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Guangping Chen - One of the best experts on this subject based on the ideXlab platform.
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rna seq analysis of glycosylation related gene expression in stz induced diabetic rat Kidney Inner Medulla
Frontiers in Physiology, 2015Co-Authors: Xiaoqian Qian, Janet D. Klein, Titilayo O Ilori, Rebecca P Hughey, Abdel A Alli, Zhengyu Guo, Xiang Song, Guangping ChenAbstract:The UT-A1 urea transporter is crucial to the Kidney’s ability to generate concentrated urine. Native UT-A1 from Kidney Inner Medulla (IM) is a heavily glycosylated protein with two glycosylation forms of 97 and 117 kDa. In diabetes, UT-A1 protein abundance, particularly the 117 kD isoform, is significantly increased corresponding to an increased urea permeability in perfused IM collecting ducts, which plays an important role in preventing the osmotic diuresis caused by glucosuria. However, how the glycan carbohydrate structure change and the glycan related enzymes regulate Kidney urea transport activity, particularly under diabetic condition, is largely unknown. In this study, using sugar-specific binding lectins, we found that the carbohydrate structure of UT-A1 is changed with increased amounts of sialic acid, fucose, and increased glycan branching under diabetic conditions. These changes were accompanied by altered UT-A1 association with the galectin proteins, α-galactoside glycan binding proteins. To explore the molecular basis of the alterations of glycan structures, the highly sensitive next generation sequencing (NGS) technology, Illumina RNA-seq, was employed to analyze genes involved in the process of UT-A1 glycosylation using streptozotocin (STZ) - induced diabetic rat Kidney. Differential gene expression analysis combining quantitative PCR revealed that expression of a number of important glycosylation related genes were changed under diabetic conditions. These genes include the glycosyltransferase genes Mgat4a, the sialylation enzymes St3gal1 and St3gal4 and glycan binding protein galectin-3, -5, -8 and -9. In contrast, although highly expressed in Kidney IM, the glycosyltransferase genes Mgat1, Mgat2, and fucosyltransferase Fut8, did not show any changes. Conclusions: In diabetes, not only is UT-A1 protein abundance increased but the protein’s glycan structure is also significantly changed. UT-A1 protein becomes highly sialylated, fucosylated and branched. Consistently, a number of crucial glycosylation related genes are changed under diabetic conditions. The alteration of these genes may contribute to changes in the UT-A1 glycan structure and therefore modulate Kidney urea transport activity and alleviate osmotic diuresis caused by glucosuria in diabetes.
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downregulation of urea transporter ut a1 activity by 14 3 3 protein
American Journal of Physiology-renal Physiology, 2015Co-Authors: Janet D. Klein, Jeff M. Sands, Xiuyan Feng, Hui Cai, Guangping ChenAbstract:Urea transporter (UT)-A1 in the Kidney Inner Medulla plays a critical role in the urinary concentrating mechanism and thereby in the regulation of water balance. The 14-3-3 proteins are a family of seven isoforms. They are multifunctional regulatory proteins that mainly bind to phosphorylated serine/threonine residues in target proteins. In the present study, we found that all seven 14-3-3 isoforms were detected in the Kidney Inner Medulla. However, only the 14-3-3 γ-isoform was specifically and highly associated with UT-A1, as demonstrated by a glutathione- S -transferase-14-3-3 pulldown assay. The cAMP/adenylyl cyclase stimulator forskolin significantly enhanced their binding. Coinjection of 14-3-3γ cRNA into oocytes resulted in a decrease of UT-A1 function. In addition, 14-3-3γ increased UT-A1 ubiquitination and protein degradation. 14-3-3γ can interact with both UT-A1 and mouse double minute 2, the E3 ubiquitin ligase for UT-A1. Thus, activation of cAMP/PKA increases 14-3-3γ interactions with UT-A1 and stimulates mouse double minute 2-mediated UT-A1 ubiquitination and degradation, thereby forming a novel regulatory mechanism of urea transport activity.
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glycoforms of ut a3 urea transporter with poly n acetyllactosamine glycosylation have enhanced transport activity
American Journal of Physiology-renal Physiology, 2012Co-Authors: Conner B Carter, Otto Fröhlich, Richard D Cummings, Guangping ChenAbstract:Urea transporters UT-A1 and UT-A3 are both expressed in the Kidney Inner Medulla. However, the function of UT-A3 remains unclear. Here, we found that UT-A3, which comprises only the NH2-terminal ha...
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the ut a1 urea transporter interacts with snapin a snare associated protein
Journal of Biological Chemistry, 2007Co-Authors: Abinash C. Mistry, Janet D. Klein, Guangping Chen, Otto Fröhlich, Rickta Mallick, Armin Rehm, Jeff M. SandsAbstract:The UT-A1 urea transporter mediates rapid transepithelial urea transport across the Inner Medullary collecting duct and plays a major role in the urinary concentrating mechanism. To transport urea, UT-A1 must be present in the plasma membrane. The purpose of this study was to screen for UT-A1-interacting proteins and to study the interactions of one of the identified potential binding partners with UT-A1. Using a yeast two-hybrid screen of a human Kidney cDNA library with the UT-A1 intracellular loop (residues 409-594) as bait, we identified snapin, a ubiquitously expressed SNARE-associated protein, as a novel UT-A1 binding partner. Deletion analysis indicated that the C-terminal coiled-coil domain (H2) of snapin is required for UT-A1 interaction. Snapin binds to the intracellular loop of UT-A1 but not to the N- or C-terminal fragments. Glutathione S-transferase pulldown experiments and co-immunoprecipitation studies verified that snapin interacts with native UT-A1, SNAP23, and syntaxin-4 (t-SNARE partners), indicating that UT-A1 participates with the SNARE machinery in rat Kidney Inner Medulla. Confocal microscopic analysis of immunofluorescent UT-A1 and snapin showed co-localization in both the cytoplasm and in the plasma membrane. When we co-injected UT-A1 with snapin cRNA in Xenopus oocytes, urea influx was significantly increased. In the absence of snapin, the influx was decreased when UT-A1 was combined with t-SNARE components syntaxin-4 and SNAP23. We conclude that UT-A1 may be linked to the SNARE machinery via snapin and that this interaction may be functionally and physiologically important for urea transport.
Janet D. Klein - One of the best experts on this subject based on the ideXlab platform.
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rna seq analysis of glycosylation related gene expression in stz induced diabetic rat Kidney Inner Medulla
Frontiers in Physiology, 2015Co-Authors: Xiaoqian Qian, Janet D. Klein, Titilayo O Ilori, Rebecca P Hughey, Abdel A Alli, Zhengyu Guo, Xiang Song, Guangping ChenAbstract:The UT-A1 urea transporter is crucial to the Kidney’s ability to generate concentrated urine. Native UT-A1 from Kidney Inner Medulla (IM) is a heavily glycosylated protein with two glycosylation forms of 97 and 117 kDa. In diabetes, UT-A1 protein abundance, particularly the 117 kD isoform, is significantly increased corresponding to an increased urea permeability in perfused IM collecting ducts, which plays an important role in preventing the osmotic diuresis caused by glucosuria. However, how the glycan carbohydrate structure change and the glycan related enzymes regulate Kidney urea transport activity, particularly under diabetic condition, is largely unknown. In this study, using sugar-specific binding lectins, we found that the carbohydrate structure of UT-A1 is changed with increased amounts of sialic acid, fucose, and increased glycan branching under diabetic conditions. These changes were accompanied by altered UT-A1 association with the galectin proteins, α-galactoside glycan binding proteins. To explore the molecular basis of the alterations of glycan structures, the highly sensitive next generation sequencing (NGS) technology, Illumina RNA-seq, was employed to analyze genes involved in the process of UT-A1 glycosylation using streptozotocin (STZ) - induced diabetic rat Kidney. Differential gene expression analysis combining quantitative PCR revealed that expression of a number of important glycosylation related genes were changed under diabetic conditions. These genes include the glycosyltransferase genes Mgat4a, the sialylation enzymes St3gal1 and St3gal4 and glycan binding protein galectin-3, -5, -8 and -9. In contrast, although highly expressed in Kidney IM, the glycosyltransferase genes Mgat1, Mgat2, and fucosyltransferase Fut8, did not show any changes. Conclusions: In diabetes, not only is UT-A1 protein abundance increased but the protein’s glycan structure is also significantly changed. UT-A1 protein becomes highly sialylated, fucosylated and branched. Consistently, a number of crucial glycosylation related genes are changed under diabetic conditions. The alteration of these genes may contribute to changes in the UT-A1 glycan structure and therefore modulate Kidney urea transport activity and alleviate osmotic diuresis caused by glucosuria in diabetes.
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downregulation of urea transporter ut a1 activity by 14 3 3 protein
American Journal of Physiology-renal Physiology, 2015Co-Authors: Janet D. Klein, Jeff M. Sands, Xiuyan Feng, Hui Cai, Guangping ChenAbstract:Urea transporter (UT)-A1 in the Kidney Inner Medulla plays a critical role in the urinary concentrating mechanism and thereby in the regulation of water balance. The 14-3-3 proteins are a family of seven isoforms. They are multifunctional regulatory proteins that mainly bind to phosphorylated serine/threonine residues in target proteins. In the present study, we found that all seven 14-3-3 isoforms were detected in the Kidney Inner Medulla. However, only the 14-3-3 γ-isoform was specifically and highly associated with UT-A1, as demonstrated by a glutathione- S -transferase-14-3-3 pulldown assay. The cAMP/adenylyl cyclase stimulator forskolin significantly enhanced their binding. Coinjection of 14-3-3γ cRNA into oocytes resulted in a decrease of UT-A1 function. In addition, 14-3-3γ increased UT-A1 ubiquitination and protein degradation. 14-3-3γ can interact with both UT-A1 and mouse double minute 2, the E3 ubiquitin ligase for UT-A1. Thus, activation of cAMP/PKA increases 14-3-3γ interactions with UT-A1 and stimulates mouse double minute 2-mediated UT-A1 ubiquitination and degradation, thereby forming a novel regulatory mechanism of urea transport activity.
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Cyclooxygenase-2 in the Kidney: good, BAD, or both?
Kidney international, 2011Co-Authors: S. Russ Price, Janet D. KleinAbstract:Hypertonic stress in the Kidney Inner Medulla is common, yet Inner Medullary cells adapt to limit cell death. Kuper et al. have identified a cell-survival response by which increased cyclooxygenase-2 (COX-2) stimulates a prostaglandin E 2 (PGE 2 )/protein kinase A (PKA)-mediated inactivation of the pro-apoptotic protein BAD. However, the PGE 2 /PKA pathway is not the only means to inactivate BAD and limit cell death. This Commentary shows a broader picture of this pathway to examine the Kidney's BAD options.
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the ut a1 urea transporter interacts with snapin a snare associated protein
Journal of Biological Chemistry, 2007Co-Authors: Abinash C. Mistry, Janet D. Klein, Guangping Chen, Otto Fröhlich, Rickta Mallick, Armin Rehm, Jeff M. SandsAbstract:The UT-A1 urea transporter mediates rapid transepithelial urea transport across the Inner Medullary collecting duct and plays a major role in the urinary concentrating mechanism. To transport urea, UT-A1 must be present in the plasma membrane. The purpose of this study was to screen for UT-A1-interacting proteins and to study the interactions of one of the identified potential binding partners with UT-A1. Using a yeast two-hybrid screen of a human Kidney cDNA library with the UT-A1 intracellular loop (residues 409-594) as bait, we identified snapin, a ubiquitously expressed SNARE-associated protein, as a novel UT-A1 binding partner. Deletion analysis indicated that the C-terminal coiled-coil domain (H2) of snapin is required for UT-A1 interaction. Snapin binds to the intracellular loop of UT-A1 but not to the N- or C-terminal fragments. Glutathione S-transferase pulldown experiments and co-immunoprecipitation studies verified that snapin interacts with native UT-A1, SNAP23, and syntaxin-4 (t-SNARE partners), indicating that UT-A1 participates with the SNARE machinery in rat Kidney Inner Medulla. Confocal microscopic analysis of immunofluorescent UT-A1 and snapin showed co-localization in both the cytoplasm and in the plasma membrane. When we co-injected UT-A1 with snapin cRNA in Xenopus oocytes, urea influx was significantly increased. In the absence of snapin, the influx was decreased when UT-A1 was combined with t-SNARE components syntaxin-4 and SNAP23. We conclude that UT-A1 may be linked to the SNARE machinery via snapin and that this interaction may be functionally and physiologically important for urea transport.
Shutish C. Patel - One of the best experts on this subject based on the ideXlab platform.
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Diminished expression of renal dopamine D1A receptors in the Kidney Inner Medulla of the spontaneously hypertensive rat.
Journal of Hypertension, 1998Co-Authors: Anita Sidhu, Ujendra Kumar, Shutish C. PatelAbstract:Background Dysfunctional dopamine neurotransmission and greater than normal retention of salt have been found for renal proximal tubules of the spontaneously hypertensive rat Objective To determine whether there are differences between Kidney D1 A dopamine receptor distributions of spontaneously hypertensive rats and Wistar-Kyoto rats. Methods We examined the expression of D1 A dopamine receptors in Kidneys of spontaneously hypertensive rats and the normotensive Wistar-Kyoto rat through Western blots and immunocytochemistry, using highly specific antipeptide antibodies directed against the receptor. Results The specificity of the antisera was demonstrated by Western blot studies, using proximal tubules, from Wistar-Kyoto rats. The antiserum recognized a major polypeptide with M r of 72 kDa and a minor protein of M r 66 kDa, which were not detected either by antigen-adsorbed or by preimmune sera. In renal cortex of both Wistar-Kyoto rats and spontaneously hypertensive rats, D1 A receptors were expressed at equivalent levels. In the Inner Medulla of Wistar-Kyoto rat, there was diminished (by 60%) expression of D1 A receptors compared with that of the renal cortex. However, the expression of D1 A receptors in the Inner Medulla in the spontaneously hypertensive rat was even more diminished (by 83%) relative to levels found in spontaneously hypertensive rat renal cortex. Immunocytochemical studies localized the D1 A receptor protein in renal cortex primarily to epithelia of tubules. Relative to renal cortex, there was an overall decrease in staining intensity in the Inner Medulla both of Wistar-Kyoto rats and of spontaneously hypertensive rats. Compared with that of Wistar-Kyoto rat, the intensity of staining of D1 A receptors in the Inner Medulla of spontaneously hypertensive rats was greatly diminished, confirming the Western blot analyses. The less than normal expression of D1 A receptors in the Inner Medulla of spontaneously hypertensive rats might be of physiologic importance in the etiology of greater than normal retention of salt and hypertension in spontaneously hypertensive rats.
Chinrang Yang - One of the best experts on this subject based on the ideXlab platform.
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an integrative proteogenomics approach reveals peptides encoded by annotated lincrna in the mouse Kidney Inner Medulla
Physiological Genomics, 2020Co-Authors: Cameron T Flower, Mark A Knepper, Hyun Jun Jung, Lihe Chen, Viswanathan Raghuram, Chinrang YangAbstract:Long noncoding RNAs (lncRNAs) are intracellular transcripts longer than 200 nucleotides and lack protein-coding information. A subclass of lncRNA known as long intergenic noncoding RNAs (lincRNAs) are transcribed from genomic regions that share no overlap with annotated protein-coding genes. Increasing evidence has shown that some annotated lincRNA transcripts do in fact contain open reading frames (ORFs) encoding functional short peptides in the cell. Few robust methods for lincRNA-encoded peptide identification have been reported, and the tissue-specific expression of these peptides has been largely unexplored. Here we propose an integrative workflow for lincRNA-encoded peptide discovery and test it on the mouse Kidney Inner Medulla (IM). In brief, low molecular weight protein fractions were enriched from homogenate of IMs and trypsinized into shorter peptides, which were sequenced by high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). To curate a hypothetical lincRNA-encoded peptide database for peptide-spectrum matching following LC-MS/MS, we performed RNA-Seq on IMs, computationally removed reads overlapping with annotated protein-coding genes, and remapped the remaining reads to a database of mouse noncoding transcripts to infer lincRNA expression. Expressed lincRNAs were searched for ORFs by an existing rule-based algorithm, and translated ORFs were used for peptide-spectrum matching. Peptides identified by LC-MS/MS were further evaluated by using several quality control criteria and bioinformatics methods. We discovered three novel lincRNA-encoded peptides, which are conserved in mouse, rat, and human. The workflow can be adapted for discovery of small protein-coding genes in any species or tissue where noncoding transcriptome information is available.
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an integrative proteogenomics approach reveals peptides encoded by annotated lincrna in the mouse Kidney Inner Medulla
Physiological Genomics, 2020Co-Authors: Cameron T Flower, Mark A Knepper, Hyun Jun Jung, Lihe Chen, Viswanathan Raghuram, Chinrang YangAbstract:Long noncoding RNAs (lncRNAs) are intracellular transcripts longer than 200 nucleotides and lack protein-coding information. A subclass of lncRNA known as long intergenic noncoding RNAs (lincRNAs) ...
Jeff M. Sands - One of the best experts on this subject based on the ideXlab platform.
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downregulation of urea transporter ut a1 activity by 14 3 3 protein
American Journal of Physiology-renal Physiology, 2015Co-Authors: Janet D. Klein, Jeff M. Sands, Xiuyan Feng, Hui Cai, Guangping ChenAbstract:Urea transporter (UT)-A1 in the Kidney Inner Medulla plays a critical role in the urinary concentrating mechanism and thereby in the regulation of water balance. The 14-3-3 proteins are a family of seven isoforms. They are multifunctional regulatory proteins that mainly bind to phosphorylated serine/threonine residues in target proteins. In the present study, we found that all seven 14-3-3 isoforms were detected in the Kidney Inner Medulla. However, only the 14-3-3 γ-isoform was specifically and highly associated with UT-A1, as demonstrated by a glutathione- S -transferase-14-3-3 pulldown assay. The cAMP/adenylyl cyclase stimulator forskolin significantly enhanced their binding. Coinjection of 14-3-3γ cRNA into oocytes resulted in a decrease of UT-A1 function. In addition, 14-3-3γ increased UT-A1 ubiquitination and protein degradation. 14-3-3γ can interact with both UT-A1 and mouse double minute 2, the E3 ubiquitin ligase for UT-A1. Thus, activation of cAMP/PKA increases 14-3-3γ interactions with UT-A1 and stimulates mouse double minute 2-mediated UT-A1 ubiquitination and degradation, thereby forming a novel regulatory mechanism of urea transport activity.
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the ut a1 urea transporter interacts with snapin a snare associated protein
Journal of Biological Chemistry, 2007Co-Authors: Abinash C. Mistry, Janet D. Klein, Guangping Chen, Otto Fröhlich, Rickta Mallick, Armin Rehm, Jeff M. SandsAbstract:The UT-A1 urea transporter mediates rapid transepithelial urea transport across the Inner Medullary collecting duct and plays a major role in the urinary concentrating mechanism. To transport urea, UT-A1 must be present in the plasma membrane. The purpose of this study was to screen for UT-A1-interacting proteins and to study the interactions of one of the identified potential binding partners with UT-A1. Using a yeast two-hybrid screen of a human Kidney cDNA library with the UT-A1 intracellular loop (residues 409-594) as bait, we identified snapin, a ubiquitously expressed SNARE-associated protein, as a novel UT-A1 binding partner. Deletion analysis indicated that the C-terminal coiled-coil domain (H2) of snapin is required for UT-A1 interaction. Snapin binds to the intracellular loop of UT-A1 but not to the N- or C-terminal fragments. Glutathione S-transferase pulldown experiments and co-immunoprecipitation studies verified that snapin interacts with native UT-A1, SNAP23, and syntaxin-4 (t-SNARE partners), indicating that UT-A1 participates with the SNARE machinery in rat Kidney Inner Medulla. Confocal microscopic analysis of immunofluorescent UT-A1 and snapin showed co-localization in both the cytoplasm and in the plasma membrane. When we co-injected UT-A1 with snapin cRNA in Xenopus oocytes, urea influx was significantly increased. In the absence of snapin, the influx was decreased when UT-A1 was combined with t-SNARE components syntaxin-4 and SNAP23. We conclude that UT-A1 may be linked to the SNARE machinery via snapin and that this interaction may be functionally and physiologically important for urea transport.
