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Deshmukh A, Chang K, Cuala J, Campos MJH, Mahmood S, Verma R, Georgia S, Loconte V, White KL. Secretory stimuli distinctly regulate insulin secretory granule maturation through structural remodeling. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.03.29.644646. [PMID: 40235991 PMCID: PMC11996419 DOI: 10.1101/2025.03.29.644646] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 04/17/2025]
Abstract
Insulin secretory granule (ISG) maturation is a crucial aspect of insulin secretion and glucose homeostasis. The regulation of this maturation remains poorly understood, especially how secretory stimuli affect ISG maturity and subcellular localization. In this study, we used soft X-tomography (SXT) to quantitatively map ISG morphology, density, and location in single INS-1E and mouse pancreatic β-cells under the effect of various secretory stimuli. We found that the activation of glucokinase (GK), gastric inhibitory polypeptide receptor (GIPR), glucagon-like peptide-1 receptor (GLP-1R), and G-protein coupled receptor 40 (GPR40) promote ISG maturation. Each stimulus induces unique structural remodeling in ISGs, by altering size and density, depending on the specific signaling cascades activated. These distinct ISG subpopulations mobilize and redistribute in the cell altering overall cellular structural organization. Our results provide insight into how current diabetes and obesity therapies impact ISG maturation and may inform the development of future treatments that target maturation specifically.
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Hossain S, Liu Z, Robbins N, Cowen LE. Exploring the differential localization of protein kinase A isoforms in Candida albicans. mSphere 2025; 10:e0103724. [PMID: 39998251 PMCID: PMC11934313 DOI: 10.1128/msphere.01037-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2024] [Accepted: 01/28/2025] [Indexed: 02/26/2025] Open
Abstract
The cAMP-dependent protein kinase A (PKA) plays important roles in a wide range of biological processes in eukaryotic organisms. In the fungal pathogen Candida albicans, PKA is a critical regulator of morphological transitions, which are a key virulence trait. PKA is composed of two catalytic isoforms, Tpk1 and Tpk2, which are often thought to act together in a complex with the regulatory subunit Bcy1. Although Tpk1 and Tpk2 have some redundant functions, they also have distinct cellular functions for which the mechanistic underpinnings remain largely elusive. Here, we constructed functional GFP-tagged fusion proteins for Tpk1, Tpk2, and Bcy1 to explore the localization of PKA isoforms. We observed that the PKA holoenzyme is mainly found in the cytoplasm, as Bcy1 is always excluded from the nucleus. Under glucose-replete conditions, both Tpk1 and Tpk2 translocate into the nucleus from the cytosol. In the presence of glycerol, Tpk1 resides in the cytosol, whereas Tpk2 and Bcy1 become enriched on the vacuolar membrane. As the C-terminal domains of Tpk are highly homologous, we investigated the localization and function of hybrid Tpk proteins with exchanged N-terminal domains. We found the catalytic C-terminus of Tpk1 is required for morphogenesis in solid medium, whereas the C-terminus of Tpk2 is critical for filamentation in liquid. Interestingly, the N-terminus of Tpk2 drives its localization to the vacuolar membrane. Our work highlights environmentally contingent localization patterns for the PKA subunits and suggests that the nuclear localization of Tpk is not sufficient to induce the filamentation program in a leading fungal pathogen of humans.IMPORTANCEFungal pathogens have a devastating impact on human health worldwide. They infect billions of people and kill more than 2.5 million per year. Candida albicans is a leading human fungal pathogen responsible for causing life-threatening systemic disease in immunocompromised individuals. A key virulence trait in C. albicans is the ability to switch between yeast and filamentous forms. The conserved protein kinase A (PKA) regulates diverse functions in the cell, including growth and filamentation. Although PKA has been studied in C. albicans for decades, the subcellular localization of PKA has not been thoroughly investigated. Here, we constructed functional GFP-tagged PKA subunits to explore their localization. We identified differential localization patterns for the PKA subunits that are carbon-source dependent and report that these proteins localize into foci in response to diverse environmental stresses. These findings further our understanding of a critical regulator of growth and virulence in C. albicans.
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Affiliation(s)
- Saif Hossain
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
| | - Zhongle Liu
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
| | - Nicole Robbins
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
| | - Leah E. Cowen
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
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3
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Chrysopoulou M, Rinschen MM. Metabolic Rewiring and Communication: An Integrative View of Kidney Proximal Tubule Function. Annu Rev Physiol 2024; 86:405-427. [PMID: 38012048 DOI: 10.1146/annurev-physiol-042222-024724] [Citation(s) in RCA: 14] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2023]
Abstract
The kidney proximal tubule is a key organ for human metabolism. The kidney responds to stress with altered metabolite transformation and perturbed metabolic pathways, an ultimate cause for kidney disease. Here, we review the proximal tubule's metabolic function through an integrative view of transport, metabolism, and function, and embed it in the context of metabolome-wide data-driven research. Function (filtration, transport, secretion, and reabsorption), metabolite transformation, and metabolite signaling determine kidney metabolic rewiring in disease. Energy metabolism and substrates for key metabolic pathways are orchestrated by metabolite sensors. Given the importance of renal function for the inner milieu, we also review metabolic communication routes with other organs. Exciting research opportunities exist to understand metabolic perturbation of kidney and proximal tubule function, for example, in hypertension-associated kidney disease. We argue that, based on the integrative view outlined here, kidney diseases without genetic cause should be approached scientifically as metabolic diseases.
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Affiliation(s)
| | - Markus M Rinschen
- Department of Biomedicine, Aarhus University, Aarhus, Denmark;
- III. Department of Medicine and Hamburg Center for Kidney Health, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
- Aarhus Institute of Advanced Studies, Aarhus University, Aarhus, Denmark
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Kang MJ, Ioannou S, Lougheide Q, Dittmar M, Hsu Y, Pastor-Soler NM. The study of intercalated cells using ex vivo techniques: primary cell culture, cell lines, kidney slices, and organoids. Am J Physiol Cell Physiol 2024; 326:C229-C251. [PMID: 37899748 DOI: 10.1152/ajpcell.00479.2022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2022] [Revised: 10/23/2023] [Accepted: 10/24/2023] [Indexed: 10/31/2023]
Abstract
This review summarizes methods to study kidney intercalated cell (IC) function ex vivo. While important for acid-base homeostasis, IC dysfunction is often not recognized clinically until it becomes severe. The advantage of using ex vivo techniques is that they allow for the differential evaluation of IC function in controlled environments. Although in vitro kidney tubular perfusion is a classical ex vivo technique to study IC, here we concentrate on primary cell cultures, immortalized cell lines, and ex vivo kidney slices. Ex vivo techniques are useful in evaluating IC signaling pathways that allow rapid responses to extracellular changes in pH, CO2, and bicarbonate (HCO3-). However, these methods for IC work can also be challenging, as cell lines that recapitulate IC do not proliferate easily in culture. Moreover, a "pure" IC population in culture does not necessarily replicate its collecting duct (CD) environment, where ICs are surrounded by the more abundant principal cells (PCs). It is reassuring that many findings obtained in ex vivo IC systems signaling have been largely confirmed in vivo. Some of these newly identified signaling pathways reveal that ICs are important for regulating NaCl reabsorption, thus suggesting new frontiers to target antihypertensive treatments. Moreover, recent single-cell characterization studies of kidney epithelial cells revealed a dual developmental origin of IC, as well as the presence of novel CD cell types with certain IC characteristics. These exciting findings present new opportunities for the study of IC ex vivo and will likely rediscover the importance of available tools in this field.NEW & NOTEWORTHY The study of kidney intercalated cells has been limited by current cell culture and kidney tissue isolation techniques. This review is to be used as a reference to select ex vivo techniques to study intercalated cells. We focused on the use of cell lines and kidney slices as potential useful models to study membrane transport proteins. We also review how novel collecting duct organoids may help better elucidate the role of these intriguing cells.
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Affiliation(s)
- Min Ju Kang
- Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine of USC, Los Angeles, California, United States
| | - Silvia Ioannou
- Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine of USC, Los Angeles, California, United States
| | - Quinn Lougheide
- Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine of USC, Los Angeles, California, United States
| | - Michael Dittmar
- Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine of USC, Los Angeles, California, United States
| | - Young Hsu
- Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine of USC, Los Angeles, California, United States
| | - Nuria M Pastor-Soler
- Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine of USC, Los Angeles, California, United States
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5
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Tsuchiya H, Fujinoki M, Azuma M, Koshimizu TA. Vasopressin V1a receptor and oxytocin receptor regulate murine sperm motility differently. Life Sci Alliance 2023; 6:e202201488. [PMID: 36650057 PMCID: PMC9846835 DOI: 10.26508/lsa.202201488] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2022] [Revised: 01/06/2023] [Accepted: 01/06/2023] [Indexed: 01/19/2023] Open
Abstract
Specific receptors for the neurohypophyseal hormones, arginine vasopressin (AVP) and oxytocin, are present in the male reproductive organs. However, their exact roles remain unknown. To elucidate the physiological functions of pituitary hormones in male reproduction, this study first focused on the distribution and function of one of the AVP receptors, V1a. In situ hybridization analysis revealed high expression of the Avpr1a in Leydig cells of the testes and narrow/clear cells in the epididymis, with the expression pattern differing from that of the oxytocin receptor (OTR). Notably, persistent motility and highly proportional hyperactivation were observed in spermatozoa from V1a receptor-deficient mice. In contrast, OTR blocking by antagonist atosiban decreased hyperactivation rate. Furthermore, AVP stimulation could alter the extracellular pH mediated by the V1a receptor. The results highlight the crucial role of neurohypophyseal hormones in male reproductive physiology, with potential contradicting roles of V1a and OTR in sperm maturation. Our findings suggest that V1a receptor antagonists are potential therapeutic drugs for male infertility.
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Affiliation(s)
- Hiroyoshi Tsuchiya
- Division of Molecular Pharmacology, Department of Pharmacology, Jichi Medical University, Shimotsuke, Japan
| | - Masakatsu Fujinoki
- Research Center for Laboratory Animals, Comprehensive Research Facilities for Advanced Medical Science, School of Medicine, Dokkyo Medical University, Mibu, Japan
| | - Morio Azuma
- Division of Molecular Pharmacology, Department of Pharmacology, Jichi Medical University, Shimotsuke, Japan
| | - Taka-Aki Koshimizu
- Division of Molecular Pharmacology, Department of Pharmacology, Jichi Medical University, Shimotsuke, Japan
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Al-Bataineh MM, Kinlough CL, Marciszyn A, Lam T, Ye L, Kidd K, Maggiore JC, Poland PA, Kmoch S, Bleyer A, Bain DJ, Montalbetti N, Kleyman TR, Hughey RP, Ray EC. Influence of glycoprotein MUC1 on trafficking of the Ca 2+-selective ion channels, TRPV5 and TRPV6, and on in vivo calcium homeostasis. J Biol Chem 2023; 299:102925. [PMID: 36682497 PMCID: PMC9996365 DOI: 10.1016/j.jbc.2023.102925] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2022] [Revised: 01/09/2023] [Accepted: 01/12/2023] [Indexed: 01/22/2023] Open
Abstract
Polymorphism of the gene encoding mucin 1 (MUC1) is associated with skeletal and dental phenotypes in human genomic studies. Animals lacking MUC1 exhibit mild reduction in bone density. These phenotypes could be a consequence of modulation of bodily Ca homeostasis by MUC1, as suggested by the previous observation that MUC1 enhances cell surface expression of the Ca2+-selective channel, TRPV5, in cultured unpolarized cells. Using biotinylation of cell surface proteins, we asked whether MUC1 influences endocytosis of TRPV5 and another Ca2+-selective TRP channel, TRPV6, in cultured polarized epithelial cells. Our results indicate that MUC1 reduces endocytosis of both channels, enhancing cell surface expression. Further, we found that mice lacking MUC1 lose apical localization of TRPV5 and TRPV6 in the renal tubular and duodenal epithelium. Females, but not males, lacking MUC1 exhibit reduced blood Ca2+. However, mice lacking MUC1 exhibited no differences in basal urinary Ca excretion or Ca retention in response to PTH receptor signaling, suggesting compensation by transport mechanisms independent of TRPV5 and TRPV6. Finally, humans with autosomal dominant tubulointerstitial kidney disease due to frame-shift mutation of MUC1 (ADTKD-MUC1) exhibit reduced plasma Ca concentrations compared to control individuals with mutations in the gene encoding uromodulin (ADTKD-UMOD), consistent with MUC1 haploinsufficiency causing reduced bodily Ca2+. In summary, our results provide further insight into the role of MUC1 in Ca2+-selective TRP channel endocytosis and the overall effects on Ca concentrations.
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Affiliation(s)
- Mohammad M Al-Bataineh
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Carol L Kinlough
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Allison Marciszyn
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Tracey Lam
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Lorena Ye
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Kendrah Kidd
- Section on Nephrology, Department of Medicine, Wake Forest University, Winston-Salem, North Carolina, USA; Department of Paediatrics and Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Prague, Czech Republic
| | - Joseph C Maggiore
- Department of Developmental Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Paul A Poland
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Stanislav Kmoch
- Section on Nephrology, Department of Medicine, Wake Forest University, Winston-Salem, North Carolina, USA; Department of Paediatrics and Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Prague, Czech Republic
| | - Anthony Bleyer
- Section on Nephrology, Department of Medicine, Wake Forest University, Winston-Salem, North Carolina, USA; Department of Paediatrics and Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Prague, Czech Republic
| | - Daniel J Bain
- Department of Geology and Environmental Science, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Nicolas Montalbetti
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Thomas R Kleyman
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; Department of Cell Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Rebecca P Hughey
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; Department of Cell Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
| | - Evan C Ray
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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Hotait ZS, Lo Cascio JN, Choos END, Shepard BD. The sugar daddy: the role of the renal proximal tubule in glucose homeostasis. Am J Physiol Cell Physiol 2022; 323:C791-C803. [PMID: 35912988 PMCID: PMC9448277 DOI: 10.1152/ajpcell.00225.2022] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2022] [Revised: 07/26/2022] [Accepted: 07/26/2022] [Indexed: 11/22/2022]
Abstract
Renal blood flow represents >20% of total cardiac output and with this comes the great responsibility of maintaining homeostasis through the intricate regulation of solute handling. Through the processes of filtration, reabsorption, and secretion, the kidneys ensure that solutes and other small molecules are either returned to circulation, catabolized within renal epithelial cells, or excreted through the process of urination. Although this occurs throughout the renal nephron, one segment is tasked with the bulk of solute reabsorption-the proximal tubule. Among others, the renal proximal tubule is entirely responsible for the reabsorption of glucose, a critical source of energy that fuels the body. In addition, it is the only other site of gluconeogenesis outside of the liver. When these processes go awry, pathophysiological conditions such as diabetes and acidosis result. In this review, we highlight the recent advances made in understanding these processes that occur within the renal proximal tubule. We focus on the physiological mechanisms at play regarding glucose reabsorption and glucose metabolism, emphasize the conditions that occur under diseased states, and explore the emerging class of therapeutics that are responsible for restoring homeostasis.
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Affiliation(s)
- Zahraa S Hotait
- Department of Human Science, Georgetown University, Washington, District of Columbia
| | - Julia N Lo Cascio
- Department of Human Science, Georgetown University, Washington, District of Columbia
| | - Elijah N D Choos
- Department of Human Science, Georgetown University, Washington, District of Columbia
| | - Blythe D Shepard
- Department of Human Science, Georgetown University, Washington, District of Columbia
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8
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Cheval L, Viollet B, Klein C, Rafael C, Figueres L, Devevre E, Zadigue G, Azroyan A, Crambert G, Vogt B, Doucet A. Acidosis-induced activation of distal nephron principal cells triggers Gdf15 secretion and adaptive proliferation of intercalated cells. Acta Physiol (Oxf) 2021; 232:e13661. [PMID: 33840159 DOI: 10.1111/apha.13661] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2020] [Revised: 04/03/2021] [Accepted: 04/05/2021] [Indexed: 12/11/2022]
Abstract
AIM Type A intercalated cells of the renal collecting duct participate in the maintenance of the acid/base balance through their capacity to adapt proton secretion to homeostatic requirements. We previously showed that increased proton secretion stems in part from the enlargement of the population of proton secreting cells in the outer medullary collecting duct through division of fully differentiated cells, and that this response is triggered by growth/differentiation factor 15. This study aimed at deciphering the mechanism of acid load-induced secretion of Gdf15 and its mechanism of action. METHODS We developed an original method to evaluate the proliferation of intercalated cells and applied it to genetically modified or pharmacologically treated mice under basal and acid-loaded conditions. RESULTS Gdf15 is secreted by principal cells of the collecting duct in response to the stimulation of vasopressin receptors. Vasopressin-induced production of cAMP triggers activation of AMP-stimulated kinases and of Na,K-ATPase, and induction of p53 and Gdf15. Gdf15 action on intercalated cells is mediated by ErbB2 receptors, the activation of which triggers the expression of cyclin d1, of p53 and anti-proliferative genes, and of Egr1. CONCLUSION Acidosis-induced proliferation of intercalated cells results from a cross talk with principal cells which secrete Gdf15 in response to their stimulation by vasopressin. Thus, vasopressin is a major determinant of the collecting duct cellular homeostasis as it promotes proliferation of intercalated cells under acidosis conditions and of principal cells under normal acid-base status.
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Affiliation(s)
- Lydie Cheval
- Centre de Recherche des Cordeliers INSERMSorbonne UniversitéUniversité de Paris Paris France
- CNRS ERL 8228 ‐ Laboratoire de Physiologie Rénale et Tubulopathies Paris France
| | - Benoit Viollet
- Université de ParisInstitut CochinINSERMCNRS Paris France
| | - Christophe Klein
- Centre de Recherche des Cordeliers INSERMSorbonne UniversitéUniversité de Paris Paris France
| | - Chloé Rafael
- Centre de Recherche des Cordeliers INSERMSorbonne UniversitéUniversité de Paris Paris France
- CNRS ERL 8228 ‐ Laboratoire de Physiologie Rénale et Tubulopathies Paris France
| | - Lucile Figueres
- Centre de Recherche des Cordeliers INSERMSorbonne UniversitéUniversité de Paris Paris France
- CNRS ERL 8228 ‐ Laboratoire de Physiologie Rénale et Tubulopathies Paris France
| | - Estelle Devevre
- Centre de Recherche des Cordeliers INSERMSorbonne UniversitéUniversité de Paris Paris France
| | - Georges Zadigue
- Centre de Recherche des Cordeliers INSERMSorbonne UniversitéUniversité de Paris Paris France
| | - Anie Azroyan
- Program in Membrane Biology Nephrology Division Center for Systems Biology Massachusetts General Hospital Harvard Medical School Boston MA USA
| | - Gilles Crambert
- Centre de Recherche des Cordeliers INSERMSorbonne UniversitéUniversité de Paris Paris France
- CNRS ERL 8228 ‐ Laboratoire de Physiologie Rénale et Tubulopathies Paris France
| | - Bruno Vogt
- Department of Nephrology and Hypertension, Inselspital Bern University Hospital Bern Switzerland
| | - Alain Doucet
- Centre de Recherche des Cordeliers INSERMSorbonne UniversitéUniversité de Paris Paris France
- CNRS ERL 8228 ‐ Laboratoire de Physiologie Rénale et Tubulopathies Paris France
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Manzato MC, de Santi F, da Silva AAS, Beltrame FL, Cerri PS, Sasso‐Cerri E. Cimetidine-induced androgenic failure causes cell death and changes in actin, EGF and V-ATPase immunoexpression in rat submandibular glands. J Anat 2021; 239:136-150. [PMID: 33713423 PMCID: PMC8197950 DOI: 10.1111/joa.13408] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2020] [Revised: 01/31/2021] [Accepted: 02/01/2021] [Indexed: 01/18/2023] Open
Abstract
Submandibular gland (SMG) is responsive to androgens via androgen receptor (AR). We verified whether cimetidine induces androgenic dysfunction in SMG, and evaluated the structural integrity, cell death and immunoexpression of actin, EGF and V-ATPase in androgen-deficient SMG. Male rats received cimetidine (CMTG) and control animals (CG) received saline. Granular convoluted tubules (GCTs) diameter and number of acinar cell nuclei were evaluated. TUNEL and immunofluorescence reactions for detection of AR, testosterone, actin, EGF and V-ATPase were quantitatively analysed. In CG, testosterone immunolabelling was detected in acinar and ductal cells cytoplasm. AR-immunolabelled nuclei were observed in acinar cells whereas ductal cells showed AR-immunostained cytoplasm, indicating a non-genomic AR action. In CMTG, the weak testosterone and AR immunoexpression confirmed cimetidine-induced androgenic failure. A high cell death index was correlated with decreased number of acinar cells, GCTs diameter and EGF immunoexpression under androgenic dysfunction. Actin immunofluorescence decreased in the SMG cells, but an increased and diffuse cytoplasmic V-ATPase immunolabelling was observed in striated ducts, suggesting a disruption in the actin-dependent V-ATPase recycling due to androgenic failure. Our findings reinforce the androgenic role in the maintenance of SMG histophysiology, and point to a potential clinical use of cimetidine against androgen-dependent glandular tumour cells.
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Affiliation(s)
- Mariane Castro Manzato
- Department of Morphology, Genetics, Orthodontics and Pediatric DentistrySchool of DentistrySão Paulo State University (Unesp)AraraquaraBrazil
| | - Fabiane de Santi
- Department of Morphology and GeneticsFederal University of São PauloSão PauloBrazil
| | - André Acácio Souza da Silva
- Department of Morphology, Genetics, Orthodontics and Pediatric DentistrySchool of DentistrySão Paulo State University (Unesp)AraraquaraBrazil
| | - Flávia L. Beltrame
- Department of Morphology and GeneticsFederal University of São PauloSão PauloBrazil
| | - Paulo S. Cerri
- Department of Morphology, Genetics, Orthodontics and Pediatric DentistrySchool of DentistrySão Paulo State University (Unesp)AraraquaraBrazil
| | - Estela Sasso‐Cerri
- Department of Morphology, Genetics, Orthodontics and Pediatric DentistrySchool of DentistrySão Paulo State University (Unesp)AraraquaraBrazil
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Chu A, Zirngibl RA, Manolson MF. The V-ATPase a3 Subunit: Structure, Function and Therapeutic Potential of an Essential Biomolecule in Osteoclastic Bone Resorption. Int J Mol Sci 2021; 22:ijms22136934. [PMID: 34203247 PMCID: PMC8269383 DOI: 10.3390/ijms22136934] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2021] [Revised: 06/18/2021] [Accepted: 06/21/2021] [Indexed: 12/29/2022] Open
Abstract
This review focuses on one of the 16 proteins composing the V-ATPase complex responsible for resorbing bone: the a3 subunit. The rationale for focusing on this biomolecule is that mutations in this one protein account for over 50% of osteopetrosis cases, highlighting its critical role in bone physiology. Despite its essential role in bone remodeling and its involvement in bone diseases, little is known about the way in which this subunit is targeted and regulated within osteoclasts. To this end, this review is broadened to include the three other mammalian paralogues (a1, a2 and a4) and the two yeast orthologs (Vph1p and Stv1p). By examining the literature on all of the paralogues/orthologs of the V-ATPase a subunit, we hope to provide insight into the molecular mechanisms and future research directions specific to a3. This review starts with an overview on bone, highlighting the role of V-ATPases in osteoclastic bone resorption. We then cover V-ATPases in other location/functions, highlighting the roles which the four mammalian a subunit paralogues might play in differential targeting and/or regulation. We review the ways in which the energy of ATP hydrolysis is converted into proton translocation, and go in depth into the diverse role of the a subunit, not only in proton translocation but also in lipid binding, cell signaling and human diseases. Finally, the therapeutic implication of targeting a3 specifically for bone diseases and cancer is discussed, with concluding remarks on future directions.
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11
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Nakamura M, Satoh N, Tsukada H, Mizuno T, Fujii W, Suzuki A, Horita S, Nangaku M, Suzuki M. Stimulatory effect of insulin on H+-ATPase in the proximal tubule via the Akt/mTORC2 pathway. Physiol Int 2020; 107:376-389. [PMID: 32990653 DOI: 10.1556/2060.2020.00030] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2019] [Accepted: 06/12/2020] [Indexed: 11/19/2022]
Abstract
Purpose Acid-base transport in renal proximal tubules (PTs) is mainly sodium-dependent and conducted in coordination by the apical Na+/H+ exchanger (NHE3), vacuolar H+-adenosine triphosphatase (V-ATPase), and the basolateral Na+/HCO3- cotransporter. V-ATPase on PTs is well-known to play an important role in proton excretion. Recently we reported a stimulatory effect of insulin on these transporters. However, it is unclear whether insulin is involved in acid-base balance in PTs. Thus, we assessed the role of insulin in acid-base balance in PTs. Methods V-ATPase activity was evaluated using freshly isolated PTs obtained from mice, and specific inhibitors were then used to assess the signaling pathways involved in the observed effects. Results V-ATPase activity in PTs was markedly enhanced by insulin, and its activation was completely inhibited by bafilomycin (a V-ATPase-specific inhibitor), Akt inhibitor VIII, and PP242 (an mTORC1/2 inhibitor), but not by rapamycin (an mTORC1 inhibitor). V-ATPase activity was stimulated by 1 nm insulin by approximately 20% above baseline, which was completely suppressed by Akt1/2 inhibitor VIII. PP242 completely suppressed the insulin-mediated V-ATPase stimulation in mouse PTs, whereas rapamycin failed to influence the effect of insulin. Insulin-induced Akt phosphorylation in the mouse renal cortex was completely suppressed by Akt1/2 inhibitor VIII and PP242, but not by rapamycin. Conclusion Our results indicate that stimulation of V-ATPase activity by insulin in PTs is mediated via the Akt2/mTORC2 pathway. These results reveal the mechanism underlying the complex signaling in PT acid-base balance, providing treatment targets for renal disease.
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Affiliation(s)
- M Nakamura
- 1Division of Nephrology and Endocrinology, The University of Tokyo, Tokyo, Japan
| | - N Satoh
- 1Division of Nephrology and Endocrinology, The University of Tokyo, Tokyo, Japan
| | - H Tsukada
- 1Division of Nephrology and Endocrinology, The University of Tokyo, Tokyo, Japan
| | - T Mizuno
- 1Division of Nephrology and Endocrinology, The University of Tokyo, Tokyo, Japan
| | - W Fujii
- 1Division of Nephrology and Endocrinology, The University of Tokyo, Tokyo, Japan
| | - A Suzuki
- 1Division of Nephrology and Endocrinology, The University of Tokyo, Tokyo, Japan.,2Department of Nephrology, Japan Community Health care Organization (JCHO), Tokyo Yamate Medical Center, Tokyo, Japan
| | - S Horita
- 1Division of Nephrology and Endocrinology, The University of Tokyo, Tokyo, Japan
| | - M Nangaku
- 1Division of Nephrology and Endocrinology, The University of Tokyo, Tokyo, Japan
| | - M Suzuki
- 3Health Service Center, Tokyo Gakugei University, Tokyo, Japan
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12
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Kluge M, Namkoong E, Khakipoor S, Park K, Roussa E. Differential regulation of vacuolar H + -ATPase subunits by transforming growth factor-β1 in salivary ducts. J Cell Physiol 2019; 234:15061-15079. [PMID: 30648263 DOI: 10.1002/jcp.28147] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2018] [Accepted: 12/20/2018] [Indexed: 01/24/2023]
Abstract
Bicarbonate concentration in saliva is controlled by the action of acid-base transporters in salivary duct cells. We show for the first time expression of ATP6V1B1 in submandibular gland and introduce transforming growth factor-beta (TGF-β) as a novel regulator of V-ATPase subunits. Using QRT-PCR, immunoblotting, biotinylation of surface proteins, immunofluorescence, chromatin immunoprecipitation, and intracellular H(+ ) recording with H(+ )-sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein we show that in the human submandibular gland (HSG) cell line, activation of TGF-β signaling upregulates ATP6V1E1 and ATP6V1B2, downregulates ATP6V1B1, and has no effect on ATP6V1A. TGF-β1 effects on ATP6V1B1 are mediated through the canonical, the soluble adenylate cyclase, and ERK signaling. A CREB binding sequence was identified in the ATP6V1B1 promoter and CREB binding decreased after TGF-β1 treatment. Following acidosis, a bafilomycin-sensitive and Na+ -independent cell pH recovery was observed in HSG cells, an effect that was not influenced after disruption of acidic lysosomes. Moreover, neutralization of TGF-βs, inhibition of TGF-β receptor, or inhibition of the canonical pathway decreased membrane expression of ATP6V1A and prevented the acidosis-induced increased V-ATPase activity. The results suggest multiple modes of action of TGF-β1 on V-ATPase subunits in HSG cells: TGF-β1 may regulate transcription or protein synthesis of certain subunits and trafficking of other subunits in a context-dependent manner. Moreover, surface V-ATPase is active in salivary duct cells and involved in intracellular pH regulation following acidosis.
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Affiliation(s)
- Milena Kluge
- Department of Molecular Embryology, Faculty of Medicine, Institute of Anatomy and Cell Biology, University of Freiburg, Freiburg, Germany
| | - Eun Namkoong
- Department of Physiology, School of Dentistry, Seoul National University, Seoul, South Korea
| | - Shokoufeh Khakipoor
- Department of Molecular Embryology, Faculty of Medicine, Institute of Anatomy and Cell Biology, University of Freiburg, Freiburg, Germany
| | - Kyungpyo Park
- Department of Physiology, School of Dentistry, Seoul National University, Seoul, South Korea
| | - Eleni Roussa
- Department of Molecular Embryology, Faculty of Medicine, Institute of Anatomy and Cell Biology, University of Freiburg, Freiburg, Germany
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13
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Abstract
The epithelium of the kidney collecting duct (CD) is composed mainly of two different types of cells with distinct and complementary functions. CD principal cells traditionally have been considered to have a major role in Na+ and water regulation, while intercalated cells (ICs) were thought to largely modulate acid-base homeostasis. In recent years, our understanding of IC function has improved significantly owing to new research findings. Thus, we now have a new model for CD transport that integrates mechanisms of salt and water reabsorption, K+ homeostasis, and acid-base status between principal cells and ICs. There are three main types of ICs (type A, type B, and non-A, non-B), which first appear in the late distal convoluted tubule or in the connecting segment in a species-dependent manner. ICs can be detected in CD from cortex to the initial part of the inner medulla, although some transport proteins that are key components of ICs also are present in medullary CD, cells considered inner medullary. Of the three types of ICs, each has a distinct morphology and expresses different complements of membrane transport proteins that translate into very different functions in homeostasis and contributions to CD luminal pro-urine composition. This review includes recent discoveries in IC intracellular and paracrine signaling that contributes to acid-base regulation as well as Na+, Cl-, K+, and Ca2+ homeostasis. Thus, these new findings highlight the potential role of ICs as targets for potential hypertension treatments.
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Affiliation(s)
- Renee Rao
- University of Southern California/University Kidney Research Organization, Kidney Research Center, Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine of University of Southern California, Los Angeles, CA
| | - Vivek Bhalla
- Division of Nephrology, Department of Medicine, Stanford University School of Medicine, Stanford, CA
| | - Núria M Pastor-Soler
- University of Southern California/University Kidney Research Organization, Kidney Research Center, Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine of University of Southern California, Los Angeles, CA.
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14
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Ex vivo kidney slice preparations as a model system to study signaling cascades in kidney epithelial cells. Methods Cell Biol 2019; 153:185-203. [PMID: 31395379 DOI: 10.1016/bs.mcb.2019.04.017] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
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% CO2 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.
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15
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Lu X, Chan T, Cheng Z, Shams T, Zhu L, Murray M, Zhou F. The 5'-AMP-Activated Protein Kinase Regulates the Function and Expression of Human Organic Anion Transporting Polypeptide 1A2. Mol Pharmacol 2018; 94:1412-1420. [PMID: 30348897 DOI: 10.1124/mol.118.113423] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2018] [Accepted: 09/28/2018] [Indexed: 12/14/2022] Open
Abstract
The organic anion transporting polypeptides (OATPs) are important membrane proteins that mediate the cellular uptake of drugs and endogenous substances. OATP1A2 is widely distributed in many human tissues that are targeted in drug therapy; defective OATP1A2 leads to altered drug disposition influencing therapeutic outcomes. 5'-AMP-activated protein kinase (AMPK) signaling plays an important role in the pathogenesis of the metabolic syndrome characterized by an increased incidence of type II diabetes and nonalcoholic fatty liver disease. This study investigated the regulatory role of AMPK in OATP1A2 transport function and expression. We found that the treatment of AMPK-specific inhibitor compound C (dorsomorphin dihydrochloride) decreased OATP1A2-mediated uptake of estrone-3-sulfate in a concentration- and time-dependent manner. The impaired OATP1A2 function was associated with a reduced Vmax [154.6 ± 17.9 pmol × (μg × 4 minutes)-1 in compound C-treated cells vs. 413.6 ± 52.5 pmol × (μg × 4 minutes)-1 in controls]; the Km was unchanged. The cell-surface expression of OATP1A2 was decreased by compound C treatment, but total cellular expression was unchanged. The impaired cell-surface expression of OATP1A2 was associated with accelerated internalization and impaired targeting/recycling. Silencing of the AMPK α1-subunit using specific small interfering RNA corroborated the findings with compound C and revealed a role for AMPK in regulating OATP1A2 protein stability. Overall, this study implicated AMPK in the regulation of the function and expression of OATP1A2, which potentially impacts on the disposition of OATP1A2 drug substrates that may be used to treat patients with the metabolic syndrome and other diseases.
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Affiliation(s)
- Xiaoxi Lu
- School of Pharmacy (X.L., T.C., Z.C., T.S., F.Z.), Save Sight Institute (L.Z.), and Discipline of Pharmacology (M.M.), Faculty of Medicine and Health, University of Sydney, New South Wales, Australia; and Japanese Foundation for Cancer Research, Koto-ku, Tokyo, Japan (T.C.)
| | - Ting Chan
- School of Pharmacy (X.L., T.C., Z.C., T.S., F.Z.), Save Sight Institute (L.Z.), and Discipline of Pharmacology (M.M.), Faculty of Medicine and Health, University of Sydney, New South Wales, Australia; and Japanese Foundation for Cancer Research, Koto-ku, Tokyo, Japan (T.C.)
| | - Zhengqi Cheng
- School of Pharmacy (X.L., T.C., Z.C., T.S., F.Z.), Save Sight Institute (L.Z.), and Discipline of Pharmacology (M.M.), Faculty of Medicine and Health, University of Sydney, New South Wales, Australia; and Japanese Foundation for Cancer Research, Koto-ku, Tokyo, Japan (T.C.)
| | - Tahiatul Shams
- School of Pharmacy (X.L., T.C., Z.C., T.S., F.Z.), Save Sight Institute (L.Z.), and Discipline of Pharmacology (M.M.), Faculty of Medicine and Health, University of Sydney, New South Wales, Australia; and Japanese Foundation for Cancer Research, Koto-ku, Tokyo, Japan (T.C.)
| | - Ling Zhu
- School of Pharmacy (X.L., T.C., Z.C., T.S., F.Z.), Save Sight Institute (L.Z.), and Discipline of Pharmacology (M.M.), Faculty of Medicine and Health, University of Sydney, New South Wales, Australia; and Japanese Foundation for Cancer Research, Koto-ku, Tokyo, Japan (T.C.)
| | - Michael Murray
- School of Pharmacy (X.L., T.C., Z.C., T.S., F.Z.), Save Sight Institute (L.Z.), and Discipline of Pharmacology (M.M.), Faculty of Medicine and Health, University of Sydney, New South Wales, Australia; and Japanese Foundation for Cancer Research, Koto-ku, Tokyo, Japan (T.C.)
| | - Fanfan Zhou
- School of Pharmacy (X.L., T.C., Z.C., T.S., F.Z.), Save Sight Institute (L.Z.), and Discipline of Pharmacology (M.M.), Faculty of Medicine and Health, University of Sydney, New South Wales, Australia; and Japanese Foundation for Cancer Research, Koto-ku, Tokyo, Japan (T.C.)
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16
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AMP-Activated Protein Kinase (AMPK)-Dependent Regulation of Renal Transport. Int J Mol Sci 2018; 19:ijms19113481. [PMID: 30404151 PMCID: PMC6274953 DOI: 10.3390/ijms19113481] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2018] [Revised: 10/26/2018] [Accepted: 10/30/2018] [Indexed: 02/06/2023] Open
Abstract
AMP-activated kinase (AMPK) is a serine/threonine kinase that is expressed in most cells and activated by a high cellular AMP/ATP ratio (indicating energy deficiency) or by Ca2+. In general, AMPK turns on energy-generating pathways (e.g., glucose uptake, glycolysis, fatty acid oxidation) and stops energy-consuming processes (e.g., lipogenesis, glycogenesis), thereby helping cells survive low energy states. The functional element of the kidney, the nephron, consists of the glomerulus, where the primary urine is filtered, and the proximal tubule, Henle's loop, the distal tubule, and the collecting duct. In the tubular system of the kidney, the composition of primary urine is modified by the reabsorption and secretion of ions and molecules to yield final excreted urine. The underlying membrane transport processes are mainly energy-consuming (active transport) and in some cases passive. Since active transport accounts for a large part of the cell's ATP demands, it is an important target for AMPK. Here, we review the AMPK-dependent regulation of membrane transport along nephron segments and discuss physiological and pathophysiological implications.
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Whitton B, Okamoto H, Packham G, Crabb SJ. Vacuolar ATPase as a potential therapeutic target and mediator of treatment resistance in cancer. Cancer Med 2018; 7:3800-3811. [PMID: 29926527 PMCID: PMC6089187 DOI: 10.1002/cam4.1594] [Citation(s) in RCA: 84] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2018] [Revised: 05/07/2018] [Accepted: 05/08/2018] [Indexed: 01/10/2023] Open
Abstract
Vacuolar ATPase (V-ATPase) is an ATP-dependent H+ -transporter that pumps protons across intracellular and plasma membranes. It consists of a large multi-subunit protein complex and influences a wide range of cellular processes. This review focuses on emerging evidence for the roles for V-ATPase in cancer. This includes how V-ATPase dysregulation contributes to cancer growth, metastasis, invasion and proliferation, and the potential link between V-ATPase and the development of drug resistance.
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Affiliation(s)
- Bradleigh Whitton
- Southampton Cancer Research UK CentreUniversity of SouthamptonSouthamptonUK
- Biological SciencesFaculty of Natural and Environmental SciencesUniversity of SouthamptonSouthamptonUK
| | - Haruko Okamoto
- Biological SciencesFaculty of Natural and Environmental SciencesUniversity of SouthamptonSouthamptonUK
| | - Graham Packham
- Southampton Cancer Research UK CentreUniversity of SouthamptonSouthamptonUK
| | - Simon J. Crabb
- Southampton Cancer Research UK CentreUniversity of SouthamptonSouthamptonUK
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18
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Role of AMP-activated protein kinase in kidney tubular transport, metabolism, and disease. Curr Opin Nephrol Hypertens 2017; 26:375-383. [DOI: 10.1097/mnh.0000000000000349] [Citation(s) in RCA: 49] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
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19
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Mukherjee A, Wang Z, Kinlough CL, Poland PA, Marciszyn AL, Montalbetti N, Carattino MD, Butterworth MB, Kleyman TR, Hughey RP. Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel. J Biol Chem 2017; 292:4152-4163. [PMID: 28154191 DOI: 10.1074/jbc.m117.776146] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2017] [Indexed: 11/06/2022] Open
Abstract
The epithelial sodium channel (ENaC) has an important role in regulating extracellular fluid volume and blood pressure, as well as airway surface liquid volume and mucociliary clearance. ENaC is a trimer of three homologous subunits (α, β, and γ). We previously reported that cytoplasmic residues on the β (βCys-43 and βCys-557) and γ (γCys-33 and γCys-41) subunits are palmitoylated. Mutation of Cys that blocked ENaC palmitoylation also reduced channel open probability. Furthermore, γ subunit palmitoylation had a dominant role over β subunit palmitoylation in regulating ENaC. To determine which palmitoyltransferases (termed DHHCs) regulate the channel, mouse ENaCs were co-expressed in Xenopus oocytes with each of the 23 mouse DHHCs. ENaC activity was significantly increased by DHHCs 1, 2, 3, 7, and 14. ENaC activation by DHHCs was lost when γ subunit palmitoylation sites were mutated, whereas DHHCs 1, 2, and 14 still activated ENaC lacking β subunit palmitoylation sites. β subunit palmitoylation was increased by ENaC co-expression with DHHC 7. Both wild type ENaC and channels lacking β and γ palmitoylation sites co-immunoprecipitated with the five activating DHHCs, suggesting that ENaC forms a complex with multiple DHHCs. RT-PCR revealed that transcripts for the five activating DHHCs were present in cultured mCCDcl1 cells, and DHHC 3 was expressed in aquaporin 2-positive principal cells of mouse aldosterone-sensitive distal nephron where ENaC is localized. Treatment of polarized mCCDcl1 cells with a general inhibitor of palmitoylation reduced ENaC-mediated Na+ currents within minutes. Our results indicate that specific DHHCs have a role in regulating ENaC.
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Affiliation(s)
| | | | | | | | | | | | | | | | - Thomas R Kleyman
- From the Departments of Medicine, .,Cell Biology, and.,Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
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20
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Satoh N, Suzuki M, Nakamura M, Suzuki A, Horita S, Seki G, Moriya K. Functional coupling of V-ATPase and CLC-5. World J Nephrol 2017; 6:14-20. [PMID: 28101447 PMCID: PMC5215204 DOI: 10.5527/wjn.v6.i1.14] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/12/2016] [Revised: 10/04/2016] [Accepted: 11/02/2016] [Indexed: 02/06/2023] Open
Abstract
Dent’s disease is an X-linked renal tubulopathy characterized by low molecular weight proteinuria, hypercalciuria and progressive renal failure. Disease aetiology is associated with mutations in the CLCN5 gene coding for the electrogenic 2Cl-/H+ antiporter chloride channel 5 (CLC-5), which is expressed in the apical endosomes of renal proximal tubules with the vacuolar type H+-ATPase (V-ATPase). Initially identified as a member of the CLC family of Cl- channels, CLC-5 was presumed to provide Cl- shunt into the endosomal lumen to dissipate H+ accumulation by V-ATPase, thereby facilitating efficient endosomal acidification. However, recent findings showing that CLC-5 is in fact not a Cl- channel but a 2Cl-/H+ antiporter challenged this classical shunt model, leading to a renewed and intense debate on its physiological roles. Cl- accumulation via CLC-5 is predicted to play a critical role in endocytosis, as illustrated in mice carrying an artificial Cl- channel mutation E211A that developed defective endocytosis but normal endosomal acidification. Conversely, a recent functional analysis of a newly identified disease-causing Cl- channel mutation E211Q in a patient with typical Dent’s disease confirmed the functional coupling between V-ATPase and CLC-5 in endosomal acidification, lending support to the classical shunt model. In this editorial, we will address the current recognition of the physiological role of CLC-5 with a specific focus on the functional coupling of V-ATPase and CLC-5.
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21
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Emlet DR, Pastor-Soler N, Marciszyn A, Wen X, Gomez H, Humphries WH, Morrisroe S, Volpe JK, Kellum JA. Insulin-like growth factor binding protein 7 and tissue inhibitor of metalloproteinases-2: differential expression and secretion in human kidney tubule cells. Am J Physiol Renal Physiol 2016; 312:F284-F296. [PMID: 28003188 DOI: 10.1152/ajprenal.00271.2016] [Citation(s) in RCA: 98] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2016] [Revised: 11/11/2016] [Accepted: 11/28/2016] [Indexed: 01/04/2023] Open
Abstract
We have characterized the expression and secretion of the acute kidney injury (AKI) biomarkers insulin-like growth factor binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases-2 (TIMP-2) in human kidney epithelial cells in primary cell culture and tissue. We established cell culture model systems of primary kidney cells of proximal and distal tubule origin and observed that both proteins are indeed expressed and secreted in both tubule cell types in vitro. However, TIMP-2 is both expressed and secreted preferentially by cells of distal tubule origin, while IGFBP7 is equally expressed across tubule cell types yet preferentially secreted by cells of proximal tubule origin. In human kidney tissue, strong staining of IGFBP7 was seen in the luminal brush-border region of a subset of proximal tubule cells, and TIMP-2 stained intracellularly in distal tubules. Additionally, while some tubular colocalization of both biomarkers was identified with the injury markers kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin, both biomarkers could also be seen alone, suggesting the possibility for differential mechanistic and/or temporal profiles of regulation of these early AKI biomarkers from known markers of injury. Last, an in vitro model of ischemia-reperfusion demonstrated enhancement of secretion of both markers early after reperfusion. This work provides a rationale for further investigation of these markers for their potential role in the pathogenesis of acute kidney injury.
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Affiliation(s)
- David R Emlet
- Center for Critical Care Nephrology, Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Nuria Pastor-Soler
- Renal Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and
| | - Allison Marciszyn
- Renal Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and
| | - Xiaoyan Wen
- Center for Critical Care Nephrology, Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Hernando Gomez
- Center for Critical Care Nephrology, Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | | | - Seth Morrisroe
- Center for Critical Care Nephrology, Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Jacob K Volpe
- Center for Critical Care Nephrology, Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - John A Kellum
- Center for Critical Care Nephrology, Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; .,Renal Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and
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22
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Al-Bataineh MM, Li H, Ohmi K, Gong F, Marciszyn AL, Naveed S, Zhu X, Neumann D, Wu Q, Cheng L, Fenton RA, Pastor-Soler NM, Hallows KR. Activation of the metabolic sensor AMP-activated protein kinase inhibits aquaporin-2 function in kidney principal cells. Am J Physiol Renal Physiol 2016; 311:F890-F900. [PMID: 27534994 PMCID: PMC5130465 DOI: 10.1152/ajprenal.00308.2016] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2016] [Accepted: 08/15/2016] [Indexed: 11/22/2022] Open
Abstract
Aquaporin-2 (AQP2) is essential to maintain body water homeostasis. AQP2 traffics from intracellular vesicles to the apical membrane of kidney collecting duct principal cells in response to vasopressin [arginine vasopressin (AVP)], a hormone released with low intravascular volume, which causes decreased kidney perfusion. Decreased kidney perfusion activates AMP-activated kinase (AMPK), a metabolic sensor that inhibits the activity of several transport proteins. We hypothesized that AMPK activation also inhibits AQP2 function. These putative AMPK effects could protect interstitial ionic gradients required for urinary concentration during metabolic stress when low intravascular volume induces AVP release. Here we found that short-term AMPK activation by treatment with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR; 75 min) in kidney tissue prevented baseline AQP2 apical accumulation in principal cells, but did not prevent AQP2 apical accumulation in response to the AVP analog desmopressin (dDAVP). Prolonged AMPK activation prevented AQP2 cell membrane accumulation in response to forskolin in mouse collecting duct mpkCCDc14 cells. Moreover, AMPK inhibition accelerated hypotonic lysis of Xenopus oocytes expressing AQP2. We performed phosphorylation assays to elucidate the mechanism by which AMPK regulates AQP2. Although AMPK weakly phosphorylated immunoprecipitated AQP2 in vitro, no direct AMPK phosphorylation of the AQP2 COOH-terminus was detected by mass spectrometry. AMPK promoted Ser-261 phosphorylation and antagonized dDAVP-dependent phosphorylation of other AQP2 COOH-terminal sites in cells. Our findings suggest an increasing, time-dependent antagonism of AMPK on AQP2 regulation with AICAR-dependent inhibition of cAMP-dependent apical accumulation and AVP-dependent phosphorylation of AQP2. This inhibition likely occurs via a mechanism that does not involve direct AQP2 phosphorylation by AMPK.
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Affiliation(s)
- Mohammad M Al-Bataineh
- Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Hui Li
- Department of Medicine, University of Southern California/University Kidney Research Organization, Kidney Research Center, Division of Nephrology and Hypertension, Keck School of Medicine of University of Southern California, Los Angeles, California
| | - Kazuhiro Ohmi
- Department of Medicine, University of Southern California/University Kidney Research Organization, Kidney Research Center, Division of Nephrology and Hypertension, Keck School of Medicine of University of Southern California, Los Angeles, California
| | - Fan Gong
- Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Allison L Marciszyn
- Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Sajid Naveed
- Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Xiaoqing Zhu
- Department of Molecular Genetics, School for Cardiovascular Diseases, Maastricht University, Maastricht, The Netherlands; and
| | - Dietbert Neumann
- Department of Molecular Genetics, School for Cardiovascular Diseases, Maastricht University, Maastricht, The Netherlands; and
| | - Qi Wu
- Department of Biomedicine, InterPrET Center, Aarhus University, Aarhus, Denmark
| | - Lei Cheng
- Department of Biomedicine, InterPrET Center, Aarhus University, Aarhus, Denmark
| | - Robert A Fenton
- Department of Biomedicine, InterPrET Center, Aarhus University, Aarhus, Denmark
| | - Núria M Pastor-Soler
- Department of Medicine, University of Southern California/University Kidney Research Organization, Kidney Research Center, Division of Nephrology and Hypertension, Keck School of Medicine of University of Southern California, Los Angeles, California;
| | - Kenneth R Hallows
- Department of Medicine, University of Southern California/University Kidney Research Organization, Kidney Research Center, Division of Nephrology and Hypertension, Keck School of Medicine of University of Southern California, Los Angeles, California
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Cotter K, Stransky L, McGuire C, Forgac M. Recent Insights into the Structure, Regulation, and Function of the V-ATPases. Trends Biochem Sci 2016; 40:611-622. [PMID: 26410601 DOI: 10.1016/j.tibs.2015.08.005] [Citation(s) in RCA: 240] [Impact Index Per Article: 26.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2015] [Revised: 08/06/2015] [Accepted: 08/07/2015] [Indexed: 10/23/2022]
Abstract
The vacuolar (H(+))-ATPases (V-ATPases) are ATP-dependent proton pumps that acidify intracellular compartments and are also present at the plasma membrane. They function in such processes as membrane traffic, protein degradation, virus and toxin entry, bone resorption, pH homeostasis, and tumor cell invasion. V-ATPases are large multisubunit complexes, composed of an ATP-hydrolytic domain (V1) and a proton translocation domain (V0), and operate by a rotary mechanism. This review focuses on recent insights into their structure and mechanism, the mechanisms that regulate V-ATPase activity (particularly regulated assembly and trafficking), and the role of V-ATPases in processes such as cell signaling and cancer. These developments have highlighted the potential of V-ATPases as a therapeutic target in a variety of human diseases.
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Affiliation(s)
- Kristina Cotter
- Program in Cellular and Molecular Physiology, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA
| | - Laura Stransky
- Program in Cellular and Molecular Physiology, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA
| | - Christina McGuire
- Program in Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA
| | - Michael Forgac
- Program in Cellular and Molecular Physiology, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA; Program in Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA; Department of Developmental, Molecular, and Chemical Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA.
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Al-Bataineh MM, Kinlough CL, Poland PA, Pastor-Soler NM, Sutton TA, Mang HE, Bastacky SI, Gendler SJ, Madsen CS, Singh S, Monga SP, Hughey RP. Muc1 enhances the β-catenin protective pathway during ischemia-reperfusion injury. Am J Physiol Renal Physiol 2016; 310:F569-79. [PMID: 26739894 PMCID: PMC4796271 DOI: 10.1152/ajprenal.00520.2015] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2015] [Accepted: 01/03/2016] [Indexed: 11/22/2022] Open
Abstract
The hypoxia-inducible factor (HIF)-1 and β-catenin protective pathways represent the two most significant cellular responses that are activated in response to acute kidney injury. We previously reported that murine mucin (Muc)1 protects kidney function and morphology in a mouse model of ischemia-reperfusion injury (IRI) by stabilizing HIF-1α, enhancing HIF-1 downstream signaling, and thereby preventing metabolic stress (Pastor-Soler et al. Muc1 is protective during kidney ischemia-reperfusion injury. Am J Physiol Renal Physiol 308: F1452-F1462, 2015). We asked if Muc1 regulates the β-catenin protective pathway during IRI as 1) β-catenin nuclear targeting is MUC1 dependent in cultured human cells, 2) β-catenin is found in coimmunoprecipitates with human MUC1 in extracts of both cultured cells and tissues, and 3) MUC1 prevents β-catenin phosphorylation by glycogen synthase kinase (GSK)3β and thereby β-catenin degradation. Using the same mouse model of IRI, we found that levels of active GSK3β were significantly lower in kidneys of control mice compared with Muc1 knockout (KO) mice. Consequently, β-catenin was significantly upregulated at 24 and 72 h of recovery and appeared in the nuclear fraction at 72 h in control mouse kidneys. Both β-catenin induction and nuclear targeting were absent in Muc1 KO mice. We also found downstream induction of β-catenin prosurvival factors (activated Akt, survivin, transcription factor T cell factor 4 (TCF4), and its downstream target cyclin D1) and repression of proapoptotic factors (p53, active Bax, and cleaved caspase-3) in control mouse kidneys that were absent or aberrant in kidneys of Muc1 KO mice. Altogether, the data clearly indicate that Muc1 protection during acute kidney injury proceeds by enhancing both the HIF-1 and β-catenin protective pathways.
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Affiliation(s)
- Mohammad M Al-Bataineh
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Carol L Kinlough
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Paul A Poland
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Núria M Pastor-Soler
- Division of Nephrology and Hypertension, Department of Medicine, University of Southern California/UKRO Kidney Research Center, Keck School of Medicine of the University of Southern California, Los Angeles, California
| | - Timothy A Sutton
- Division of Nephrology, Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana
| | - Henry E Mang
- Division of Nephrology, Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana
| | - Sheldon I Bastacky
- Division of Anatomic Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Sandra J Gendler
- Department of Immunology and Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Comprehensive Cancer Center, Mayo Clinic in Arizona, Scottsdale, Arizona
| | - Cathy S Madsen
- Division of Anatomic Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Sucha Singh
- Division of Experimental Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Satdarshan P Monga
- Division of Experimental Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Rebecca P Hughey
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
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Al-Bataineh MM, Alzamora R, Ohmi K, Ho PY, Marciszyn AL, Gong F, Li H, Hallows KR, Pastor-Soler NM. Aurora kinase A activates the vacuolar H+-ATPase (V-ATPase) in kidney carcinoma cells. Am J Physiol Renal Physiol 2016; 310:F1216-28. [PMID: 26911844 DOI: 10.1152/ajprenal.00061.2016] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2016] [Accepted: 02/19/2016] [Indexed: 11/22/2022] Open
Abstract
Extracellular proton-secreting transport systems that contribute to extracellular pH include the vacuolar H(+)-ATPase (V-ATPase). This pump, which mediates ATP-driven transport of H(+) across membranes, is involved in metastasis. We previously showed (Alzamora R, Thali RF, Gong F, Smolak C, Li H, Baty CJ, Bertrand CA, Auchli Y, Brunisholz RA, Neumann D, Hallows KR, Pastor-Soler NM. J Biol Chem 285: 24676-24685, 2010) that V-ATPase A subunit phosphorylation at Ser-175 is important for PKA-induced V-ATPase activity at the membrane of kidney intercalated cells. However, Ser-175 is also located within a larger phosphorylation consensus sequence for Aurora kinases, which are known to phosphorylate proteins that contribute to the pathogenesis of metastatic carcinomas. We thus hypothesized that Aurora kinase A (AURKA), overexpressed in aggressive carcinomas, regulates the V-ATPase in human kidney carcinoma cells (Caki-2) via Ser-175 phosphorylation. We found that AURKA is abnormally expressed in Caki-2 cells, where it binds the V-ATPase A subunit in an AURKA phosphorylation-dependent manner. Treatment with the AURKA activator anacardic acid increased V-ATPase expression and activity at the plasma membrane of Caki-2 cells. In addition, AURKA phosphorylates the V-ATPase A subunit at Ser-175 in vitro and in Caki-2 cells. Immunolabeling revealed that anacardic acid induced marked membrane accumulation of the V-ATPase A subunit in transfected Caki-2 cells. However, anacardic acid failed to induce membrane accumulation of a phosphorylation-deficient Ser-175-to-Ala (S175A) A subunit mutant. Finally, S175A-expressing cells had decreased migration in a wound-healing assay compared with cells expressing wild-type or a phospho-mimetic Ser-175-to-Asp (S175D) mutant A subunit. We conclude that AURKA activates the V-ATPase in kidney carcinoma cells via phosphorylation of Ser-175 in the V-ATPase A subunit. This regulation contributes to kidney carcinoma V-ATPase-mediated extracellular acidification and cell migration.
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Affiliation(s)
- Mohammad M Al-Bataineh
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and
| | - Rodrigo Alzamora
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and
| | - Kazuhiro Ohmi
- Department of Medicine, USC/UKRO Kidney Research Center, Division of Nephrology and Hypertension, Keck School of Medicine of USC, Los Angeles, California
| | - Pei-Yin Ho
- Department of Medicine, USC/UKRO Kidney Research Center, Division of Nephrology and Hypertension, Keck School of Medicine of USC, Los Angeles, California
| | - Allison L Marciszyn
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and
| | - Fan Gong
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and
| | - Hui Li
- Department of Medicine, USC/UKRO Kidney Research Center, Division of Nephrology and Hypertension, Keck School of Medicine of USC, Los Angeles, California
| | - Kenneth R Hallows
- Department of Medicine, USC/UKRO Kidney Research Center, Division of Nephrology and Hypertension, Keck School of Medicine of USC, Los Angeles, California
| | - Núria M Pastor-Soler
- Department of Medicine, USC/UKRO Kidney Research Center, Division of Nephrology and Hypertension, Keck School of Medicine of USC, Los Angeles, California
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Abstract
The H(+) concentration in human blood is kept within very narrow limits, ~40 nmol/L, despite the fact that dietary metabolism generates acid and base loads that are added to the systemic circulation throughout the life of mammals. One of the primary functions of the kidney is to maintain the constancy of systemic acid-base chemistry. The kidney has evolved the capacity to regulate blood acidity by performing three key functions: (i) reabsorb HCO3(-) that is filtered through the glomeruli to prevent its excretion in the urine; (ii) generate a sufficient quantity of new HCO3(-) to compensate for the loss of HCO3(-) resulting from dietary metabolic H(+) loads and loss of HCO3(-) in the urea cycle; and (iii) excrete HCO3(-) (or metabolizable organic anions) following a systemic base load. The ability of the kidney to perform these functions requires that various cell types throughout the nephron respond to changes in acid-base chemistry by modulating specific ion transport and/or metabolic processes in a coordinated fashion such that the urine and renal vein chemistry is altered appropriately. The purpose of the article is to provide the interested reader with a broad review of a field that began historically ~60 years ago with whole animal studies, and has evolved to where we are currently addressing questions related to kidney acid-base regulation at the single protein structure/function level.
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Affiliation(s)
- Ira Kurtz
- Division of Nephrology, David Geffen School of Medicine, Los Angeles, CA; Brain Research Institute, UCLA, Los Angeles, CA
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Sun X, Stephens L, DuBose TD, Petrovic S. Adaptation by the collecting duct to an exogenous acid load is blunted by deletion of the proton-sensing receptor GPR4. Am J Physiol Renal Physiol 2015; 309:F120-36. [DOI: 10.1152/ajprenal.00507.2014] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2014] [Accepted: 05/05/2015] [Indexed: 12/14/2022] Open
Abstract
We previously reported that the deletion of the pH sensor GPR4 causes a non-gap metabolic acidosis and defective net acid excretion (NAE) in the GPR4 knockout mouse (GPR4−/−) (Sun X, Yang LV, Tiegs BC, Arend LJ, McGraw DW, Penn RB, and Petrovic S. J Am Soc Nephrol 21: 1745–1755, 2010). Since the major regulatory site of NAE in the kidney is the collecting duct (CD), we examined acid-base transport proteins in intercalated cells (ICs) of the CD and found comparable mRNA expression of kidney anion exchanger 1 (kAE1), pendrin, and the a4 subunit of H+-ATPase in GPR4−/− vs. +/+. However, NH4Cl loading elicited adaptive doubling of AE1 mRNA in GPR4+/+, but a 50% less pronounced response in GPR4−/−. In GPR4+/+, NH4Cl loading evoked a cellular response characterized by an increase in AE1-labeled and a decrease in pendrin-labeled ICs similar to what was reported in rabbits and rats. This response did not occur in GPR4−/−. Microperfusion experiments demonstrated that the activity of the basolateral Cl−/HCO3− exchanger, kAE1, in CDs isolated from GPR4−/− failed to increase with NH4Cl loading, in contrast to the increase observed in GPR4+/+. Therefore, the deficiency of GPR4 blunted, but did not eliminate the adaptive response to an acid load, suggesting a compensatory response from other pH/CO2/bicarbonate sensors. Indeed, the expression of the calcium-sensing receptor (CaSR) was nearly doubled in GPR4−/− kidneys, in the absence of apparent disturbances of Ca2+ homeostasis. In summary, the expression and activity of the key transport proteins in GPR4−/− mice are consistent with spontaneous metabolic acidosis, but the adaptive response to a superimposed exogenous acid load is blunted and might be partially compensated for by CaSR.
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Affiliation(s)
- Xuming Sun
- Department of Physiology and Pharmacology, Wake Forest School of Medicine, Winston-Salem, North Carolina
| | - Lisa Stephens
- Department of Internal Medicine, University of Cincinnati, Cincinnati, Ohio
| | - Thomas D. DuBose
- Section on Nephrology, Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina
| | - Snezana Petrovic
- Department of Physiology and Pharmacology, Wake Forest School of Medicine, Winston-Salem, North Carolina
- Section on Nephrology, Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina
- Claude D. Pepper Older Americans Independence Center, Wake Forest School of Medicine, Winston-Salem, North Carolina
- Hypertension and Vascular Research Center, Wake Forest School of Medicine, Winston-Salem, North Carolina; and
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28
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Roy A, Al-bataineh MM, Pastor-Soler NM. Collecting duct intercalated cell function and regulation. Clin J Am Soc Nephrol 2015; 10:305-24. [PMID: 25632105 DOI: 10.2215/cjn.08880914] [Citation(s) in RCA: 171] [Impact Index Per Article: 17.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Intercalated cells are kidney tubule epithelial cells with important roles in the regulation of acid-base homeostasis. However, in recent years the understanding of the function of the intercalated cell has become greatly enhanced and has shaped a new model for how the distal segments of the kidney tubule integrate salt and water reabsorption, potassium homeostasis, and acid-base status. These cells appear in the late distal convoluted tubule or in the connecting segment, depending on the species. They are most abundant in the collecting duct, where they can be detected all the way from the cortex to the initial part of the inner medulla. Intercalated cells are interspersed among the more numerous segment-specific principal cells. There are three types of intercalated cells, each having distinct structures and expressing different ensembles of transport proteins that translate into very different functions in the processing of the urine. This review includes recent findings on how intercalated cells regulate their intracellular milieu and contribute to acid-base regulation and sodium, chloride, and potassium homeostasis, thus highlighting their potential role as targets for the treatment of hypertension. Their novel regulation by paracrine signals in the collecting duct is also discussed. Finally, this article addresses their role as part of the innate immune system of the kidney tubule.
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Affiliation(s)
- Ankita Roy
- Renal-Electrolyte Division, Department of Medicine; and
| | | | - Núria M Pastor-Soler
- Renal-Electrolyte Division, Department of Medicine; and Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania A.R. and M.M.A. contributed equally to this work.
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