concentration for each drug (IC 20 ) on renal proximal tubule cells derived from Pkd1 flox/- :TSLargeT mice. Next, we synthesized and characterized the RGD micelles encapsulated with drug combinations and measured their in vitro therapeutic effects via a 3D PKD growth model. Upon both IV and IP injections in vivo , RGD micelles showed a significantly higher accumulation in the kidneys compared to NT micelles, and the renal access of RGD micelles was significantly reduced after the inhibition of renal secretion. Specifically, both Bar+Oct and Bar+Sal in the RGD micelle treatment showed enhanced therapeutic efficacy in ADPKD mice ( Pkd1 fl/fl ;Pax8-rtTA;Tet-O-Cre ) with a significantly lower KW/BW ratio and cyst index as compared to PBS and free drug-treated controls, while other combinations did not show a significant difference. Hence, we demonstrate that renal targeting through basolateral targeting micelles enhances the therapeutic potential of combination therapy in genetic kidney disease.

Chronic and genetic kidney diseases such as autosomal dominant polycystic kidney disease (ADPKD) have few therapeutic options, and clinical trials testing small molecule drugs have been unfavorable due to low kidney bioavailability and adverse side effects. Although nanoparticles can be designed to deliver drugs directly to the diseased site, there are no kidney-targeted nanomedicines clinically available, and most FDA-approved nanoparticles are administered intravenously which is not ideal for chronic diseases. To meet these challenges of chronic diseases, we developed a biomaterials-based strategy using chitosan particles (CP) for oral delivery of therapeutic, kidney-targeting peptide amphiphile micelles (KMs). We hypothesized that encapsuling KMs into CP would enhance the bioavailability of KMs upon oral administration given the high stability of chitosan in acidic conditions and mucoadhesive properties enabling absorption within the intestines. To test this, we evaluated the mechanism of KM access to the kidneys via intravital imaging and investigated the KM biodistribution in a porcine model. Next, we loaded KMs carrying the ADPKD drug metformin into CP (KM-CP -met ) and measured in vitro therapeutic effect. Upon oral administration in vivo, KM-CP -met showed significantly greater bioavailability and accumulation in the kidneys as compared to KM only or free drug. As such, KM-CP -met treatment in ADPKD mice ( Pkd1 fl/fl ; Pax8-rtTA ; Tet-O-Cre which develops the disease over 120 days and mimics the slow development of ADPKD) showed enhanced therapeutic efficacy without affecting safety despite repeated treatment. Herein, we demonstrate the potential of KM-CP as a nanomedicine strategy for oral delivery for the long-term treatment of chronic kidney diseases., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic kidney disease and is characterized by the formation of renal cysts and the eventual development of end-stage kidney disease. One approach to treating ADPKD is through inhibition of the mammalian target of rapamycin (mTOR) pathway, which has been implicated in cell overproliferation, contributing to renal cyst expansion. However, mTOR inhibitors, including rapamycin, everolimus, and RapaLink-1, have off-target side effects including immunosuppression. Thus, we hypothesized that the encapsulation of mTOR inhibitors in drug delivery carriers that target the kidneys would provide a strategy that would enable therapeutic efficacy while minimizing off-target accumulation and associated toxicity. Toward eventual in vivo application, we synthesized cortical collecting duct (CCD) targeted peptide amphiphile micelle (PAM) nanoparticles and show high drug encapsulation efficiency (>92.6%). In vitro analysis indicated that drug encapsulation into PAMs enhanced the anti-proliferative effect of all three drugs in human CCD cells. Analysis of in vitro biomarkers of the mTOR pathway via western blotting confirmed that PAM encapsulation of mTOR inhibitors did not reduce their efficacy. These results indicate that PAM encapsulation is a promising way to deliver mTOR inhibitors to CCD cells and potentially treat ADPKD. Future studies will evaluate the therapeutic effect of PAM-drug formulations and ability to prevent off-target side effects associated with mTOR inhibitors in mouse models of ADPKD., Competing Interests: Declaration of competing interests The authors declare no financial interests/personal relationships which may be considered as potential competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Dysregulated cellular metabolism contributes to autosomal dominant polycystic kidney disease (ADPKD) pathogenesis. The Trial of Administration of Metformin in Polycystic Kidney Disease (TAME-PKD) tested the effects of metformin treatment over 2 years in adult ADPKD patients with mild-moderate disease severity. Metformin was found to be safe and tolerable with an insignificant trend toward reduced estimated glomerular filtration rate (eGFR) decline compared to placebo. Here we tested whether targeted urinary metabolic biomarkers measured in TAME-PKD participants correlated with disease progression, severity, and metformin treatment in cross-sectional and longitudinal analyses., Methods: Concentrations of total protein, targeted metabolites (lactate, pyruvate, and succinate), and glycolytic enzymes (pyruvate kinase-M2, lactate dehydrogenase-A, and pyruvate dehydrogenase kinase-1) were measured and normalized by creatinine or osmolality in urine specimens and compared with height-adjusted total kidney volume (htTKV) and eGFR at the different study timepoints., Results: In cross-sectional analyses utilizing placebo group data, urinary succinate normalized by creatinine negatively correlated with ln (htTKV), whereas protein excretion strongly positively correlated with ln (htTKV), and negatively correlated with eGFR. Significant time-varying negative associations occurred with eGFR and the lactate/pyruvate ratio and with urine protein normalized by osmolality, indicating correlations of these biomarkers with disease progression. In secondary analyses, urinary pyruvate normalized by osmolality was preserved in metformin-treated participants but declined in placebo over the 2-year study period with a significant between-arm difference, suggesting time-dependent urinary pyruvate changes may serve as a discriminator for metformin treatment effects in this study population., Conclusion: Proteinuria with enhanced glycolytic and reduced oxidative metabolic markers generally correlated with disease severity and risk of progression in the TAME-PKD study population., (© 2022 Published by Elsevier Inc. on behalf of the International Society of Nephrology.)

ADPKD has few therapeutic options. Tolvaptan slows disease but has side effects limiting its tolerability. Bempedoic acid (BA), an ATP citrate-lyase (ACLY) inhibitor FDA-approved for hypercholesterolemia, catalyzes a key step in fatty acid/sterol synthesis important for cell proliferation. BA is activated by very long-chain acyl-CoA synthetase (FATP2) expressed primarily in kidney and liver. BA also activates AMPK. We hypothesized that BA could be a novel ADPKD therapy by inhibiting cyst growth, proliferation, injury, and metabolic dysregulation via ACLY inhibition and AMPK activation. Pkd1 -null kidney cell lines derived from mouse proximal tubule (PT) and collecting duct (IMCD) were grown in 2D or 3D Matrigel cultures and treated ± BA, ± SB-204990 (another ACLY inhibitor) or with Acly shRNA before cyst analysis, immunoblotting or mitochondrial assays using MitoSox and MitoTracker staining. Pkd1 fl/fl ; Pax8-rtTA; Tet-O-Cre C57BL/6J mice were induced with doxycycline injection on postnatal days 10 and 11 (P10-P11) and then treated ± BA (30 mg/kg/d) ± tolvaptan (30-100 mg/kg/d) by gavage from P12-21. Disease severity was determined by % total-kidney-weight-to-bodyweight (%TKW/BW) and BUN levels at euthanasia (P22). Kidney and liver homogenates were immunoblotted for expression of key biomarkers. ACLY expression and activity were upregulated in Pkd1 -null PT and IMCD-derived cells vs. controls. Relative to controls, both BA and SB-204990 inhibited cystic growth in Pkd1 -null kidney cells, as did Acly knockdown. BA inhibited mitochondrial superoxide production and promoted mitochondrial elongation, suggesting improved mitochondrial function. In ADPKD mice, BA reduced %TKW/BW and BUN to a similar extent as tolvaptan vs. untreated controls. Addition of BA to tolvaptan caused a further reduction in %TKW/BW and BUN vs. tolvaptan alone. BA generally reduced ACLY and stimulated AMPK activity in kidneys and livers vs. controls. BA also inhibited mTOR and ERK signaling and reduced kidney injury markers. In liver, BA treatment, both alone and together with tolvaptan, increased mitochondrial biogenesis while inhibiting apoptosis. We conclude that BA and ACLY inhibition inhibited cyst growth in vitro , and BA decreased ADPKD severity in vivo . Combining BA with tolvaptan further improved various ADPKD disease parameters. Repurposing BA may be a promising new ADPKD therapy, having beneficial effects alone and along with tolvaptan., Competing Interests: The authors declare that this study received funding from Esperion Therapeutics. The funder had the following involvement in the study: data review and paper review as per author contributions of SP., (Copyright © 2022 Hallows, Li, Saitta, Sepehr, Huang, Pham, Wang, Mancino, Chung, Pinkosky and Pastor-Soler.)

Current kidney organoids model development and diseases of the nephron but not the contiguous epithelial network of the kidney's collecting duct (CD) system. Here, we report the generation of an expandable, 3D branching ureteric bud (UB) organoid culture model that can be derived from primary UB progenitors from mouse and human fetal kidneys, or generated de novo from human pluripotent stem cells. In chemically-defined culture conditions, UB organoids generate CD organoids, with differentiated principal and intercalated cells adopting spatial assemblies reflective of the adult kidney's collecting system. Aggregating 3D-cultured nephron progenitor cells with UB organoids in vitro results in a reiterative process of branching morphogenesis and nephron induction, similar to kidney development. Applying an efficient gene editing strategy to remove RET activity, we demonstrate genetically modified UB organoids can model congenital anomalies of kidney and urinary tract. Taken together, these platforms will facilitate an enhanced understanding of development, regeneration and diseases of the mammalian collecting duct system.

The metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na + channel (ENaC), a key regulator of salt reabsorption by the kidney and thus total body volume and blood pressure. Recent studies have suggested that AMPK promotes the association of p21-activated kinase-interacting exchange factor-β 1 β 1 Pix, 14-3-3 proteins, and the ubiquitin ligase neural precursor cell expressed developmentally downregulated protein (Nedd)4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and ENaC degradation. Functional β 1 Pix is required for ENaC inhibition by AMPK and promotes Nedd4-2 phosphorylation and stability in mouse kidney cortical collecting duct cells. Here, we report that AMPK directly phosphorylates β 1 Pix in vitro. Among several AMPK phosphorylation sites on β 1 Pix detected by mass spectrometry, Ser 71 was validated as functionally significant. Compared with wild-type β 1 Pix, overexpression of a phosphorylation-deficient β 1 Pix-S71A mutant attenuated ENaC inhibition and the AMPK-activated interaction of both β 1 Pix and Nedd4-2 to 14-3-3 proteins in cortical collecting duct cells. Similarly, overexpression of a β 1 Pix-Δ602-611 deletion tract mutant unable to bind 14-3-3 proteins decreased the interaction between Nedd4-2 and 14-3-3 proteins, suggesting that 14-3-3 binding to β 1 Pix is critical for the formation of a β 1 Pix/Nedd4-2/14-3-3 complex. With expression of a general peptide inhibitor of 14-3-3-target protein interactions (R18), binding of both β 1 Pix and Nedd4-2 to 14-3-3 proteins was reduced, and AMPK-dependent ENaC inhibition was also attenuated. Altogether, our results demonstrate the importance of AMPK-mediated phosphorylation of β 1 Pix at Ser 71 , which promotes 14-3-3 interactions with β 1 Pix and Nedd4-2 to form a tripartite ENaC inhibitory complex, in the mechanism of ENaC regulation by AMPK.

Several model systems have been used to study signaling cascades in kidney epithelial cells, including kidney histology after systemic treatments, ex vivo isolated tubule perfusion, epithelial cell lines in culture, kidney micropuncture, and ex vivo kidney slices. We and others have found the ex vivo kidney slice method useful to study the signaling cascades involved in the regulation of kidney transport proteins. In this chapter we describe our adaptations to this classic method for the study of the regulation of kinases and endocytosis in rodent kidney epithelial cells. Briefly, slices are obtained by sectioning of freshly harvested rat or mouse kidneys using a Stadie-Riggs tissue slicer. Alternatively, a vibratome can be used to obtain slices at a more consistent and finer thickness. The harvested kidney and kidney slices are kept viable in either cell culture media or in buffers that mimic physiological conditions equilibrated with 5% CO 2 at body temperature (37°C). These buffers keep the slices viable during hours for incubations in the presence/absence of different pharmacological agents. After the incubation period the slices can be used for biochemistry experiments by preparing tissue lysates or for histological evaluation after fixation. Moreover, the fixed slices can be used to evaluate changes in subcellular trafficking of epithelial proteins or endosomes via immunolabeling followed by confocal microscopy. The resulting micrographs can then be used for systematic quantification of protein- or compartment-specific changes in subcellular localization under each condition., (© 2019 Elsevier Inc. All rights reserved.)

Our previous work has established that the metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na + channel (ENaC) by promoting its binding to neural precursor cell-expressed, developmentally down-regulated 4-2, E3 ubiquitin protein ligase (Nedd4-2). Here, using MS analysis and in vitro phosphorylation, we show that AMPK phosphorylates Nedd4-2 at the Ser-444 ( Xenopus Nedd4-2) site critical for Nedd4-2 stability. We further demonstrate that the Pak-interacting exchange factor β 1 Pix is required for AMPK-mediated inhibition of ENaC-dependent currents in both CHO and murine kidney cortical collecting duct (CCD) cells. Short hairpin RNA-mediated knockdown of β 1 Pix expression in CCD cells attenuated the inhibitory effect of AMPK activators on ENaC currents. Moreover, overexpression of a β 1 Pix dimerization-deficient mutant unable to bind 14-3-3 proteins (Δ602-611) increased ENaC currents in CCD cells, whereas overexpression of WT β 1 Pix had the opposite effect. Using additional immunoblotting and co-immunoprecipitation experiments, we found that treatment with AMPK activators promoted the binding of β 1 Pix to 14-3-3 proteins in CCD cells. However, the association between Nedd4-2 and 14-3-3 proteins was not consistently affected by AMPK activation, β 1 Pix knockdown, or overexpression of WT β 1 Pix or the β 1 Pix-Δ602-611 mutant. Moreover, we found that β 1 Pix is important for phosphorylation of the aforementioned Nedd4-2 site critical for its stability. Overall, these findings elucidate novel molecular mechanisms by which AMPK regulates ENaC. Specifically, they indicate that AMPK promotes the assembly of β 1 Pix, 14-3-3 proteins, and Nedd4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and its degradation., (© 2018 Ho et al.)

Purpose of Review: AMP-activated protein kinase (AMPK) is a metabolic sensor that regulates cellular energy balance, transport, growth, inflammation, and survival functions. This review explores recent work in defining the effects of AMPK on various renal tubular epithelial ion transport proteins as well as its role in kidney injury and repair in normal and disease states., Recent Findings: Recently, several groups have uncovered additional functions of AMPK in the regulation of kidney and transport proteins. These new studies have focused on the role of AMPK in the kidney in the setting of various diseases such as diabetes, which include evaluation of the effects of the hyperglycemic state on podocyte and tubular cell function. Other recent studies have investigated how reduced kidney mass, polycystic kidney disease (PKD), and fibrosis affect AMPK activation status. A general theme of several conditions that lead to chronic kidney disease (CKD) is that AMPK activity is abnormally suppressed relative to that in normal kidneys. Thus, the idea that AMPK activation may be a therapeutic strategy to slow down the progression of CKD has emerged. In addition to drugs such as metformin and 5-aminoimidazole-4-carboxamide ribonucleotide that are classically used as AMPK activators, recent studies have identified the therapeutic potential of other compounds that function at least partly as AMPK activators, such as salicylates, statins, berberine, and resveratrol, in preventing the progression of CKD., Summary: AMPK in the kidney plays a unique role at the crossroads of energy metabolism, ion and water transport, inflammation, and stress. Its potential role in modulating recovery from vs. progression of acute and chronic kidney injury has been the topic of recent research findings. The continued study of AMPK in kidney physiology and disease has improved our understanding of these physiological and pathological processes and offers great hope for therapeutic avenues for the increasing population at risk to develop kidney failure.

Renal hypoxia contributes to chronic kidney disease (CKD) progression, as validated in experimental and human CKD. In the early stages, increased oxygen consumption causes oxygen demand/supply mismatch, leading to hypoxia. Hence, early targeting of the determinants and regulators of oxygen consumption in CKD may alter the disease course before permanent damage ensues. Here, we focus on hypoxia inducible factor-1α (HIF-1α) and AMP-activated protein kinase (AMPK) and on the mechanisms by which they may facilitate cellular hypoxia adaptation. We found that HIF-1α activation in the subtotal nephrectomy (STN) model of CKD limits protein synthesis, inhibits apoptosis, and activates autophagy, presumably for improved cell survival. AMPK activation was diminished in the STN kidney and was remarkably restored by HIF-1α activation, demonstrating a novel role for HIF-1α in the regulation of AMPK activity. We also investigated the independent and combined effects of HIF-1α and AMPK on cell survival and death pathways by utilizing pharmacological and knockdown approaches in cell culture models. We found that the effect of HIF-1α activation on autophagy is independent of AMPK, but on apoptosis it is partially AMPK dependent. The effects of HIF-1α and AMPK activation on inhibiting protein synthesis via the mTOR pathway appear to be additive. These various effects were also observed under hypoxic conditions. In conclusion, HIF-1α and AMPK appear to be linked at a molecular level and may act as components of a concerted cellular response to hypoxic stress in the pathophysiology of CKD., (Copyright © 2015 the American Physiological Society.)

Despite the importance of ADAM17-dependent cleavage in normal biology and disease, the physiological cues that trigger its activity, the effector pathways that promote its function, and the mechanisms that control its activity, particularly the role of phosphorylation, remain unresolved. Using native bladder epithelium, in some cases transduced with adenoviruses encoding small interfering RNA, we observe that stimulation of apically localized A1 adenosine receptors (A1ARs) triggers a Gi-Gβγ-phospholipase C-protein kinase C (PKC) cascade that promotes ADAM17-dependent HB-EGF cleavage, EGFR transactivation, and apical exocytosis. We further show that the cytoplasmic tail of rat ADAM17 contains a conserved serine residue at position 811, which resides in a canonical PKC phosphorylation site, and is phosphorylated in response to A1AR activation. Preventing this phosphorylation event by expression of a nonphosphorylatable ADAM17(S811A) mutant or expression of a tail-minus construct inhibits A1AR-stimulated, ADAM17-dependent HB-EGF cleavage. Furthermore, expression of ADAM17(S811A) in bladder tissues impairs A1AR-induced apical exocytosis. We conclude that adenosine-stimulated exocytosis requires PKC- and ADAM17-dependent EGFR transactivation and that the function of ADAM17 in this pathway depends on the phosphorylation state of Ser-811 in its cytoplasmic domain., (© 2014 Prakasam et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel mutations cause cystic fibrosis lung disease. A better understanding of CFTR regulatory mechanisms could suggest new therapeutic strategies. AMP-activated protein kinase (AMPK) binds to and phosphorylates CFTR, attenuating PKA-activated CFTR gating. However, the requirement for AMPK binding to CFTR and the potential role of other proteins in this regulation are unclear. We report that nucleoside diphosphate kinase A (NDPK-A) interacts with both AMPK and CFTR in overlay blots of airway epithelial cell lysates. Binding studies in Xenopus oocytes and transfected HEK-293 cells revealed that a CFTR peptide fragment that binds AMPK (CFTR-1420-57) disrupted the AMPK-CFTR interaction. Introduction of CFTR-1420-57 into human bronchial Calu-3 cells enhanced forskolin-stimulated whole cell conductance in patch clamp measurements. Similarly, injection of CFTR-1420-57 into Xenopus oocytes blocked the inhibition of cAMP-stimulated CFTR conductance by AMPK in two-electrode voltage clamp studies. AMPK also inhibited CFTR conductance with co-expression of WT NDPK-A in two-electrode voltage clamp studies, but co-expression of a catalytically inactive H118F mutant or various Ser-120 NDPK-A mutants prevented this inhibition. In vitro phosphorylation of WT NDPK-A was enhanced by purified active AMPK, but phosphorylation was prevented in H118F and phosphomimic Ser-120 NDPK-A mutants. AMPK does not appear to phosphorylate NDPK-A directly but rather promotes an NDPK-A autophosphorylation event that involves His-118 and Ser-120. Taken together, these results suggest that NDPK-A exists in a functional cellular complex with AMPK and CFTR in airway epithelia, and NDPK-A catalytic function is required for the AMPK-dependent regulation of CFTR.