Sterile kidney tissue injury induces neutrophil swarming in lung alveolar capillaries.

Sterile tissue injury, such as by acute kidney injury, is common in the clinic and frequently associated with respiratory compromise and hypoxemia. We previously described signaling components released by the injured kidney that drive a remote inflammatory response in the lung. How this caused the resultant hypoxemia remained unclear. Here, we report that sterile kidney tissue injury induces rapid intravascular "neutrophil train" formation in lung capillaries, a novel form of neutrophil swarming. Rapid swarming is enhanced by decreased deformability of circulating neutrophils that impedes their lung capillary passage. Classical lung monocytes are required for neutrophil train formation and release CXCL2 to attract and retain stiffened neutrophils in lung capillaries which reduces capillary perfusion. We thus discovered a novel feature of kidney-lung crosstalk after sterile kidney tissue injury, capillary perfusion deficits that lead to reduced oxygenation despite proper alveolar function and ventilation, unlike in infectious inflammatory lung processes, such as bacterial pneumonia.

Cep120 is essential for kidney stromal progenitor cell growth and differentiation.

Mutations in genes that disrupt centrosome structure or function can cause congenital kidney developmental defects and lead to fibrocystic pathologies. Yet, it is unclear how defective centrosome biogenesis impacts renal progenitor cell physiology. Here, we examined the consequences of impaired centrosome duplication on kidney stromal progenitor cell growth, differentiation, and fate. Conditional deletion of the ciliopathy gene Cep120, which is essential for centrosome duplication, in the stromal mesenchyme resulted in reduced abundance of interstitial lineages including pericytes, fibroblasts and mesangial cells. These phenotypes were caused by a combination of delayed mitosis, activation of the mitotic surveillance pathway leading to apoptosis, and changes in both Wnt and Hedgehog signaling that are key for differentiation of stromal cells. Cep120 ablation resulted in small hypoplastic kidneys with medullary atrophy and delayed nephron maturation. Finally, Cep120 and centrosome loss in the interstitium sensitized kidneys of adult mice, causing rapid fibrosis after renal injury via enhanced TGF-ß/Smad3-Gli2 signaling. Our study defines the cellular and developmental defects caused by loss of Cep120 and aberrant centrosome biogenesis in the embryonic kidney stroma.

Acute Kidney Injury-Induced Circulating TNFR1/2 Elevations Correlate with Persistent Kidney Injury and Progression to Fibrosis.

Elevated levels of circulating tumor necrosis factor receptors 1 and 2 (cTNFR1/2) predict chronic kidney disease (CKD) progression; however, the mechanisms of their release remain unknown. Whether acute kidney injury (AKI) drives cTNFR1/2 elevations and whether they predict disease outcomes after AKI remain unknown. In this study, we used AKI patient serum and urine samples, mouse models of kidney injury (ischemic, obstructive, and toxic), and progression to fibrosis, nephrectomy, and related single-cell RNA-sequencing datasets to experimentally test the role of kidney injury on cTNFR1/2 levels. We show that TNFR1/2 serum and urine levels are highly elevated in all of the mouse models of kidney injury tested, beginning within one hour post injury, and correlate with its severity. Consistent with this, serum and urine TNFR1/2 levels are increased in AKI patients and correlate with the severity of kidney failure. Kidney tissue expression of TNFR1/2 after AKI is only slightly increased and bilateral nephrectomies lead to strong cTNFR1/2 elevations, suggesting the release of these receptors by extrarenal sources. The injection of the uremic toxin indoxyl sulfate in healthy mice induces moderate cTNFR1/2 elevations. Moreover, TNF neutralization does not affect early cTNFR1/2 elevations after AKI. These data suggest that cTNFR1/2 levels in AKI do not reflect injury-induced TNF activity, but rather a rapid response to loss of kidney function and uremia. In contrast to traditional disease biomarkers, such as serum creatinine or BUN, cTNFR1/2 levels remain elevated for weeks after severe kidney injury. At these later timepoints, cTNFR1/2 levels positively correlate with remaining kidney injury. During the AKI-to-CKD transition, elevations of TNFR1/2 kidney expression and of cTNFR2 levels correlate with kidney fibrosis levels. In conclusion, our data demonstrate that kidney injury drives acute increases in cTNFR1/2 serum levels, which negatively correlate with kidney function. Sustained TNFR1/2 elevations after kidney injury during AKI-to-CKD transition reflect persistent tissue injury and progression to kidney fibrosis.

Impaired centrosome biogenesis in kidney stromal progenitors reduces abundance of interstitial lineages and accelerates injury-induced fibrosis.

Defective centrosome function can disrupt embryonic kidney development, by causing changes to the renal interstitium that leads to fibrocystic disease pathologies. Yet, it remains unknown how mutations in centrosome genes impact kidney interstitial cells. Here, we examined the consequences of defective centrosome biogenesis on stromal progenitor cell growth, differentiation and fate. Conditional deletion of Cep120 , a ciliopathy gene essential for centrosome duplication, in the stromal mesenchyme resulted in reduced abundance of pericytes, interstitial fibroblasts and mesangial cells. This was due to delayed mitosis, increased apoptosis, and changes in Wnt and Hedgehog signaling essential for differentiation of stromal lineages. Cep120 ablation resulted in hypoplastic kidneys with medullary atrophy and delayed nephron maturation. Finally, centrosome loss in the interstitium sensitized kidneys of adult mice, causing rapid fibrosis via enhanced TGF-ß/Smad3-Gli2 signaling after renal injury. Our study defines the cellular and developmental defects caused by centrosome dysfunction in embryonic kidney stroma. Highlights: Defective centrosome biogenesis in kidney stroma causes:Reduced abundance of stromal progenitors, interstitial and mesangial cell populationsDefects in cell-autonomous and paracrine signalingAbnormal/delayed nephrogenesis and tubular dilationsAccelerates injury-induced fibrosis via defective TGF-ß/Smad3-Gli2 signaling axis.

TNF or EGFR inhibition equally block AKI-to-CKD transition: opportunities for etanercept treatment.

BACKGROUND: Inflammation is a key driver of the transition of acute kidney injury to progressive fibrosis and chronic kidney disease (AKI-to-CKD transition). Blocking a-disintegrin-and-metalloprotease-17 (ADAM17)-dependent ectodomain shedding, in particular of epidermal growth factor receptor (EGFR) ligands and of the type 1 inflammatory cytokine tumor necrosis factor (TNF), reduces pro-inflammatory and pro-fibrotic responses after ischemic AKI or unilateral ureteral obstruction (UUO), a classical fibrosis model. Metalloprotease or EGFR inhibition show significant undesirable side effects in humans. In retrospective studies anti-TNF biologics reduce the incidence and progression of CKD in humans. Whether TNF has a role in AKI-to-CKD transition and how TNF inhibition compares to EGFR inhibition is largely unknown. METHODS: Mice were subjected to bilateral renal ischemia-reperfusion injury or unilateral ureteral obstruction. Kidneys were analyzed by histology, immunohistochemistry, qPCR, western blot, mass cytometry, scRNA sequencing, and cytokine profiling. RESULTS: Here we show that TNF or EGFR inhibition reduce AKI-to-CKD transition and fibrosis equally by about 25%, while combination has no additional effect. EGFR inhibition reduced kidney TNF expression by about 50% largely by reducing accumulation of TNF expressing immune cells in the kidney early after AKI, while TNF inhibition did not affect EGFR activation or immune cell accumulation. Using scRNAseq data we show that TNF is predominantly expressed by immune cells in AKI but not in proximal tubule cells (PTC), and PTC-TNF knockout did not affect AKI-to-CKD transition in UUO. Thus, the anti-inflammatory and anti-fibrotic effects of the anti-TNF biologic etanercept in AKI-to-CKD transition rely on blocking TNF that is released from immune cells recruited or accumulating in response to PTC-EGFR signals. CONCLUSION: Short-term anti-TNF biologics during or after AKI could be helpful in the prevention of AKI-to-CKD transition.

Identification of kidney injury released circulating osteopontin as causal agent of respiratory failure.

Tissue injury can drive secondary organ injury; however, mechanisms and mediators are not well understood. To identify interorgan cross-talk mediators, we used acute kidney injury (AKI)-induced acute lung injury (ALI) as a clinically important example. Using kidney and lung single-cell RNA sequencing after AKI in mice followed by ligand-receptor pairing analysis across organs, kidney ligands to lung receptors, we identify kidney-released circulating osteopontin (OPN) as a novel AKI-ALI mediator. OPN release from kidney tubule cells triggered lung endothelial leakage, inflammation, and respiratory failure. Pharmacological or genetic OPN inhibition prevented AKI-ALI. Transplantation of ischemic wt kidneys caused AKI-ALI, but not of ischemic OPN-global knockout kidneys, identifying kidney-released OPN as necessary interorgan signal to cause AKI-ALI. We show that OPN serum levels are elevated in patients with AKI and correlate with kidney injury. Our results demonstrate feasibility of using ligand-receptor analysis across organs to identify interorgan cross-talk mediators and may have important therapeutic implications in human AKI-ALI and multiorgan failure.

Soluble forms of cytokine and growth factor receptors: mechanisms of generation and modes of action in the regulation of local and systemic inflammation.

Cytokine and growth factor receptors are usually transmembrane proteins, but they can also exist in soluble forms, either through cleavage and release of their ligand-binding extracellular domain or through the secretion of a soluble isoform. As an extension of this concept, transmembrane receptors on exosomes released into the circulation may act similarly to circulating soluble receptors. These soluble receptors add to the complexity of cytokine and growth factor signalling: they can function as decoy receptor that compete for ligand binding with their respective membrane-bound forms thereby attenuating signalling, or stabilize their ligands, or activate additional signalling events through interactions with other cell-surface proteins. Their soluble nature allows for a functional role away from the production sites, in remote cell types and organs. Accumulating evidence demonstrates that soluble receptors participate in the regulation and orchestration of various key cellular processes, particularly inflammatory responses. In this review, we will discuss release mechanisms of soluble cytokine and growth factor receptors, their mechanisms of action and strategies for targeting their pathways in disease.

FOXM1 drives proximal tubule proliferation during repair from acute ischemic kidney injury.

The proximal tubule has a remarkable capacity for repair after acute injury, but the cellular lineage and molecular mechanisms underlying this repair response are incompletely understood. Here, we developed a Kim1-GFPCreERt2 knockin mouse line (Kim1-GCE) in order to perform genetic lineage tracing of dedifferentiated cells while measuring the cellular transcriptome of proximal tubule during repair. Acutely injured genetically labeled clones coexpressed KIM1, VIMENTIN, SOX9, and KI67, indicating a dedifferentiated and proliferative state. Clonal analysis revealed clonal expansion of Kim1+ cells, indicating that acutely injured, dedifferentiated proximal tubule cells, rather than fixed tubular progenitor cells, account for repair. Translational profiling during injury and repair revealed signatures of both successful and unsuccessful maladaptive repair. The transcription factor Foxm1 was induced early in injury, was required for epithelial proliferation in vitro, and was dependent on epidermal growth factor receptor (EGFR) stimulation. In conclusion, dedifferentiated proximal tubule cells effect proximal tubule repair, and we reveal an EGFR/FOXM1-dependent signaling pathway that drives proliferative repair after injury.

Proximal Tubule-Derived Amphiregulin Amplifies and Integrates Profibrotic EGF Receptor Signals in Kidney Fibrosis.

BACKGROUND: Sustained activation of EGF receptor (EGFR) in proximal tubule cells is a hallmark of progressive kidney fibrosis after AKI and in CKD. However, the molecular mechanisms and particular EGFR ligands involved are unknown. METHODS: We studied EGFR activation in proximal tubule cells and primary tubular cells isolated from injured kidneys in vitro. To determine in vivo the role of amphiregulin, a low-affinity EGFR ligand that is highly upregulated with injury, we used ischemia-reperfusion injury or unilateral ureteral obstruction in mice with proximal tubule cell-specific knockout of amphiregulin. We also injected soluble amphiregulin into knockout mice with proximal tubule cell-specific deletion of amphiregulin's releasing enzyme, the transmembrane cell-surface metalloprotease, a disintegrin and metalloprotease-17 (ADAM17), and into ADAM17 hypomorphic mice. RESULTS: Yes-associated protein 1 (YAP1)-dependent upregulation of amphiregulin transcript and protein amplifies amphiregulin signaling in a positive feedback loop. YAP1 also integrates signals of other moderately injury-upregulated, low-affinity EGFR ligands (epiregulin, epigen, TGFα), which also require soluble amphiregulin and YAP1 to induce sustained EGFR activation in proximal tubule cells in vitro. In vivo, soluble amphiregulin injection sufficed to reverse protection from fibrosis after ischemia-reperfusion injury in ADAM17 hypomorphic mice; injected soluble amphiregulin also reversed the corresponding protective proximal tubule cell phenotype in injured proximal tubule cell-specific ADAM17 knockout mice. Moreover, the finding that proximal tubule cell-specific amphiregulin knockout mice were protected from fibrosis after ischemia-reperfusion injury or unilateral ureteral obstruction demonstrates that amphiregulin was necessary for the development of fibrosis. CONCLUSIONS: Our results identify amphiregulin as a key player in injury-induced kidney fibrosis and suggest therapeutic or diagnostic applications of soluble amphiregulin in kidney disease.

Functional Genomics Approach Identifies Novel Signaling Regulators of TGFα Ectodomain Shedding.

Ectodomain shedding of cell-surface precursor proteins by metalloproteases generates important cellular signaling molecules. Of importance for disease is the release of ligands that activate the EGFR, such as TGFα, which is mostly carried out by ADAM17 [a member of the A-disintegrin and metalloprotease (ADAM) domain family]. EGFR ligand shedding has been linked to many diseases, in particular cancer development, growth and metastasis, as well as resistance to cancer therapeutics. Excessive EGFR ligand release can outcompete therapeutic EGFR inhibition or the inhibition of other growth factor pathways by providing bypass signaling via EGFR activation. Drugging metalloproteases directly have failed clinically because it indiscriminately affected shedding of numerous substrates. It is therefore essential to identify regulators for EGFR ligand cleavage. Here, integration of a functional shRNA genomic screen, computational network analysis, and dedicated validation tests succeeded in identifying several key signaling pathways as novel regulators of TGFα shedding in cancer cells. Most notably, a cluster of genes with NFκB pathway regulatory functions was found to strongly influence TGFα release, albeit independent of their NFκB regulatory functions. Inflammatory regulators thus also govern cancer cell growth-promoting ectodomain cleavage, lending mechanistic understanding to the well-known connection between inflammation and cancer.Implications: Using genomic screens and network analysis, this study defines targets that regulate ectodomain shedding and suggests new treatment opportunities for EGFR-driven cancers. Mol Cancer Res; 16(1); 147-61. ©2017 AACR.

ADAM17 substrate release in proximal tubule drives kidney fibrosis.

Kidney fibrosis following kidney injury is an unresolved health problem and causes significant morbidity and mortality worldwide. In a study into its molecular mechanism, we identified essential causative features. Acute or chronic kidney injury causes sustained elevation of a disintegrin and metalloprotease 17 (ADAM17); of its cleavage-activated proligand substrates, in particular of pro-TNFα and the EGFR ligand amphiregulin (pro-AREG); and of the substrates' receptors. As a consequence, EGFR is persistently activated and triggers the synthesis and release of proinflammatory and profibrotic factors, resulting in macrophage/neutrophil ingress and fibrosis. ADAM17 hypomorphic mice, specific ADAM17 inhibitor-treated WT mice, or mice with inducible KO of ADAM17 in proximal tubule (Slc34a1-Cre) were significantly protected against these effects. In vitro, in proximal tubule cells, we show that AREG has unique profibrotic actions that are potentiated by TNFα-induced AREG cleavage. In vivo, in acute kidney injury (AKI) and chronic kidney disease (CKD, fibrosis) patients, soluble AREG is indeed highly upregulated in human urine, and both ADAM17 and AREG expression show strong positive correlation with fibrosis markers in related kidney biopsies. Our results indicate that targeting of the ADAM17 pathway represents a therapeutic target for human kidney fibrosis.

Targeted Delivery of Immunomodulators to Lymph Nodes.

Active-targeted delivery to lymph nodes represents a major advance toward more effective treatment of immune-mediated disease. The MECA79 antibody recognizes peripheral node addressin molecules expressed by high endothelial venules of lymph nodes. By mimicking lymphocyte trafficking to the lymph nodes, we have engineered MECA79-coated microparticles containing an immunosuppressive medication, tacrolimus. Following intravenous administration, MECA79-bearing particles showed marked accumulation in the draining lymph nodes of transplanted animals. Using an allograft heart transplant model, we show that targeted lymph node delivery of microparticles containing tacrolimus can prolong heart allograft survival with negligible changes in tacrolimus serum level. Using MECA79 conjugation, we have demonstrated targeted delivery of tacrolimus to the lymph nodes following systemic administration, with the capacity for immune modulation in vivo.

PI3kα and STAT1 Interplay Regulates Human Mesenchymal Stem Cell Immune Polarization.

The immunomodulatory capacity of mesenchymal stem cells (MSCs) is critical for their use in therapeutic applications. MSC response to specific inflammatory cues allows them to switch between a proinflammatory (MSC1) or anti-inflammatory (MSC2) phenotype. Regulatory mechanisms controlling this switch remain to be defined. One characteristic feature of MSC2 is their ability to respond to IFNγ with induction of indoleamine 2,3-dioxygenase (IDO), representing the key immunoregulatory molecule released by human MSC. Here, we show that STAT1 and PI3Kα pathways interplay regulates IFNγ-induced IDO production in MSC. Chemical phosphoinositide 3-kinase (PI3K) pan-inhibition, PI3Kα-specific inhibition or shRNA knockdown diminished IFNγ-induced IDO production. This effect involved PI3Kα-mediated upregulation of STAT1 protein levels and phosphorylation at Ser727. Overexpression of STAT1 or of a constitutively active PI3Kα mutant failed to induce basal IDO production, but shifted MSC into an MSC2-like phenotype by strongly enhancing IDO production in response to IFNγ as compared to controls. STAT1 overexpression strongly enhanced MSC-mediated T-cell suppression. The same effect could be induced using short-term pretreatment of MSC with a chemical inhibitor of the counter player of PI3K, phosphatase and tensin homolog. Finally, downregulation of STAT1 abrogated the immunosuppressive capacity of MSC. Our results for the first time identify critical upstream signals for the induced production of IDO in MSCs that could be manipulated therapeutically to enhance their immunosuppressive phenotype.

Co-transplantation of autologous MSCs delays islet allograft rejection and generates a local immunoprivileged site.

AIMS: Mesenchymal stem cells (MSCs) are multipotent cells with immunomodulatory properties. We tested the ability of MSCs to delay islet allograft rejection. METHODS: Mesenchymal stem cells were generated in vitro from C57BL/6 and BALB/c mice bone marrow, and their immunomodulatory properties were tested in vitro. We then tested the effect of a local or systemic administration of heterologous and autologous MSCs on graft survival in a fully allogeneic model of islet transplantation (BALB/c islets into C57BL/6 mice). RESULTS: In vitro, autologous, but not heterologous, MSCs abrogated immune cell proliferation in response to alloantigens and skewed the immune response toward a Th2 profile. A single dose of autologous MSCs co-transplanted under the kidney capsule with allogeneic islets delayed islet rejection, reduced graft infiltration, and induced long-term graft function in 30 % of recipients. Based on ex vivo analysis of recipient splenocytes, the use of autologous MSCs did not appear to have any systemic effect on the immune response toward graft alloantigens. The systemic injection of autologous MSCs or the local injection of heterologous MSCs failed to delay islet graft rejection. CONCLUSION: Autologous, but not heterologous, MSCs showed multiple immunoregulatory properties in vitro and delayed allograft rejection in vivo when co-transplanted with islets; however, they failed to prevent rejection when injected systemically. Autologous MSCs thus appear to produce a local immunoprivileged site, which promotes graft survival.

Comparative proteomic analysis of the ATP-sensitive K+ channel complex in different tissue types.

ATP-sensitive K(+) (K(ATP)) channels are expressed ubiquitously, but have diverse roles in various organs and cells. Their diversity can partly be explained by distinct tissue-specific compositions of four copies of the pore-forming inward rectifier potassium channel subunits (Kir6.1 and/or Kir6.2) and four regulatory sulfonylurea receptor subunits (SUR1 and/or SUR2). Channel function and/or subcellular localization also can be modified by the proteins with which they transiently or permanently interact to generate even more diversity. We performed a quantitative proteomic analysis of K(ATP) channel complexes in the heart, endothelium, insulin-secreting min6 cells (pancreatic ß-cell like), and the hypothalamus to identify proteins with which they interact in different tissues. Glycolysis is an overrepresented pathway in identified proteins of the heart, min6 cells, and the endothelium. Proteins with other energy metabolic functions were identified in the hypothalamic samples. These data suggest that the metabolo-electrical coupling conferred by K(ATP) channels is conferred partly by proteins with which they interact. A large number of identified cytoskeletal and trafficking proteins suggests endocytic recycling may help control K(ATP) channel surface density and/or subcellular localization. Overall, our data demonstrate that K(ATP) channels in different tissues may assemble with proteins having common functions, but that tissue-specific complex organization also occurs.

Heterogeneity of ATP-sensitive K+ channels in cardiac myocytes: enrichment at the intercalated disk.

Ventricular ATP-sensitive potassium (K(ATP)) channels link intracellular energy metabolism to membrane excitability and contractility. Our recent proteomics experiments identified plakoglobin and plakophilin-2 (PKP2) as putative K(ATP) channel-associated proteins. We investigated whether the association of K(ATP) channel subunits with junctional proteins translates to heterogeneous subcellular distribution within a cardiac myocyte. Co-immunoprecipitation experiments confirmed physical interaction between K(ATP) channels and PKP2 and plakoglobin in rat heart. Immunolocalization experiments demonstrated that K(ATP) channel subunits (Kir6.2 and SUR2A) are expressed at a higher density at the intercalated disk in mouse and rat hearts, where they co-localized with PKP2 and plakoglobin. Super-resolution microscopy demonstrate that K(ATP) channels are clustered within nanometer distances from junctional proteins. The local K(ATP) channel density, recorded in excised inside-out patches, was larger at the cell end when compared with local currents recorded from the cell center. The K(ATP) channel unitary conductance, block by MgATP and activation by MgADP, did not differ between these two locations. Whole cell K(ATP) channel current density (activated by metabolic inhibition) was ∼40% smaller in myocytes from mice haploinsufficient for PKP2. Experiments with excised patches demonstrated that the regional heterogeneity of K(ATP) channels was absent in the PKP2 deficient mice, but the K(ATP) channel unitary conductance and nucleotide sensitivities remained unaltered. Our data demonstrate heterogeneity of K(ATP) channel distribution within a cardiac myocyte. The higher K(ATP) channel density at the intercalated disk implies a possible role at the intercellular junctions during cardiac ischemia.

Measuring and evaluating the role of ATP-sensitive K+ channels in cardiac muscle.

Since ion channels move electrical charge during their activity, they have traditionally been studied using electrophysiological approaches. This was sometimes combined with mathematical models, for example with the description of the ionic mechanisms underlying the initiation and propagation of action potentials in the squid giant axon by Hodgkin and Huxley. The methods for studying ion channels also have strong roots in protein chemistry (limited proteolysis, the use of antibodies, etc.). The advent of the molecular cloning and the identification of genes coding for specific ion channel subunits in the late 1980s introduced a multitude of new techniques with which to study ion channels and the field has been rapidly expanding ever since (e.g. antibody development against specific peptide sequences, mutagenesis, the use of gene targeting in animal models, determination of their protein structures) and new methods are still in development. This review focuses on techniques commonly employed to examine ion channel function in an electrophysiological laboratory. The focus is on the K(ATP) channel, but many of the techniques described are also used to study other ion channels.

AMP-activated protein kinase connects cellular energy metabolism to KATP channel function.

AMPK is an important sensor of cellular energy levels. The aim of these studies was to investigate whether cardiac K(ATP) channels, which couple cellular energy metabolism to membrane excitability, are regulated by AMPK activity. We investigated effects of AMPK on rat ventricular K(ATP) channels using electrophysiological and biochemical approaches. Whole-cell K(ATP) channel current was activated by metabolic inhibition; this occurred more rapidly in the presence of AICAR (an AMPK activator). AICAR had no effects on K(ATP) channel activity recorded in the inside-out patch clamp configuration, but ZMP (the intracellular intermediate of AICAR) strongly activated K(ATP) channels. An AMPK-mediated effect is demonstrated by the finding that ZMP had no effect on K(ATP) channels in the presence of Compound C (an AMPK inhibitor). Recombinant AMPK activated Kir6.2/SUR2A channels in a manner that was dependent on the AMP concentration, whereas heat-inactivated AMPK was without effect. Using mass-spectrometry and co-immunoprecipitation approaches, we demonstrate that the AMPK α-subunit physically associates with K(ATP) channel subunits. Our data demonstrate that the cardiac K(ATP) channel function is directly regulated by AMPK activation. During metabolic stress, a small change in cellular AMP that activates AMPK can be a potential trigger for K(ATP) channel opening. This article is part of a Special Issue entitled "Local Signaling in Myocytes".