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Jeff M. Sands - One of the best experts on this subject based on the ideXlab platform.
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inhibition of Urea Transporter ameliorates uremic cardiomyopathy in chronic kidney disease
The FASEB Journal, 2020Co-Authors: Akihiro Kuma, Janet D. Klein, Xiaonan H Wang, Lin Tan, Nawazish Naqvi, Fitra Rianto, Ying Huang, Jeff M. SandsAbstract:Uremic cardiomyopathy, characterized by hypertension, cardiac hypertrophy, and fibrosis, is a complication of chronic kidney disease (CKD). Urea Transporter (UT) inhibition increases the excretion of water and Urea, but the effect on uremic cardiomyopathy has not been studied. We tested UT inhibition by dimethylthioUrea (DMTU) in 5/6 nephrectomy mice. This treatment suppressed CKD-induced hypertension and cardiac hypertrophy. In CKD mice, cardiac fibrosis was associated with upregulation of UT and vimentin abundance. Inhibition of UT suppressed vimentin amount. Left ventricular mass index in DMTU-treated CKD was less compared with non-treated CKD mice as measured by echocardiography. Nephrectomy was performed in UT-A1/A3 knockout (UT-KO) to further confirm our finding. UT-A1/A3 deletion attenuates the CKD-induced increase in cardiac fibrosis and hypertension. The amount of α-smooth muscle actin and tgf-β were significantly less in UT-KO with CKD than WT/CKD mice. To study the possibility that UT inhibition could benefit heart, we measured the mRNA of renin and angiotensin-converting enzyme (ACE), and found both were sharply increased in CKD heart; DMTU treatment and UT-KO significantly abolished these increases. Conclusion: Inhibition of UT reduced hypertension, cardiac fibrosis, and improved heart function. These changes are accompanied by inhibition of renin and ACE.
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E3 ligase MDM2 mediates Urea Transporter-A1 ubiquitination under either constitutive or stimulatory conditions.
American Journal of Physiology-Renal Physiology, 2019Co-Authors: Jeff M. Sands, Chun ZhangAbstract:Posttranslational modifications are essential for the regulation of Urea Transporter-A1 (UT-A1), among which ubiquitination is a rather attractive and complex issue. Previously, our group reported ...
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identification of a novel ut b Urea Transporter in human urothelial cancer
Frontiers in Physiology, 2017Co-Authors: Baoxue Yang, Jeff M. Sands, Ruida Hou, Mehrdad Alemozaffar, Xiangbo Kong, Guangping ChenAbstract:The Urea Transporter UT-B is widely expressed and has been studied in erythrocyte, kidney, brain and intestines. Interestingly, UT-B gene has been found more abundant in bladder than any other tissue. Recently, gene analyses demonstrate that SLC14A1 (UT-B) gene mutations are associated with bladder cancer, suggesting that Urea Transporter UT-B may play an important role in bladder carcinogenesis. In this study, we examined UT-B expression in bladder cancer with human primary bladder cancer tissues and cancer derived cell lines. Human UT-B has two isoforms. We found that normal bladder expresses long form of UT-B2 but was lost in 8 of 24 (33%) or significantly downregulated in 16 of 24 (67%) of primary bladder cancer patients. In contrast, the short form of UT-B1 lacking exon 3 was detected in 20 bladder cancer samples. Surprisingly, a 24-nt in-frame deletion in exon 4 in UT-B1 (UT-B1Δ24) was identified in 11 of 20 (55%) bladder tumors. This deletion caused a functional defect of UT-B1. Immunohistochemistry revealed that UT-B protein levels were significantly decreased in bladder cancers. Western blot analysis showed a weak UT-B band of 40 kDa in some tumors, consistent with UT-B1 gene expression detected by RT-PCR. Interestingly, bladder cancer associate UT-B1Δ24 was barely sialylated, reflecting impaired glycosylation of UT-B1 in bladder tumors. In conclusion, SLC14A1 gene and UT-B protein expression are significantly changed in bladder cancers. The aberrant UT-B expression may promote bladder cancer development or facilitate carcinogenesis induced by other carcinogens.
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Transgenic Restoration of Urea Transporter A1 Confers Maximal Urinary Concentration in the Absence of Urea Transporter A3
Journal of the American Society of Nephrology, 2015Co-Authors: Janet D. Klein, Yanhua Wang, Abinash C. Mistry, Lauren M. Larocque, Patrick A. Molina, Richard T. Rogers, Mitsi A. Blount, Jeff M. SandsAbstract:Urea has a critical role in urinary concentration. Mice lacking the inner medullary collecting duct (IMCD) Urea Transporter A1 (UT-A1) and Urea Transporter A3 (UT-A3) have very low levels of Urea permeability and are unable to concentrate urine. To investigate the role of UT-A1 in the concentration of urine, we transgenically expressed UT-A1 in knockout mice lacking UT-A1 and UT-A3 using a construct with a UT-A1 gene that cannot be spliced to produce UT-A3. This construct was inserted behind the original UT-A promoter to yield a mouse expressing only UT-A1 (UT-A1(+/+)/UT-A3(-/-)). Western blot analysis demonstrated UT-A1 in the inner medulla of UT-A1(+/+)/UT-A3(-/-) and wild-type mice, but not in UT-A1/UT-A3 knockout mice, and an absence of UT-A3 in UT-A1(+/+)/UT-A3(-/-) and UT-A1/UT-A3 knockout mice. Immunohistochemistry in UT-A1(+/+)/UT-A3(-/-) mice also showed negative UT-A3 staining in kidney and other tissues and positive UT-A1 staining only in the IMCD. Urea permeability in isolated perfused IMCDs showed basal permeability in the UT-A1(+/+)/UT-A3(-/-) mice was similar to levels in wild-type mice, but vasopressin stimulation of Urea permeability in wild-type mice was significantly greater (100% increase) than in UT-A1(+/+)/UT-A3(-/-) mice (8% increase). Notably, basal urine osmolalities in both wild-type and UT-A1(+/+)/UT-A3(-/-) mice increased upon overnight water restriction. We conclude that transgenic expression of UT-A1 restores basal Urea permeability to the level in wild-type mice but does not restore vasopressin-stimulated levels of Urea permeability. This information suggests that transgenic expression of UT-A1 alone in mice lacking UT-A1 and UT-A3 is sufficient to restore urine-concentrating ability.
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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.
Janet D. Klein - One of the best experts on this subject based on the ideXlab platform.
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inhibition of Urea Transporter ameliorates uremic cardiomyopathy in chronic kidney disease
The FASEB Journal, 2020Co-Authors: Akihiro Kuma, Janet D. Klein, Xiaonan H Wang, Lin Tan, Nawazish Naqvi, Fitra Rianto, Ying Huang, Jeff M. SandsAbstract:Uremic cardiomyopathy, characterized by hypertension, cardiac hypertrophy, and fibrosis, is a complication of chronic kidney disease (CKD). Urea Transporter (UT) inhibition increases the excretion of water and Urea, but the effect on uremic cardiomyopathy has not been studied. We tested UT inhibition by dimethylthioUrea (DMTU) in 5/6 nephrectomy mice. This treatment suppressed CKD-induced hypertension and cardiac hypertrophy. In CKD mice, cardiac fibrosis was associated with upregulation of UT and vimentin abundance. Inhibition of UT suppressed vimentin amount. Left ventricular mass index in DMTU-treated CKD was less compared with non-treated CKD mice as measured by echocardiography. Nephrectomy was performed in UT-A1/A3 knockout (UT-KO) to further confirm our finding. UT-A1/A3 deletion attenuates the CKD-induced increase in cardiac fibrosis and hypertension. The amount of α-smooth muscle actin and tgf-β were significantly less in UT-KO with CKD than WT/CKD mice. To study the possibility that UT inhibition could benefit heart, we measured the mRNA of renin and angiotensin-converting enzyme (ACE), and found both were sharply increased in CKD heart; DMTU treatment and UT-KO significantly abolished these increases. Conclusion: Inhibition of UT reduced hypertension, cardiac fibrosis, and improved heart function. These changes are accompanied by inhibition of renin and ACE.
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Transgenic Restoration of Urea Transporter A1 Confers Maximal Urinary Concentration in the Absence of Urea Transporter A3
Journal of the American Society of Nephrology, 2015Co-Authors: Janet D. Klein, Yanhua Wang, Abinash C. Mistry, Lauren M. Larocque, Patrick A. Molina, Richard T. Rogers, Mitsi A. Blount, Jeff M. SandsAbstract:Urea has a critical role in urinary concentration. Mice lacking the inner medullary collecting duct (IMCD) Urea Transporter A1 (UT-A1) and Urea Transporter A3 (UT-A3) have very low levels of Urea permeability and are unable to concentrate urine. To investigate the role of UT-A1 in the concentration of urine, we transgenically expressed UT-A1 in knockout mice lacking UT-A1 and UT-A3 using a construct with a UT-A1 gene that cannot be spliced to produce UT-A3. This construct was inserted behind the original UT-A promoter to yield a mouse expressing only UT-A1 (UT-A1(+/+)/UT-A3(-/-)). Western blot analysis demonstrated UT-A1 in the inner medulla of UT-A1(+/+)/UT-A3(-/-) and wild-type mice, but not in UT-A1/UT-A3 knockout mice, and an absence of UT-A3 in UT-A1(+/+)/UT-A3(-/-) and UT-A1/UT-A3 knockout mice. Immunohistochemistry in UT-A1(+/+)/UT-A3(-/-) mice also showed negative UT-A3 staining in kidney and other tissues and positive UT-A1 staining only in the IMCD. Urea permeability in isolated perfused IMCDs showed basal permeability in the UT-A1(+/+)/UT-A3(-/-) mice was similar to levels in wild-type mice, but vasopressin stimulation of Urea permeability in wild-type mice was significantly greater (100% increase) than in UT-A1(+/+)/UT-A3(-/-) mice (8% increase). Notably, basal urine osmolalities in both wild-type and UT-A1(+/+)/UT-A3(-/-) mice increased upon overnight water restriction. We conclude that transgenic expression of UT-A1 restores basal Urea permeability to the level in wild-type mice but does not restore vasopressin-stimulated levels of Urea permeability. This information suggests that transgenic expression of UT-A1 alone in mice lacking UT-A1 and UT-A3 is sufficient to restore urine-concentrating ability.
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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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activation of protein kinase c α and src kinase increases Urea Transporter a1 α 2 6 sialylation
Journal of The American Society of Nephrology, 2015Co-Authors: Baoxue Yang, Janet D. Klein, Jeff M. Sands, Minguang Chen, Guangping ChenAbstract:The Urea Transporter A1 (UT-A1) is a glycosylated protein with two glycoforms: 117 and 97 kD. In diabetes, the increased abundance of the heavily glycosylated 117-kD UT-A1 corresponds to an increase of kidney tubule Urea permeability. We previously reported that diabetes not only causes an increase of UT-A1 protein abundance but also, results in UT-A1 glycan changes, including an increase of sialic acid content. Because activation of the diacylglycerol (DAG)-protein kinase C (PKC) pathway is elevated in diabetes and PKC- α regulates UT-A1 Urea transport activity, we explored the role of PKC in UT-A1 glycan sialylation. We found that activation of PKC specifically promotes UT-A1 glycan sialylation in both UT-A1-MDCK cells and rat kidney inner medullary collecting duct suspensions, and inhibition of PKC activity blocks high glucose-induced UT-A1 sialylation. Overexpression of PKC- α promoted UT-A1 sialylation and membrane surface expression. Conversely, PKC- α –deficient mice had significantly less sialylated UT-A1 compared with wild-type mice. Furthermore, the effect of PKC- α –induced UT-A1 sialylation was mainly mediated by Src kinase but not Raf-1 kinase. Functionally, increased UT-A1 sialylation corresponded with enhanced Urea transport activity. Thus, our results reveal a novel mechanism by which PKC regulates UT-A1 function by increasing glycan sialylation through Src kinase pathways, which may have an important role in preventing the osmotic diuresis caused by glucosuria under diabetic conditions.
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mature n linked glycans facilitate ut a1 Urea Transporter lipid raft compartmentalization
The FASEB Journal, 2011Co-Authors: Guangping Chen, Janet D. Klein, Otto Fröhlich, Ashley G Howe, Jeff M. SandsAbstract:The UT-A1 Urea Transporter is a glycoprotein with two different glycosylated forms of 97 and 117 kDa. In this study, we found the 117-kDa UT-A1 preferentially resides in lipid rafts, suggesting that the glycosylation status may interfere with UT-A1 lipid raft trafficking. This was confirmed by a site-directed mutagenesis study in MDCK cells. The nonglycosylated UT-A1 showed reduced localization in lipid rafts. By using sugar-specific binding lectins, we further found that the UT-A1 in nonlipid rafts contained a high amount of mannose, as detected by concanavalin A, while the UT-A1 in lipid rafts was the mature N-acetylglucosamine-containing form, as detected by wheat germ agglutinin. In the inner medulla (IM) of diabetic rats, the more abundant 117-kDa UT-A1 in lipid rafts was the mature glycosylation form, with high amounts of N-acetylglucosamine and sialic acid. In contrast, in the IM of normal rats, the predominant 97-kDa UT-A1 was the form enriched in mannose. Functionally, inhibition of glycosylation...
Guangping Chen - One of the best experts on this subject based on the ideXlab platform.
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The Urea Transporter UT-A1 plays a predominant role in a Urea-dependent urine-concentrating mechanism.
Journal of Biological Chemistry, 2020Co-Authors: Xiaoqiang Geng, Hong Zhou, Guangping Chen, Shun Zhang, Yang BaoxueAbstract:Urea Transporters are a family of Urea-selective channel proteins expressed in multiple tissues and play an important role in the urine-concentrating mechanism of the mammalian kidney. Previous studies have shown that knockout of Urea Transporter-B (UT-B), UT-A1/A3, or all-UT lead to Urea-selective diuresis, indicating that Urea Transporters have important roles in urine concentration. Here, we sought to determine the role of UT-A1 in the urine-concentrating mechanism in a newly developed UT-A1-knockout mouse model. Phenotypically, daily urine output in UT-A1-knockout mice was nearly 3-fold that of wild-type mice and 82% of all-UT-knockout mice, and the UT-A1-knockout mice had significantly lower urine osmolality than wild-type mice. After 24 h water restriction, acute Urea loading, or high-protein (40%) intake, UT-A1 knockout mice were unable to increase urine-concentrating ability. Compared with all-UT-knockout mice, the UT-A1-null mice exhibited similarly elevated daily urine output and decreased urine osmolality, indicating impaired Urea-selective urine concentration. Our experimental findings reveal that UT-A1 has a predominant role in Urea-dependent urine-concentrating mechanisms, suggesting that UT-A1 represents a promising diuretic target.
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identification of a novel ut b Urea Transporter in human urothelial cancer
Frontiers in Physiology, 2017Co-Authors: Baoxue Yang, Jeff M. Sands, Ruida Hou, Mehrdad Alemozaffar, Xiangbo Kong, Guangping ChenAbstract:The Urea Transporter UT-B is widely expressed and has been studied in erythrocyte, kidney, brain and intestines. Interestingly, UT-B gene has been found more abundant in bladder than any other tissue. Recently, gene analyses demonstrate that SLC14A1 (UT-B) gene mutations are associated with bladder cancer, suggesting that Urea Transporter UT-B may play an important role in bladder carcinogenesis. In this study, we examined UT-B expression in bladder cancer with human primary bladder cancer tissues and cancer derived cell lines. Human UT-B has two isoforms. We found that normal bladder expresses long form of UT-B2 but was lost in 8 of 24 (33%) or significantly downregulated in 16 of 24 (67%) of primary bladder cancer patients. In contrast, the short form of UT-B1 lacking exon 3 was detected in 20 bladder cancer samples. Surprisingly, a 24-nt in-frame deletion in exon 4 in UT-B1 (UT-B1Δ24) was identified in 11 of 20 (55%) bladder tumors. This deletion caused a functional defect of UT-B1. Immunohistochemistry revealed that UT-B protein levels were significantly decreased in bladder cancers. Western blot analysis showed a weak UT-B band of 40 kDa in some tumors, consistent with UT-B1 gene expression detected by RT-PCR. Interestingly, bladder cancer associate UT-B1Δ24 was barely sialylated, reflecting impaired glycosylation of UT-B1 in bladder tumors. In conclusion, SLC14A1 gene and UT-B protein expression are significantly changed in bladder cancers. The aberrant UT-B expression may promote bladder cancer development or facilitate carcinogenesis induced by other carcinogens.
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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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activation of protein kinase c α and src kinase increases Urea Transporter a1 α 2 6 sialylation
Journal of The American Society of Nephrology, 2015Co-Authors: Baoxue Yang, Janet D. Klein, Jeff M. Sands, Minguang Chen, Guangping ChenAbstract:The Urea Transporter A1 (UT-A1) is a glycosylated protein with two glycoforms: 117 and 97 kD. In diabetes, the increased abundance of the heavily glycosylated 117-kD UT-A1 corresponds to an increase of kidney tubule Urea permeability. We previously reported that diabetes not only causes an increase of UT-A1 protein abundance but also, results in UT-A1 glycan changes, including an increase of sialic acid content. Because activation of the diacylglycerol (DAG)-protein kinase C (PKC) pathway is elevated in diabetes and PKC- α regulates UT-A1 Urea transport activity, we explored the role of PKC in UT-A1 glycan sialylation. We found that activation of PKC specifically promotes UT-A1 glycan sialylation in both UT-A1-MDCK cells and rat kidney inner medullary collecting duct suspensions, and inhibition of PKC activity blocks high glucose-induced UT-A1 sialylation. Overexpression of PKC- α promoted UT-A1 sialylation and membrane surface expression. Conversely, PKC- α –deficient mice had significantly less sialylated UT-A1 compared with wild-type mice. Furthermore, the effect of PKC- α –induced UT-A1 sialylation was mainly mediated by Src kinase but not Raf-1 kinase. Functionally, increased UT-A1 sialylation corresponded with enhanced Urea transport activity. Thus, our results reveal a novel mechanism by which PKC regulates UT-A1 function by increasing glycan sialylation through Src kinase pathways, which may have an important role in preventing the osmotic diuresis caused by glucosuria under diabetic conditions.
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activation of the camp pka pathway induces ut a1 Urea Transporter monoubiquitination and targets it for lysosomal degradation
American Journal of Physiology-renal Physiology, 2013Co-Authors: Minguang Chen, Jeff M. Sands, Guangping ChenAbstract:Regulation of Urea Transporter UT-A1 in the kidney is important for the urinary concentrating mechanism. We previously reported that activation of the cAMP/PKA pathway by forskolin (FSK) leads to UT-A1 ubiquitination, endocytosis, and degradation. In this study, we discovered that FSK-induced UT-A1 ubiquitination is monoubiquitination as judged by immunoblotting with specific ubiquitin antibodies to the different linkages of the ubiquitin chain. UT-A1 monoubiquitination induced by FSK was processed mainly on the cell plasma membrane. Monoubiquitination facilitates UT-A1 endocytosis, and internalized UT-A1 is accumulated in the early endosome. Inhibition of ubiquitination by E1 ubiquitin-activating enzyme inhibitor PYR-41 significantly reduced FSK-induced UT-A1 endocytosis and degradation. Interestingly, FSK-stimulated UT-A1 degradation occurs through a lysosomal protein degradation system. We further found that the PKA phosphorylation sites of UT-A1 at Ser486 and Ser499 are required for FSK-induced UT-A1 monoubiquitination. The physiological significance was confirmed using rat kidney inner medullary collecting duct suspensions, which showed that vasopressin treatment promotes UT-A1 ubiquitination. We conclude that unlike under basal conditions in which UT-A1 is subject to polyubiquitination and proteasome-mediated protein degradation, activation of UT-A1 by FSK induces UT-A1 monoubiquitination and protein lysosomal degradation.
Pascal Bailly - One of the best experts on this subject based on the ideXlab platform.
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UT-B1 Urea Transporter is expressed along the urinary and gastrointestinal tracts of the mouse
American Journal of Physiology-Regulatory Integrative and Comparative Physiology, 2005Co-Authors: Nicole Lucien, Pascal Bailly, Jean-pierre Cartron, Patrick Bruneval, François Lasbennes, Marie-france Belair, Chantal Mandet, Marie-marcelle Trinh-trang-tanAbstract:Selective Transporters account for rapid Urea transport across plasma membranes of several cell types. UT-B1 Urea Transporter is widely distributed in rat and human tissues. Because mice exhibit hi...
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antigenic and functional properties of the human red blood cell Urea Transporter hut b1
Journal of Biological Chemistry, 2002Co-Authors: Nicole Lucien, Jean-pierre Cartron, Pierre Ripoche, Frederic Sidouxwalter, Nathalie Roudier, Martine Huet, Mariemarcelle Trinhtrangtan, Pascal BaillyAbstract:Abstract The Kidd (JK) blood group locus encodes the Urea Transporter hUT-B1, which is expressed on human red blood cells and other tissues. The common JK*A/JK*B blood group polymorphism is caused by a single nucleotide transition G838A changing Asp-280 to Asn-280 on the polypeptide, and transfection of erythroleukemic K562 cells with hUT-B1 cDNAs carrying either the G838 or the A838 nucleotide substitutions resulted in the isolation of stable clones that expressed the Jka or Jkb antigens, respectively, thus providing the first direct demonstration that the hUT-B1 gene encodes the Kidd blood group antigens. In addition, immunochemical analysis of red blood cells demonstrated that hUT-B1 also exhibits ABO determinants attached to the single N-linked sugar chain at Asn-211. Moreover, immunoadsorption studies, using inside-out and right-side-out red cell membrane vesicles as competing antigen, demonstrated that the C- and N-terminal ends of hUT-B1 are oriented intracellularly. Mutagenesis and functional studies by expression in Xenopus oocytes revealed that both cysteines Cys-25 and Cys-30 (but not alone) are essential for plasma membrane addressing. Conversely, the transport function was not affected by the JK*A/JK*Bpolymorphism, C-terminal deletion (residues 360–389), or mutation of the extracellular N-glycosylation consensus site and remains poorly para-chloromercuribenzene sulfonate (pCMBS)-sensitive. However, transport studies by stopped flow light scattering using Jk-K562 transfectants demonstrated that the hUT-B1-mediated Urea transport is pCMBS-sensitive in an erythroid context, as reported previously for the Transporter of human red blood cells. Mutagenesis analysis also indicated that Cys-151 and Cys-236, at least alone, are not involved in pCMBS inhibition. Altogether, these antigenic, topologic, and functional properties might have implications into the physiology of hUT-B1 and other members of the Urea Transporter family.
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At physiological expression levels the Kidd blood group/Urea Transporter protein is not a water channel.
Journal of Biological Chemistry, 1999Co-Authors: Frédéric Sidoux-walter, Bernadette Olives, Jean-pierre Cartron, Pierre Ripoche, Germain Rousselet, Nicole Lucien, Renée Gobin, Erik-jan Kamsteeg, Peter M. T. Deen, Pascal BaillyAbstract:The Kidd (JK) blood group locus encodes a Urea Transporter that is expressed on human red cells and on endothelial cells of the vasa recta in the kidney. Here, we report the identification in human erythroblasts of a novel cDNA, designated HUT11A, which encodes a protein identical to the previously reported erythroid HUT11 Urea Transporter, except for a Lys(44) --> Glu substitution and a Val-Gly dipeptide deletion after proline 227, which leads to a polypeptide of 389 residues versus 391 in HUT11. Genomic typing by polymerase chain reaction and transcript analysis by ribonuclease protection assay demonstrated that HUT11A encodes the true Kidd blood group/Urea Transporter protein, which carries only 2 Val-Gly motifs. Upon expression at high levels in Xenopus oocytes, the physiological Kidd/Urea Transporter HUT11A conferred a rapid transfer of Urea (which was insensitive to p-chloromercuribenzene sulfonate or phloretin), a high water permeability, and a selective uptake of small solutes including amides and diols, but not glycerol and meso-erythritol. However, at plasma membrane expression levels close to the level observed in the red cell membrane, HUT11A-mediated water transport and small solutes uptake were absent and the Urea transport was poorly inhibited by p-chloromercuribenzene sulfonate, but strongly inhibited by phloretin. These findings show that, at physiological expression levels, the HUT11A Transporter confers Urea permeability but not water permeability, and that the observed water permeability is a feature of the red cell Urea Transporter when expressed at unphysiological high levels.
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at physiological expression levels the kidd blood group Urea Transporter protein is not a water channel
Journal of Biological Chemistry, 1999Co-Authors: Frederic Sidouxwalter, Bernadette Olives, Jean-pierre Cartron, Pierre Ripoche, Germain Rousselet, Nicole Lucien, Renée Gobin, Erik-jan Kamsteeg, Peter M. T. Deen, Pascal BaillyAbstract:The Kidd (JK) blood group locus encodes a Urea Transporter that is expressed on human red cells and on endothelial cells of the vasa recta in the kidney. Here, we report the identification in human erythroblasts of a novel cDNA, designated HUT11A, which encodes a protein identical to the previously reported erythroid HUT11 Urea Transporter, except for a Lys(44) --> Glu substitution and a Val-Gly dipeptide deletion after proline 227, which leads to a polypeptide of 389 residues versus 391 in HUT11. Genomic typing by polymerase chain reaction and transcript analysis by ribonuclease protection assay demonstrated that HUT11A encodes the true Kidd blood group/Urea Transporter protein, which carries only 2 Val-Gly motifs. Upon expression at high levels in Xenopus oocytes, the physiological Kidd/Urea Transporter HUT11A conferred a rapid transfer of Urea (which was insensitive to p-chloromercuribenzene sulfonate or phloretin), a high water permeability, and a selective uptake of small solutes including amides and diols, but not glycerol and meso-erythritol. However, at plasma membrane expression levels close to the level observed in the red cell membrane, HUT11A-mediated water transport and small solutes uptake were absent and the Urea transport was poorly inhibited by p-chloromercuribenzene sulfonate, but strongly inhibited by phloretin. These findings show that, at physiological expression levels, the HUT11A Transporter confers Urea permeability but not water permeability, and that the observed water permeability is a feature of the red cell Urea Transporter when expressed at unphysiological high levels.
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Molecular and functional characterization of an amphibian Urea Transporter
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1999Co-Authors: Cécile Couriaud, Pascal Bailly, Pierre Ripoche, Christine Leroy, Matthieu Simon, Claudia Silberstein, Germain RousseletAbstract:We report the characterization of a frog (Rana esculenta) Urea Transporter (fUT). The cloned cDNA is 1.4 kb long and contains a putative open reading frame of 1203 bp. In frog urinary bladder, the gene is expressed as two mRNAs of 4.3 and 1.6 kb. The fUT protein is 63.1 and 56.3% identical to rat UT-A2 and UT-B1, respectively. The internal duplication of UT-A2 and UT-B, as well as the double LP box Urea Transporter signature sequence were found in this amphibian Urea Transporter. When expressed in Xenopus oocytes, fUT induced a 10-fold increase in Urea permeability, which was blocked by both phloretin and mercurial reagents. The fUT protein did not transport thioUrea, but the fUT-mediated Urea transport was strongly inhibited by this compound. Thus, this amphibian Urea Transporter displays transport characteristics in between those of UT-A2 and UT-B.
Jean-pierre Cartron - One of the best experts on this subject based on the ideXlab platform.
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UT-B1 Urea Transporter is expressed along the urinary and gastrointestinal tracts of the mouse
AJP - Regulatory Integrative and Comparative Physiology, 2005Co-Authors: N. Lucien, Jean-pierre Cartron, Patrick Bruneval, François Lasbennes, Marie-france Belair, Chantal Mandet, P. Bailly, Marie-marcelle Trinh-trang-tanAbstract:Selective Transporters account for rapid Urea transport across plasma membranes of several cell types. UT-B1 Urea Transporter is widely distributed in rat and human tissues. Because mice exhibit high Urea turnover and are the preferred species for gene engineering, we have delineated UT-B1 tissue expression in murine tissues. A cDNA was cloned from BALB/c mouse kidney, encoding a polypeptide that differed from C57BL/6 mouse UT-B1 by one residue (Val-8-Ala). UT-B1 mRNA was detected by RT-PCR in brain, kidney, bladder, testis, lung, spleen, and digestive tract (liver, stomach, jejunum, colon). Northern blotting revealed seven UT-B1 transcripts in mouse tissues. Immunoblots identified a nonglycosylated UT-B1 protein of 29 kDa in most tissues and of 36 and 32 kDa in testis and liver, respectively. UT-B1 protein of gastrointestinal tract did not undergo N-glycosylation. Immunohistochemistry and in situ hybridization localized UT-B1 in urinary tract urothelium (papillary surface, ureter, bladder, and urethra), prominently on plasma membranes and restricted to the basolateral area in umbrella cells. UT-B1 was found in endothelial cells of descending vasa recta in kidney medulla and in astrocyte processes in brain. Dehydration induced by water deprivation for 2 days caused a tissue-specific decrease in UT-B1 abundance in the urinary bladder and the ureter.
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UT-B1 Urea Transporter is expressed along the urinary and gastrointestinal tracts of the mouse
American Journal of Physiology-Regulatory Integrative and Comparative Physiology, 2005Co-Authors: Nicole Lucien, Pascal Bailly, Jean-pierre Cartron, Patrick Bruneval, François Lasbennes, Marie-france Belair, Chantal Mandet, Marie-marcelle Trinh-trang-tanAbstract:Selective Transporters account for rapid Urea transport across plasma membranes of several cell types. UT-B1 Urea Transporter is widely distributed in rat and human tissues. Because mice exhibit hi...
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antigenic and functional properties of the human red blood cell Urea Transporter hut b1
Journal of Biological Chemistry, 2002Co-Authors: Nicole Lucien, Jean-pierre Cartron, Pierre Ripoche, Frederic Sidouxwalter, Nathalie Roudier, Martine Huet, Mariemarcelle Trinhtrangtan, Pascal BaillyAbstract:Abstract The Kidd (JK) blood group locus encodes the Urea Transporter hUT-B1, which is expressed on human red blood cells and other tissues. The common JK*A/JK*B blood group polymorphism is caused by a single nucleotide transition G838A changing Asp-280 to Asn-280 on the polypeptide, and transfection of erythroleukemic K562 cells with hUT-B1 cDNAs carrying either the G838 or the A838 nucleotide substitutions resulted in the isolation of stable clones that expressed the Jka or Jkb antigens, respectively, thus providing the first direct demonstration that the hUT-B1 gene encodes the Kidd blood group antigens. In addition, immunochemical analysis of red blood cells demonstrated that hUT-B1 also exhibits ABO determinants attached to the single N-linked sugar chain at Asn-211. Moreover, immunoadsorption studies, using inside-out and right-side-out red cell membrane vesicles as competing antigen, demonstrated that the C- and N-terminal ends of hUT-B1 are oriented intracellularly. Mutagenesis and functional studies by expression in Xenopus oocytes revealed that both cysteines Cys-25 and Cys-30 (but not alone) are essential for plasma membrane addressing. Conversely, the transport function was not affected by the JK*A/JK*Bpolymorphism, C-terminal deletion (residues 360–389), or mutation of the extracellular N-glycosylation consensus site and remains poorly para-chloromercuribenzene sulfonate (pCMBS)-sensitive. However, transport studies by stopped flow light scattering using Jk-K562 transfectants demonstrated that the hUT-B1-mediated Urea transport is pCMBS-sensitive in an erythroid context, as reported previously for the Transporter of human red blood cells. Mutagenesis analysis also indicated that Cys-151 and Cys-236, at least alone, are not involved in pCMBS inhibition. Altogether, these antigenic, topologic, and functional properties might have implications into the physiology of hUT-B1 and other members of the Urea Transporter family.
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At physiological expression levels the Kidd blood group/Urea Transporter protein is not a water channel.
Journal of Biological Chemistry, 1999Co-Authors: Frédéric Sidoux-walter, Bernadette Olives, Jean-pierre Cartron, Pierre Ripoche, Germain Rousselet, Nicole Lucien, Renée Gobin, Erik-jan Kamsteeg, Peter M. T. Deen, Pascal BaillyAbstract:The Kidd (JK) blood group locus encodes a Urea Transporter that is expressed on human red cells and on endothelial cells of the vasa recta in the kidney. Here, we report the identification in human erythroblasts of a novel cDNA, designated HUT11A, which encodes a protein identical to the previously reported erythroid HUT11 Urea Transporter, except for a Lys(44) --> Glu substitution and a Val-Gly dipeptide deletion after proline 227, which leads to a polypeptide of 389 residues versus 391 in HUT11. Genomic typing by polymerase chain reaction and transcript analysis by ribonuclease protection assay demonstrated that HUT11A encodes the true Kidd blood group/Urea Transporter protein, which carries only 2 Val-Gly motifs. Upon expression at high levels in Xenopus oocytes, the physiological Kidd/Urea Transporter HUT11A conferred a rapid transfer of Urea (which was insensitive to p-chloromercuribenzene sulfonate or phloretin), a high water permeability, and a selective uptake of small solutes including amides and diols, but not glycerol and meso-erythritol. However, at plasma membrane expression levels close to the level observed in the red cell membrane, HUT11A-mediated water transport and small solutes uptake were absent and the Urea transport was poorly inhibited by p-chloromercuribenzene sulfonate, but strongly inhibited by phloretin. These findings show that, at physiological expression levels, the HUT11A Transporter confers Urea permeability but not water permeability, and that the observed water permeability is a feature of the red cell Urea Transporter when expressed at unphysiological high levels.
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at physiological expression levels the kidd blood group Urea Transporter protein is not a water channel
Journal of Biological Chemistry, 1999Co-Authors: Frederic Sidouxwalter, Bernadette Olives, Jean-pierre Cartron, Pierre Ripoche, Germain Rousselet, Nicole Lucien, Renée Gobin, Erik-jan Kamsteeg, Peter M. T. Deen, Pascal BaillyAbstract:The Kidd (JK) blood group locus encodes a Urea Transporter that is expressed on human red cells and on endothelial cells of the vasa recta in the kidney. Here, we report the identification in human erythroblasts of a novel cDNA, designated HUT11A, which encodes a protein identical to the previously reported erythroid HUT11 Urea Transporter, except for a Lys(44) --> Glu substitution and a Val-Gly dipeptide deletion after proline 227, which leads to a polypeptide of 389 residues versus 391 in HUT11. Genomic typing by polymerase chain reaction and transcript analysis by ribonuclease protection assay demonstrated that HUT11A encodes the true Kidd blood group/Urea Transporter protein, which carries only 2 Val-Gly motifs. Upon expression at high levels in Xenopus oocytes, the physiological Kidd/Urea Transporter HUT11A conferred a rapid transfer of Urea (which was insensitive to p-chloromercuribenzene sulfonate or phloretin), a high water permeability, and a selective uptake of small solutes including amides and diols, but not glycerol and meso-erythritol. However, at plasma membrane expression levels close to the level observed in the red cell membrane, HUT11A-mediated water transport and small solutes uptake were absent and the Urea transport was poorly inhibited by p-chloromercuribenzene sulfonate, but strongly inhibited by phloretin. These findings show that, at physiological expression levels, the HUT11A Transporter confers Urea permeability but not water permeability, and that the observed water permeability is a feature of the red cell Urea Transporter when expressed at unphysiological high levels.
