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1.
J Clin Invest ; 130(7): 3734-3749, 2020 07 01.
Article in English | MEDLINE | ID: mdl-32287042

ABSTRACT

Ischemic acute kidney injury (AKI), a complication that frequently occurs in hospital settings, is often associated with hemodynamic compromise, sepsis, cardiac surgery, or exposure to nephrotoxins. Here, using a murine renal ischemia/reperfusion injury (IRI) model, we show that intercalated cells (ICs) rapidly adopted a proinflammatory phenotype after IRI. Wwe demonstrate that during the early phase of AKI either blockade of the proinflammatory P2Y14 receptor located on the apical membrane of ICs or ablation of the gene encoding the P2Y14 receptor in ICs (a) inhibited IRI-induced increase of chemokine expression in ICs, (b) reduced neutrophil and monocyte renal infiltration, (c) reduced the extent of kidney dysfunction, and (d) attenuated proximal tubule damage. These observations indicate that the P2Y14 receptor participates in the very first inflammatory steps associated with ischemic AKI. In addition, we show that the concentration of the P2Y14 receptor ligand UDP-glucose (UDP-Glc) was higher in urine samples from intensive care unit patients who developed AKI compared with patients without AKI. In particular, we observed a strong correlation between UDP-Glc concentration and the development of AKI in cardiac surgery patients. Our study identifies the UDP-Glc/P2Y14 receptor axis as a potential target for the prevention and/or attenuation of ischemic AKI.


Subject(s)
Acute Kidney Injury , Ischemia , Kidney , Receptors, Purinergic P2Y/metabolism , Acute Kidney Injury/genetics , Acute Kidney Injury/metabolism , Acute Kidney Injury/pathology , Acute Kidney Injury/prevention & control , Animals , Chemokines/biosynthesis , Chemokines/genetics , Ischemia/genetics , Ischemia/metabolism , Ischemia/pathology , Ischemia/prevention & control , Kidney/blood supply , Kidney/metabolism , Kidney/pathology , Mice , Mice, Knockout , Monocytes/metabolism , Monocytes/pathology , Neutrophil Infiltration , Neutrophils/metabolism , Neutrophils/pathology , Receptors, Purinergic P2Y/genetics
2.
J Am Soc Nephrol ; 29(2): 545-556, 2018 02.
Article in English | MEDLINE | ID: mdl-29222395

ABSTRACT

Acidosis is an important complication of AKI and CKD. Renal intercalated cells (ICs) express the proton pumping vacuolar H+-ATPase (V-ATPase) and are extensively involved in acid-base homeostasis. H+ secretion in type A intercalated cells (A-ICs) is regulated by apical vesicle recycling and stimulated by cAMP. In other cell types, cAMP is increased by extracellular agonists, including adenosine, through purinergic receptors. Adenosine is a Food and Drug Administration-approved drug, but very little is known about the effect of adenosine on IC function. Therefore, we investigated the role of adenosine in the regulation of V-ATPase in ICs. Intravenous treatment of mice with adenosine or agonists of ADORA2A and ADORA2B purinergic P1 receptors induced V-ATPase apical membrane accumulation in medullary A-ICs but not in cortical A-ICs or other IC subtypes. Both receptors are located in A-IC apical membranes, and adenosine injection increased urine adenosine concentration and decreased urine pH. Cell fractionation showed that adenosine or an ADORA2A or ADORA2B agonist induced V-ATPase translocation from vesicles to the plasma membrane and increased protein kinase A (PKA)-dependent protein phosphorylation in purified medullary ICs that were isolated from mice. Either ADORA2A or ADORA2B antagonists or the PKA inhibitor mPKI blocked these effects. Finally, a fluorescence pH assay showed that adenosine activates V-ATPase in isolated medullary ICs. Our study shows that medullary A-ICs respond to luminal adenosine through ADORA2A and ADORA2B receptors in a cAMP/PKA pathway-dependent mechanism to induce V-ATPase-dependent H+ secretion.


Subject(s)
Adenosine A2 Receptor Agonists/pharmacology , Adenosine/metabolism , Adenosine/pharmacology , Epithelial Cells/enzymology , Vacuolar Proton-Translocating ATPases/metabolism , Acid-Base Equilibrium , Adenosine A2 Receptor Antagonists/pharmacology , Animals , Cell Membrane/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Homeostasis , Kidney/cytology , Male , Mice , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Transport/drug effects , Receptor, Adenosine A2A , Receptor, Adenosine A2B , Transport Vesicles , Urinalysis
3.
Oncotarget ; 7(29): 46142-46157, 2016 Jul 19.
Article in English | MEDLINE | ID: mdl-27323815

ABSTRACT

The vacuolar (H+)-ATPases (V-ATPases) are a family of ATP-driven proton pumps that acidify intracellular compartments and transport protons across the plasma membrane. Previous work has demonstrated that plasma membrane V-ATPases are important for breast cancer invasion in vitro and that the V-ATPase subunit a isoform a3 is upregulated in and critical for MDA-MB231 and MCF10CA1a breast cancer cell invasion. It has been proposed that subunit a3 is present on the plasma membrane of invasive breast cancer cells and is overexpressed in human breast cancer. To test this, we used an a3-specific antibody to assess localization in breast cancer cells. Subunit a3 localizes to the leading edge of migrating breast cancer cells, but not the plasma membrane of normal breast epithelial cells. Furthermore, invasive breast cancer cells express a3 throughout all intracellular compartments tested, including endosomes, the Golgi, and lysosomes. Moreover, subunit a3 knockdown in MB231 breast cancer cells reduces in vitro migration. This reduction is not enhanced upon addition of a V-ATPase inhibitor, suggesting that a3-containing V-ATPases are critical for breast cancer migration. Finally, we have tested a3 expression in human breast cancer tissue and mRNA prepared from normal and cancerous breast tissue. a3 mRNA was upregulated 2.5-47 fold in all breast tumor cDNA samples tested relative to normal tissue, with expression generally correlated to cancer stage. Furthermore, a3 protein expression was increased in invasive breast cancer tissue relative to noninvasive cancer and normal breast tissue. These studies suggest that subunit a3 plays an important role in invasive human breast cancer.


Subject(s)
Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Vacuolar Proton-Translocating ATPases/metabolism , Cell Line, Tumor , Cell Membrane/enzymology , Cell Movement/physiology , Female , Humans , Isoenzymes
4.
PLoS One ; 10(3): e0121419, 2015.
Article in English | MEDLINE | ID: mdl-25799465

ABSTRACT

Uncontrolled inflammation is one of the leading causes of kidney failure. Pro-inflammatory responses can occur in the absence of infection, a process called sterile inflammation. Here we show that the purinergic receptor P2Y14 (GPR105) is specifically and highly expressed in collecting duct intercalated cells (ICs) and mediates sterile inflammation in the kidney. P2Y14 is activated by UDP-glucose, a damage-associated molecular pattern molecule (DAMP) released by injured cells. We found that UDP-glucose increases pro-inflammatory chemokine expression in ICs as well as MDCK-C11 cells, and UDP-glucose activates the MEK1/2-ERK1/2 pathway in MDCK-C11 cells. These effects were prevented following inhibition of P2Y14 with the small molecule PPTN. Tail vein injection of mice with UDP-glucose induced the recruitment of neutrophils to the renal medulla. This study identifies ICs as novel sensors, mediators and effectors of inflammation in the kidney via P2Y14.


Subject(s)
Inflammation/metabolism , Kidney Tubules, Collecting/pathology , Receptors, Purinergic P2Y/metabolism , Uridine Diphosphate Glucose/pharmacology , Animals , Cells, Cultured , Dogs , Inflammation/immunology , Inflammation/pathology , Kidney Tubules, Collecting/cytology , Kidney Tubules, Collecting/metabolism , MAP Kinase Signaling System/drug effects , Madin Darby Canine Kidney Cells , Male , Mice , Neutrophils/metabolism
5.
J Biol Chem ; 289(3): 1355-63, 2014 Jan 17.
Article in English | MEDLINE | ID: mdl-24273170

ABSTRACT

The vacuolar (H(+))-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation.


Subject(s)
Bone Marrow Cells/enzymology , Dendritic Cells/enzymology , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction/physiology , TOR Serine-Threonine Kinases/metabolism , Vacuolar Proton-Translocating ATPases/metabolism , Androstadienes/pharmacology , Animals , Bone Marrow Cells/cytology , Dendritic Cells/cytology , Female , Immunosuppressive Agents/pharmacology , Lysosomes/enzymology , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Inbred BALB C , Multiprotein Complexes/antagonists & inhibitors , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Wortmannin
6.
J Biol Chem ; 288(31): 22798-808, 2013 Aug 02.
Article in English | MEDLINE | ID: mdl-23740254

ABSTRACT

The vacuolar ATPase (V-ATPase) is a multisubunit complex that carries out ATP-driven proton transport. It is composed of a peripheral V1 domain that hydrolyzes ATP and an integral V0 domain that translocates protons. Subunit a is a 100-kDa integral membrane protein (part of V0) that possesses an N-terminal cytoplasmic domain and a C-terminal hydrophobic domain. Although the C-terminal domain functions in proton transport, the N-terminal domain is critical for intracellular targeting and regulation of V-ATPase assembly. Despite its importance, there is currently no high resolution structure for subunit a of the V-ATPase. Recently, the crystal structure of the N-terminal domain of the related subunit I from the archaebacterium Meiothermus ruber was reported. We have used homology modeling to construct a model of the N-terminal domain of Vph1p, one of two isoforms of subunit a expressed in yeast. To test this model, unique cysteine residues were introduced into a Cys-less form of Vph1p and their accessibility to modification by the sulfhydryl reagent 3-(N-maleimido-propionyl) biocytin (MPB) was determined. In addition, accessibility of introduced cysteine residues to MPB modification was compared in the V1V0 complex and the free V0 domain to identify residues protected from modification by the presence of V1. The results provide an experimental test of the proposed model and have identified regions of the N-terminal domain of subunit a that likely serve as interfacial contact sites with the peripheral V1 domain. The possible significance of these results for in vivo regulation of V-ATPase assembly is discussed.


Subject(s)
Cysteine/chemistry , Saccharomyces cerevisiae/enzymology , Vacuolar Proton-Translocating ATPases/chemistry , Amino Acid Sequence , Models, Molecular , Molecular Sequence Data , Mutagenesis , Protein Conformation , Sequence Homology, Amino Acid , Vacuolar Proton-Translocating ATPases/genetics
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