scholarly journals ClC-5: role in endocytosis in the proximal tubule

2005 ◽  
Vol 289 (4) ◽  
pp. F850-F862 ◽  
Author(s):  
Yinghong Wang ◽  
Hui Cai ◽  
Liudmila Cebotaru ◽  
Deanne H. Hryciw ◽  
Edward J. Weinman ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Molecular Mass ◽  
Primary Cultures ◽  
Apical Surface ◽  
Wild Type ◽  
Proximal Tubule Cells ◽  
Dent’S Disease ◽  
Receptor Mediated Endocytosis ◽  
Dent's Disease ◽  
Tubule Cells

The proper functioning of the Cl−channel, ClC-5, is essential for the uptake of low molecular mass proteins through receptor-mediated endocytosis in the proximal tubule. Dent’s disease patients with mutant ClC-5 channels and ClC-5 knockout (KO) mice both have low molecular mass proteinuria. To further understand the function of ClC-5, endocytosis was studied in LLC-PK1cells and primary cultures of proximal tubule cells from wild-type (WT) and ClC-5 KO kidneys. Endocytosis in the proximal tubule cells from KO mice was reduced compared with that in WT animals. Endocytosis in WT but not in KO cells was inhibited by bafilomycin A-1 and Cl−depletion, whereas endocytosis in both WT and KO cells was inhibited by the NHE3 blocker, S3226. Infection with adenovirus containing WT ClC-5 rescued receptor-mediated endocytosis in KO cells, whereas infection with any of the three disease-causing mutants, myc-W22G-ClC-5, myc-S520P-ClC-5, or myc-R704X-ClC-5, did not. WT and the three mutants all trafficked to the apical surface, as assessed by surface biotinylation. WT-ClC-5 and the W22G mutant were internalized similarly, whereas neither the S520P nor the R704X mutants was. These data indicate that ClC-5 is important for Cl−and proton pump-mediated endocytosis. However, not all receptor-mediated endocytosis in the proximal tubule is dependent on ClC-5. There is a significant fraction that can be inhibited by an NHE3 blocker. Our data from the mutants suggest that defective targeting and trafficking of mutant ClC-5 to the endosomes are a major determinant in the lack of normal endocytosis in Dent’s disease.

Defective PTH regulation of sodium-dependent phosphate transport in NHERF-1−/− renal proximal tubule cells and wild-type cells adapted to low-phosphate media

2005 ◽  
Vol 289 (4) ◽  
pp. F933-F938 ◽  
Author(s):  
Rochelle Cunningham ◽  
Xiaofei E ◽  
Deborah Steplock ◽  
Shirish Shenolikar ◽  
Edward J. Weinman
Keyword(s):  
Proximal Tubule ◽  
Primary Cultures ◽  
Phosphate Transport ◽  
Inhibitory Response ◽  
Renal Proximal Tubule ◽  
Wild Type ◽  
Proximal Tubule Cells ◽  
Sodium Dependent ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

The present experiments using primary cultures from renal proximal tubule cells examine two aspects of the regulation of sodium-dependent phosphate transport and membrane sodium-dependent phosphate transporter (Npt2a) expression by parathyroid hormone (PTH). Sodium-dependent phosphate transport in proximal tubule cells from wild-type mice grown in normal-phosphate media averaged 4.4 ± 0.5 nmol·mg protein−1·10 min−1 and was inhibited by 30.5 ± 8.6% by PTH (10−7 M). This was associated with a 32.7 ± 5.2% decrease in Npt2a expression in the plasma membrane. Proximal tubule cells from Na+/H+ exchanger regulatory factor-1 (NHERF-1)−/− mice had a lower rate of phosphate transport compared with wild-type cells and a significantly reduced inhibitory response to PTH. Wild-type cells incubated in low-phosphate media for 24 h had a higher rate of phosphate transport compared with wild-type cells grown in normal-phosphate media but a significantly blunted inhibitory response to PTH. These data indicate a role for NHERF-1 in mediating the membrane retrieval of Npt2a and the subsequent inhibition of phosphate transport in renal proximal tubules. These studies also suggest that there is a blunted phosphaturic effect of PTH in cells adapted to low-phosphate media.

Nephrotoxicity Testing In Vitro: The Current Situation

1997 ◽  
Vol 25 (5) ◽  
pp. 497-503
Author(s):  
Jean-Paul Morin ◽  
Marc E. De Broe ◽  
Walter Pfaller ◽  
Gabriele Schmuck
Keyword(s):  
Proximal Tubule ◽  
Culture Media ◽  
Task Force ◽  
Primary Cultures ◽  
Phenotypic Expression ◽  
Transepithelial Transport ◽  
Specific Cell ◽  
Proximal Tubule Cells ◽  
Tubule Cells

An ECVAM task force on nephrotoxicity has been established to advise, in particular, on the follow-up to recommendations made in the ECVAM workshop report on nephrotoxicity testing in vitro. Since this workshop was held, in 1994, there have been several improvements in the techniques used. For example, the duration of renal slice viability, and the maintenance of functional activities in slices, have been improved by using dynamic incubation systems with higher oxygen tensions and more-appropriate cell culture media. Highly differentiated primary cultures of pig, human and rabbit proximal tubule cells have been established by using specific cell isolation procedures and/or selective culture media. To date, the most comparable phenotypic expression and transepithelial transport capacities to proximal tubules in vivo have been obtained with primary cultures of rabbit proximal tubule cells which are grown on bicompartmental supports; in this system, transepithelial substrate gradients are generated and the transepithelial transport of both organic anions and cations is highly active. This in vitro system has been selected by ECVAM for further evaluation and prevalidation. Industrial needs in the area of nephrotoxicity testing have been identified, and recommendations are made at the end of this report concerning possible future initiatives.

Primary cultures of renal proximal tubule cells derived from individuals with primary hyperoxaluria

2009 ◽  
Vol 37 (3) ◽  
pp. 127-132 ◽  
Author(s):  
Karen L. Price ◽  
Sally-Anne Hulton ◽  
William G. van’t Hoff ◽  
John R. Masters ◽  
Gill Rumsby
Keyword(s):  
Proximal Tubule ◽  
Primary Cultures ◽  
Primary Hyperoxaluria ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

Angiotensin II-dependent proximal tubule sodium transport requires receptor-mediated endocytosis

1994 ◽  
Vol 266 (3) ◽  
pp. C669-C675 ◽  
Author(s):  
J. R. Schelling ◽  
S. L. Linas
Keyword(s):  
Angiotensin Ii ◽  
Proximal Tubule ◽  
Receptor Internalization ◽  
Ang Ii ◽  
Na Transport ◽  
Cellular Mechanisms ◽  
Transport Pathways ◽  
Proximal Tubule Cells ◽  
Receptor Mediated Endocytosis ◽  
Tubule Cells

Angiotensin II (ANG II) receptors are present on apical and basolateral surfaces of proximal tubule cells. To determine the cellular mechanisms of proximal tubule ANG II receptor-mediated Na transport, apical-to-basolateral 22Na flux was measured in cultured proximal tubule cells. Apical ANG II caused increases in 22Na flux (maximum response: 100 nM, 30 min). Basolateral ANG II resulted in 22Na flux that was 23-56% greater than 22Na flux observed with equimolar apical ANG II. Apical ANG II-induced 22Na flux was prevented by preincubation with amiloride, ouabain, and the AT1 receptor antagonist losartan. Because apical ANG II signaling was previously shown to be endocytosis dependent, we questioned whether endocytosis was required for ANG II-stimulated proximal tubule Na transport as well. Apical (but not basolateral) ANG II-dependent 22Na flux was inhibited by phenylarsine oxide, an agent which prevents ANG II receptor internalization. In conclusion, apical and basolateral ANG II caused proximal tubule Na transport. Apical ANG II-dependent Na flux was mediated by AT1 receptors, transcellular transport pathways, and receptor-mediated endocytosis.

scholarly journals PTH transiently increases the percent mobile fraction of Npt2a in OK cells as determined by FRAP

2009 ◽  
Vol 297 (6) ◽  
pp. F1560-F1565 ◽  
Author(s):  
Edward J. Weinman ◽  
Deborah Steplock ◽  
Boyoung Cha ◽  
Olga Kovbasnjuk ◽  
Nicholas A. Frost ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Binding Proteins ◽  
Apical Membrane ◽  
Wild Type ◽  
Lateral Mobility ◽  
Proximal Tubule Cells ◽  
Ok Cells ◽  
Mobile Fraction ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

Renal sodium-dependent phosphate transporter 2a (Npt2a) binds to a number of PDZ adaptor proteins including sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), which regulates its retention in the apical membrane of renal proximal tubule cells and the response to parathyroid hormone (PTH). The present experiments were designed to study the lateral mobility of enhanced green fluorescent protein (EGFP)-Npt2a in proximal tubule-like opossum kidney (OK) cells using fluorescence recovery after photobleaching (FRAP) and to determine the role of PDZ binding proteins in mediating the effects of PTH. The mobile fraction of wild-type Npt2a (EGFP-Npt2a-TRL) under basal conditions was ∼17%. Treatment of the cells with Bis(sulfosuccinimidyl) suberate, a water-soluble cross-linker, abolished recovery nearly completely, indicating that recovery represented lateral diffusion in the plasma membrane and not the exocytosis or synthesis of unbleached transporter. Substitution of the C-terminal amino acid PDZ binding sequence TRL with AAA (EGFP-Npt2a-AAA) resulted in a nearly twofold increase in percent mobile fraction of Npt2a. Treatment of cells with PTH resulted in a rapid increase in the percent mobile fraction to >30% followed by a time-dependent decrease to baseline or below. PTH had no effect on the mobility of EGFP-Npt2a-AAA expressed in native OK cells or on wild-type EGFP-Npt2a-TRL expressed in OK-H cells deficient in NHERF-1. These findings indicate that the association of Npt2a with PDZ binding proteins limits the lateral mobility of the transporter in the apical membrane of renal proximal tubule cells. Treatment with PTH, presumably by dissociating NHERF-1/Npt2a complexes, transiently increases the mobility of Npt2a, suggesting that freeing of Npt2a from the cytoskeleton precedes PTH-mediated endocytosis.

Cyclosporin A and vehicle toxicity in primary cultures of rabbit renal proximal tubule cells

1990 ◽  
Vol 183 (6) ◽  
pp. 2438
Author(s):  
P.P. Sokol ◽  
L.C. Capodagli ◽  
M. Dixon ◽  
P.D. Holohan ◽  
C.R. Ross ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Cyclosporin A ◽  
Primary Cultures ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

Uptake and trafficking of fluorescent conjugates of folic acid in intact kidney determined using intravital two-photon microscopy

2004 ◽  
Vol 287 (2) ◽  
pp. C517-C526 ◽  
Author(s):  
Ruben M. Sandoval ◽  
Michael D. Kennedy ◽  
Philip S. Low ◽  
Bruce A. Molitoris
Keyword(s):  
Folic Acid ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Proximal Tubules ◽  
Apical Surface ◽  
Proximal Tubule Cells ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Tubule Cells ◽  
Folate Conjugate

Intravital two-photon microscopy was used to follow the uptake and trafficking of fluorescent conjugates of folic acid in the rat kidney. Intravenously administered folate-linked dye molecules quickly filled the plasma volume but not cellular components of the blood. Glomerular filtration occurred immediately and binding to proximal tubule cells was seen within seconds. Fluorescence from a pH-insensitive conjugate of folic acid, folate Texas red (FTR), was readily observed on the apical surface of the proximal tubules and in multiple cellular compartments, but little binding or uptake could be detected in any other kidney cells. Fluorescence from a pH-sensitive conjugate of folic acid, folate fluorescein, was seen only on the apical surface of proximal tubule cells, suggesting that internalized folate conjugates are localized to acidic compartments. The majority of the FTR conjugate internalized by proximal tubules accumulated within a lysosomal pool, as determined by colocalization studies. However, portions of FTR were also shown by electron microscopy to undergo transcytosis from apical to basal domains. Additional studies with colchicine, which is known to depolymerize microtubules and interrupt transcytosis, produced a marked reduction in endocytosis of FTR, with accumulation limited to the subapical region of the cell. No evidence of cytosolic release of either folate conjugate was observed, which may represent a key difference from the cytosolic deposition seen in neoplastic cells. Together, these data support the argument that folate conjugates (and, by extrapolation, physiological folate) bind to the apical surface of proximal tubule cells and are transported into and across the cells in endocytic compartments.

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