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Rikke Nielsen - One of the best experts on this subject based on the ideXlab platform.
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endocytic receptors in the renal Proximal Tubule
Physiology, 2012Co-Authors: Erik Ilsø Christensen, Henrik Birn, Tina Storm, Kathrin Weyer, Rikke NielsenAbstract:Protein reabsorption is a predominant feature of the renal Proximal Tubule. Animal studies show that the ability to rescue plasma proteins relies on the endocytic receptors megalin and cubilin. Recently, studies of patients with syndromes caused by dysfunctional receptors have supported the importance of these for protein clearance of human ultrafiltrate. This review focuses on the molecular biology and physiology of the receptors and their involvement in renal pathological conditions.
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cubilin is essential for albumin reabsorption in the renal Proximal Tubule
Journal of The American Society of Nephrology, 2010Co-Authors: Sabine Amsellem, Ghislaine Hamard, Jakub Gburek, Rikke Nielsen, Thomas E Willnow, Erik Ilsø Christensen, Olivier Devuyst, Ebba Nexo, Renata KozyrakiAbstract:Receptor-mediated endocytosis is responsible for protein reabsorption in the Proximal Tubule. This process involves two interacting receptors, megalin and cubilin, which form a complex with amnionless. Whether these proteins function in parallel or as part of an integrated system is not well understood. Here, we report the renal effects of genetic ablation of cubilin, with or without concomitant ablation of megalin, using a conditional Cre-loxP system. We observed that Proximal Tubule cells did not localize amnionless to the plasma membrane in the absence of cubilin, indicating a mutual dependency of cubilin and amnionless to form a functional membrane receptor complex. The cubilin-amnionless complex mediated internalization of intrinsic factor-vitamin B12 complexes, but megalin considerably increased the uptake. Furthermore, cubilin-deficient mice exhibited markedly decreased uptake of albumin by Proximal Tubule cells and resultant albuminuria. Inactivation of both megalin and cubilin did not increase albuminuria, indicating that the main role of megalin in albumin reabsorption is to drive the internalization of cubilin-albumin complexes. In contrast, cubulin deficiency did not affect urinary tubular uptake or excretion of vitamin D-binding protein (DBP), which binds cubilin and megalin. In addition, we observed cubilin-independent reabsorption of the "specific" cubilin ligands transferrin, CC16, and apoA-I, suggesting a role for megalin and perhaps other receptors in their reabsorption. In summary, with regard to albumin, cubilin is essential for its reabsorption by Proximal Tubule cells, and megalin drives internalization of cubilin-albumin complexes. These genetic models will allow further analysis of protein trafficking in the progression of proteinuric renal diseases.
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Receptor-mediated endocytosis in renal Proximal Tubule
Pflügers Archiv - European Journal of Physiology, 2009Co-Authors: Erik Ilsø Christensen, Pierre J. Verroust, Rikke NielsenAbstract:Proteins filtered in renal glomeruli are removed from the tubular fluid by endocytosis in the Proximal Tubule mediated by the two receptors megalin and cubilin. After endocytic uptake, the proteins are transferred to lysosomes for degradation, while the receptors are returned to the apical cell membrane by receptor recycling in dense apical Tubules. In the renal Proximal Tubule, there is no significant transcellular transport of protein. The reabsorptive process is extremely efficient as evidenced by the virtual protein free urine in humans. The two receptors bind a variety of ligands. The process serves not only to remove the proteins from the ultrafiltrate but also to conserve a variety of essential substances such as vitamins and trace elements carried by plasma proteins. The endocytic apparatus is highly developed in the Proximal Tubule demonstrating the high capacity of the cells; however, under certain circumstances like diseases affecting the glomeruli, the system is overloaded resulting in proteinuria.
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transcellular transport of vitamin b 12 in llc pk1 renal Proximal Tubule cells
Journal of The American Society of Nephrology, 2001Co-Authors: Rikke Nielsen, Boe Sandahl Sorensen, Henrik Birn, Erik Ilsø Christensen, Ebba NexoAbstract:Abstract . The transcobalamin-vitamin B 12 complex is responsible for the transport of B 12 from plasma and into the tissues. The complex is filtered in the renal glomeruli and is a high-affinity ligand for the endocytic receptor megalin expressed in the Proximal Tubule. This study shows by the use of the Proximal Tubule LLC-PK1 cell line that transcobalamin-B 12 is internalized by megalin-mediated endocytosis. After endocytosis and accumulation in endosomes, transcobalamin is degraded and the B 12 molecule is released from the cells in complex with newly synthesized proteins. The release is polarized in such a way that vitamin in the apical medium is bound to proteins with the size of haptocorrin, whereas the B 12 released at the basolateral side is complexed to two different proteins with the sizes of transcobalamin and haptocorrin. Furthermore, transcobalamin mRNA was identified by reverse transcription-PCR in LLC-PK1 cells and human and pig kidney, whereas haptocorrin mRNA was identified only in LLC-PK1 cells. The results strongly suggest that megalin located in the Proximal Tubule cells is important for receptor-mediated tubular reabsorption followed by transcellular transport and release of vitamin B 12 complexed to newly synthesized carrier proteins. This mechanism is likely to play a significant role in the maintenance of B 12 homeostasis by returning filtered B 12 to the pool of circulating vitamin.
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cubilin and megalin mediated uptake of albumin in cultured Proximal Tubule cells of opossum kidney
Kidney International, 2000Co-Authors: Rikke Nielsen, Henrik Birn, Xiao-yue Zhai, Karina DrummAbstract:Cubilin- and megalin-mediated uptake of albumin in cultured Proximal Tubule cells of opossum kidney. Background Reabsorption of albumin from the glomerular filtrate occurs via receptor-mediated endocytosis in the Proximal Tubule. This process is initiated by binding of albumin in apical clathrin-coated pits, followed by endocytosis and degradation in lysosomes. Although binding sites have been characterized by kinetic studies, the receptors responsible for the binding of albumin have not been fully identified. Two giant glycoproteins, cubilin and megalin, constitute important endocytic receptors localized to the kidney Proximal Tubule. Methods In the present study, we examined the colocalization of cubilin and megalin in the endocytic pathway and the relationship between the uptake of albumin and the expression of cubilin and megalin in opossum kidney (OK) Proximal Tubule cells by immunocytochemistry and immunoblotting. Results OK cells expressed both cubilin and megalin. The light microscope labeling patterns for cubilin and megalin were almost identical and were mainly located at the surface area of the cells. Cubilin and megalin were also shown to colocalize on cell surface microvilli, in coated pits, and in endocytic compartments at the electron microscope level. Endocytosed bovine serum albumin (BSA) was identified exclusively in cells expressing megalin and cubilin. Uptake of BSA-FITC was saturable and inhibited by receptor-associated protein (RAP) and by intrinsic factor-vitamin B 12 complex (IF-B 12 ) at high concentrations. Significant inhibition was also observed by specific antibodies to cubilin, and megalin and cubilin antisense oligonucleotides likewise significantly reduced albumin uptake. Egg albumin did not affect the uptake of BSA. Conclusion The present observations suggest that the two receptors cubilin and megalin are both involved in the endocytic uptake of albumin in renal Proximal Tubule cells.
Erik Ilsø Christensen - One of the best experts on this subject based on the ideXlab platform.
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tubular proteinuria in patients with hnf1α mutations hnf1α drives endocytosis in the Proximal Tubule
Kidney International, 2016Co-Authors: Sara Terryn, Renata Kozyraki, Karo Tanaka, Jeanphilippe Lengele, Daniele Duboislaforgue, Serge Garbay, Eric Olinger, Patrick Van Der Smissen, Erik Ilsø Christensen, Pierre J CourtoyAbstract:Hepatocyte nuclear factor 1α (HNF1α) is a transcription factor expressed in the liver, pancreas, and Proximal Tubule of the kidney. Mutations of HNF1α cause an autosomal dominant form of diabetes mellitus (MODY-HNF1A) and tubular dysfunction. To gain insights into the role of HNF1α in the Proximal Tubule, we analyzed Hnf1a- deficient mice. Compared with wild-type littermates, Hnf1a knockout mice showed low-molecular-weight proteinuria and a 70% decrease in the uptake of β 2 -microglobulin, indicating a major endocytic defect due to decreased expression of megalin/cubilin receptors. We identified several binding sites for HNF1α in promoters of Lrp2 and Cubn genes encoding megalin and cubilin, respectively. The functional interaction of HNF1α with these promoters was shown in C33 epithelial cells lacking endogenous HNF1α. Defective receptor-mediated endocytosis was confirmed in Proximal Tubule cells from these knockout mice and could be rescued by transfection of wild-type but not mutant HNF1α. Transfection of human Proximal Tubule HK2 cells with HNF1α was able to upregulate megalin and cubilin expression and to increase endocytosis of albumin. Low-molecular-weight proteinuria was consistently detected in individuals with HNF1A mutations compared with healthy controls and patients with non–MODY-HNF1A diabetes mellitus. Thus, HNF1α plays a key role in the constitutive expression of megalin and cubilin, hence regulating endocytosis in the Proximal Tubule of the kidney. These findings provide new insight into the renal phenotype of individuals with mutations of HNF1A .
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endocytic receptors in the renal Proximal Tubule
Physiology, 2012Co-Authors: Erik Ilsø Christensen, Henrik Birn, Tina Storm, Kathrin Weyer, Rikke NielsenAbstract:Protein reabsorption is a predominant feature of the renal Proximal Tubule. Animal studies show that the ability to rescue plasma proteins relies on the endocytic receptors megalin and cubilin. Recently, studies of patients with syndromes caused by dysfunctional receptors have supported the importance of these for protein clearance of human ultrafiltrate. This review focuses on the molecular biology and physiology of the receptors and their involvement in renal pathological conditions.
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cubilin is essential for albumin reabsorption in the renal Proximal Tubule
Journal of The American Society of Nephrology, 2010Co-Authors: Sabine Amsellem, Ghislaine Hamard, Jakub Gburek, Rikke Nielsen, Thomas E Willnow, Erik Ilsø Christensen, Olivier Devuyst, Ebba Nexo, Renata KozyrakiAbstract:Receptor-mediated endocytosis is responsible for protein reabsorption in the Proximal Tubule. This process involves two interacting receptors, megalin and cubilin, which form a complex with amnionless. Whether these proteins function in parallel or as part of an integrated system is not well understood. Here, we report the renal effects of genetic ablation of cubilin, with or without concomitant ablation of megalin, using a conditional Cre-loxP system. We observed that Proximal Tubule cells did not localize amnionless to the plasma membrane in the absence of cubilin, indicating a mutual dependency of cubilin and amnionless to form a functional membrane receptor complex. The cubilin-amnionless complex mediated internalization of intrinsic factor-vitamin B12 complexes, but megalin considerably increased the uptake. Furthermore, cubilin-deficient mice exhibited markedly decreased uptake of albumin by Proximal Tubule cells and resultant albuminuria. Inactivation of both megalin and cubilin did not increase albuminuria, indicating that the main role of megalin in albumin reabsorption is to drive the internalization of cubilin-albumin complexes. In contrast, cubulin deficiency did not affect urinary tubular uptake or excretion of vitamin D-binding protein (DBP), which binds cubilin and megalin. In addition, we observed cubilin-independent reabsorption of the "specific" cubilin ligands transferrin, CC16, and apoA-I, suggesting a role for megalin and perhaps other receptors in their reabsorption. In summary, with regard to albumin, cubilin is essential for its reabsorption by Proximal Tubule cells, and megalin drives internalization of cubilin-albumin complexes. These genetic models will allow further analysis of protein trafficking in the progression of proteinuric renal diseases.
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Receptor-mediated endocytosis in renal Proximal Tubule
Pflügers Archiv - European Journal of Physiology, 2009Co-Authors: Erik Ilsø Christensen, Pierre J. Verroust, Rikke NielsenAbstract:Proteins filtered in renal glomeruli are removed from the tubular fluid by endocytosis in the Proximal Tubule mediated by the two receptors megalin and cubilin. After endocytic uptake, the proteins are transferred to lysosomes for degradation, while the receptors are returned to the apical cell membrane by receptor recycling in dense apical Tubules. In the renal Proximal Tubule, there is no significant transcellular transport of protein. The reabsorptive process is extremely efficient as evidenced by the virtual protein free urine in humans. The two receptors bind a variety of ligands. The process serves not only to remove the proteins from the ultrafiltrate but also to conserve a variety of essential substances such as vitamins and trace elements carried by plasma proteins. The endocytic apparatus is highly developed in the Proximal Tubule demonstrating the high capacity of the cells; however, under certain circumstances like diseases affecting the glomeruli, the system is overloaded resulting in proteinuria.
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protein reabsorption in renal Proximal Tubule function and dysfunction in kidney pathophysiology
Pediatric Nephrology, 2004Co-Authors: Erik Ilsø Christensen, Jakub GburekAbstract:The endocytic receptors megalin and cubilin are highly expressed in the early parts of the endocytic apparatus of the renal Proximal Tubule. The two receptors appear to be responsible for the tubular clearance of most proteins filtered in the glomeruli. Since cubilin is a peripheral membrane protein it has no endocytosis signaling sequence. Cubilin binds to megalin and it appears that megalin is responsible for internalization of cubilin and its ligands, in addition to internalizing its own ligands. The importance of the receptors is underscored by the proteinuria observed in megalin-deficient mice, in dogs lacking functional cubilin, and in patients with distinct mutations of the cubilin gene. In this review we focus on the role of megalin- and cubilin-mediated endocytosis in renal pathophysiology. Association between disorders characterized by tubular proteinuria, such as megaloblastic anemia type-1, Dent disease, cystinosis, and Fabry disease and the dysfunction of Proximal tubular endocytosis is discussed. The correlation between the high capacity of endocytosis in the Proximal Tubule and progressive renal disease in overload proteinuria is considered.
Peter J. Evans - One of the best experts on this subject based on the ideXlab platform.
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UTILIZATION OF AMINO ACIDS IN GROWING KIDNEY Proximal Tubule CELL CULTURES
Cell biology international, 2001Co-Authors: Abdi Rashid Ali, Peter J. EvansAbstract:The growth of rat kidney Proximal Tubule cells was monitored continuously by the cellular incorporation of [methyl-14C] thymidine using scintillating microplates. The radioisotope had no effect on cell proliferation over a 5 day period, neither was it extensively converted to thymine. Leibovitz L-15 medium supplemented with bicarbonate proved a good growth medium and its high levels of carbohydrates and amino acids facilitated the appearance of intermediates in the cells' metabolism of additional radioactive amino acids. Kidney Proximal Tubule cells had a greater potential to process amino acids than BHK-21 cells. The utilization of amino acids by Proximal Tubule cells differed from that of other organs. The amino acids could be classified into three classes. Members of the first type were only used for protein synthesis (arginine, lysine, histidine and tyrosine). The second class of amino acids yielded only one or two metabolites (leucine and isoleucine), while the last type gave more than two metabolites (alanine, aspartate, glycine, methionine, proline and valine).
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protein degradation in kidney Proximal Tubule cell monolayers
Biochimica et Biophysica Acta, 1994Co-Authors: Peter J. EvansAbstract:Abstract Isolated Proximal Tubule cells have been labelled with l -[4,5-3]Hleucine prior to cell division. Histochemical staining demonstrated the purity of the cultures. The bicarbonate ion or a collagen support was required for cell growth. Different culture growth rates were established by varying these parameters. The Proximal Tubule marker enzyme, γ-glutamyl transpeptidase, was expressed throughout the culture period (7–10 days) and the cells undergo a glycolytic shift, shown by an increase in the levels of lactate dehydrogenase. The specific activities of these enzymes were related to the growth conditions. Exponential rates of protein degradation were observed. The uptake of labelled exogenous hepatocyte proteins in Proximal Tubule cell cultures was completely suppressed in the presence of serum (10%, v/v) showing that endocytosis did not contribute to the observed measurements of intracellular protein degradation. The increased growth rates seen in cultures were accompanied by decreased rates of protein degradation. Use of the inhibitors of proteolysis, leupeptin and ammonium chloride, showed that the decrease was at the lysosomal level. The results suggest that targeting of inhibitors of lysosomal proteolysis, via low-molecular-weight proteins, may be useful in stimulating tubular regeneration in kidney disease.
Mary Taub - One of the best experts on this subject based on the ideXlab platform.
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Data on Na,K-ATPase in primary cultures of renal Proximal Tubule cells treated with catecholamines
Elsevier, 2016Co-Authors: Mary Taub, Facundo CutuliAbstract:This data article is concerned with chronic regulation of Na,K-ATPase by catecholamines. After a chronic treatment, inhibition of Na,K-ATPase activity was observed in cultures with dopamine, while a stimulation was observed in cultures treated with norepinephrine. Following a chronic incubation with guanabenz, an α adrenergic agonist, an increase in Na,K-ATPase α and β subunit mRNAs was observed. This data supports the research article entitled, “Renal Proximal Tubule Na, K-ATPase is controlled by CREB regulated transcriptional coactivators as well as salt inducible kinase 1” (Taub et al. 2015) [1]. Keywords: Catecholamines, Kidney, Proximal Tubule, Na,K-ATPase, Chroni
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Growth and function of primary rabbit kidney Proximal Tubule cells in glucose-free serum-free medium.
Journal of cellular physiology, 1992Co-Authors: Jee Chang Jung, Sang‐mog Lee, Nina Kadakia, Mary TaubAbstract:The properties of primary rabbit kidney Proximal Tubule cells in glucose-free serum-free medium have been examined. Primary rabbit kidney Proximal Tubule cells were observed to grow at the same rate, 1.0 doublings/day, both in glucose-free and in glucose-supplemented medium. Growth in glucose-free medium was dependent upon the presence of an additional nutritional supplement, such as glutamine, pyruvate, palmitate, lactate, or beta hydroxybutyrate. Lactate, pyruvate, and glutamate are utilized for renal gluconeogenesis in vivo. The growth of the primary rabbit kidney Proximal Tubule cells in glucose-free medium was also dependent upon the presence of the three growth supplements insulin, transferrin, and hydrocortisone. Insulin was growth stimulatory to the primary Proximal Tubule cells in glucose-free medium, although insulin causes a reduction in the phosphoenolpyruvate carboxykinase (PEPCK) activity in these cells. PEPCK is a key regulatory enzyme in the gluconeogenic pathway. In order to evaluate whether or not the primary cells have gluconeogenic capacity, their glucose content was determined. The cells contained 5 pmoles D-glucose/mg protein. However, no significant glucose was detected in the medium. Presumably, the primary cells were either utilizing or storing the glucose made by the gluconeogenic pathway. Consistent with this latter possibility, cellular glycogen levels were observed to increase with time in culture. The effect of glucose on the expression of the alpha I(IV) collagen and laminin B1 chain genes was examined. Northern analysis indicated that the level of alpha I(IV) collagen mRNA was significantly elevated in glucose containing, as compared with glucose deficient, medium. In contrast, laminin B1 chain mRNA levels were not significantly affected by the glucose content of the medium.
Michael Gekle - One of the best experts on this subject based on the ideXlab platform.
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mesna or cysteine prevents chloroacetaldehyde induced cell death of human Proximal Tubule cells
Pediatric Nephrology, 2007Co-Authors: Gerald Schwerdt, Antje Kirchhoff, Ruth Freudinger, Brigitte Wollny, Andreas Benesic, Michael GekleAbstract:Chloroacetaldehyde (CAA) is formed in the body from the chemotherapeutically used drug ifosfamide (IFO). CAA leads to cell death in Proximal Tubule cells mainly through the mechanism of necrosis rather than apoptosis. During chemotherapy, 2-mercaptosulfonic acid (mesna) is used with IFO to protect the urothel from cell damage. Little is known of the effect of mesna on renal Proximal Tubule cells, the primary site of damage after IFO treatment. Mesna contains a sulfhydryl (SH) group. To clarify whether SH-group-containing molecules can prevent CAA-induced cell death, we studied the effect of mesna and cysteine on necrosis, apoptosis, and protein content in a human Proximal Tubule-derived cell line (IHKE cells) treated with CAA. Both substances prevented CAA-induced necrotic cell death and protein loss and restored CAA-inhibited caspase-3 activity. CAA also prevented cisplatin-induced apoptosis. This inhibition was reversible in the presence of glutathione (GSH). We conclude that SH-containing molecules can protect Proximal Tubule cells from cell death because they interact with CAA before CAA can disturb other important cellular SH groups. A sufficient supply of intra- and extracellular SH groups during IFO chemotherapy may therefore have the ability to protect renal Tubule cells from cell death.
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the nephrotoxin ochratoxin a induces apoptosis in cultured human Proximal Tubule cells
Cell Biology and Toxicology, 1999Co-Authors: Gerald Schwerdt, R Freudinger, Sigrid Mildenberger, S Silbernagl, Michael GekleAbstract:To test the apoptotic potential of the nephrotoxic mycotoxin ochratoxin A (OTA), we exposed human Proximal Tubule-derived cells (IHKE cells) for various times to OTA concentrations close to those occurring during dietary exposure (from 2 to 100 nmol/L) and investigated caspase 3 activation, chromatin condensation, and DNA fragmentation. OTA induced a time- and concentration-dependent activation of caspase 3: concentrations as low as 5 nmol/L OTA caused a slight but significant increase in caspase 3 activity after 7 days of OTA exposure. Exposure to 10 nmol/L OTA for 72 or 24 h led to a significantly increased activity of caspase 3 in human Proximal Tubule-derived cells. Radical scavengers such as N-acetylcysteine had no effect on OTA-induced caspase 3 activation. Chelation of intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethylester) (BAPTA-AM) also showed no effect. Exposure to 30 nmol/L or more OTA led to DNA fragmentation and chromatin condensation in IHKE cells. Cultured renal epithelial MDCK-C7 and MDCK-C11 or OK cells also showed increased caspase 3 activity after OTA exposure. We conclude that exposure to low OTA concentrations can lead to direct or indirect caspase 3 activation and subsequently to apoptosis in cultured human Proximal Tubule cells and in other renal epithelial cell lines of different origins.
