P-Glycoprotein-Mediated Drug Secretion in Mouse Proximal Tubule Perfused In Vitro

Abstract. To examine the functional significance of durg-transporting P-glycoprotein (P-gp), studies were conducted in the isolated and perfused proximal tubule S2 segment from mice disrupting both mdr1a and mdr1b genes [mdr1a/1b(—)(—)] and their wild-type mice. Efflux of the intracellular fluorescence of rhodamine 123, a fluorescence substrate of P-gp, into the lumen was measured, and the decay half-time of the intracellular fluorescence (T1/2) as an index of the drug-transporting P-gp activity was regarded. In the wild-type mice, the T1/2 was 34 ± 4 s (n = 36) at the basal period and was increased to 434 ± 41 s by the addition of luminal verapamil, a P-gp inhibitor. In the mdr1a/1b(—)(—) mice, the T1/2 was 407 ± 16 s (n = 10) at the basal period and was no longer affected by the luminal addition of verapamil. The digoxin content in the kidney after a repeated administration of the drug was markedly elevated in the mdr1a/1b(—)(—) mice. In conclusion, P-gp—mediated drug efflux capacity indeed exists in the apical membrane of proximal tubule cells from the wild-type mice, whereas it is absent in that of the mdr1a/1b(—)(—) mice.

Download Full-text

P-gp-induced modulation of regulatory volume increase occurs via PKC in mouse proximal tubule

AJP Renal Physiology ◽

10.1152/ajprenal.0036.2001 ◽

2002 ◽

Vol 282 (1) ◽

pp. F65-F76 ◽

Cited By ~ 5

Author(s):

Yukio Miyata ◽

Koji Okada ◽

Shun Ishibashi ◽

Yasushi Asano ◽

Shigeaki Muto

Keyword(s):

Proximal Tubule ◽

Wild Type ◽

Phosphatidylinositol 3 Kinase ◽

Glycoprotein P ◽

Volume Increase ◽

Regulatory Volume Increase ◽

Kinase C ◽

P Glycoprotein ◽

Pkc Inhibitors

The present study examined the role of protein kinase C (PKC) in the P-glycoprotein (P-gp)-induced modulation of regulatory volume increase (RVI) in the isolated nonperfused proximal tubule S2 segments from mice lacking both mdr1a and mdr1b genes (KO) and wild-type (WT) mice. The hyperosmotic solution (500 mosmol/kgH2O) involving 200 mM mannitol activated PKC and elicited RVI in the tubules from KO mice but not from WT mice. The addition of the hyperosmotic solution including the PKC activator phorbol 12-myristate 13-acetate (PMA) to the tubules of the WT mice activated PKC and elicited RVI. The hyperosmotic solution in the presence of the P-gp inhibitors (verapamil or cyclosporin A) elicited RVI in the tubules from the WT mice but not from the KO mice. The PMA- and the P-gp inhibitors-induced RVI was abolished by cotreatment with the PKC inhibitors (staurosporine or calphostin C). In the tubules of the KO mice, the PKC inhibitors abolished RVI, but PMA did not. In the tubules of the WT mice, the microtubule disruptor (colchicine), the microfilament disruptor (cytochalasin B), the phosphatidylinositol 3-kinase (PI 3-kinase) blocker (wortmannin), but not another PI 3-kinase blocker (LY-294002), inhibited the PMA-induced RVI. In the tubules of the KO mice, colchicine, cytochalsin B, and wortmannin abolished RVI, but LY-294002 did not. We conclude that 1) in the mouse proximal tubule, P-gp-induced modulation of RVI occurs via PKC; and 2) the microtubule, microfilament, and wortmannin-sensitive, LY-294002-insensitive PI 3-kinase contribute to the PKC-induced RVI.

Download Full-text

Nephrotoxicity Testing In Vitro: The Current Situation

Alternatives to Laboratory Animals ◽

10.1177/026119299702500506 ◽

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.

Download Full-text

P-Glycoprotein and Breast Cancer Resistance Protein Restrict Apical-to-Basolateral Permeability of Human Brain Endothelium to Amyloid-β

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2009.42 ◽

2009 ◽

Vol 29 (6) ◽

pp. 1079-1083 ◽

Cited By ~ 73

Author(s):

Leon M Tai ◽

A Jane Loughlin ◽

David K Male ◽

Ignacio A Romero

Keyword(s):

Breast Cancer ◽

Human Brain ◽

Breast Cancer Resistance Protein ◽

Amyloid Β ◽

Cancer Resistance ◽

Glycoprotein P ◽

P Glycoprotein ◽

Resistance Protein ◽

The Brain

The clearance of amyloid beta (Aβ) from the brain represents a novel therapeutic target for Alzheimer's disease. Conflicting data exist regarding the contribution of adenosine triphosphatebinding cassette transporters to the clearance of Aβ through the blood-brain barrier. Therefore, we investigated whether Aβ could be a substrate for P-glycoprotein (P-gp) and/or for breast cancer resistance protein (BCRP) using a human brain endothelial cell line, hCMEC/D3. Inhibition of P-gp and BCRP increased apical-to-basolateral, but not basolateral-to-apical, permeability of hCMEC/D3 cells to 125l Aβ 1–40. Our in vitro data suggest that P-gp and BCRP might act to prevent the blood-borne Aβ 1–40 from entering the brain.

Download Full-text

Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis

Blood ◽

10.1182/blood-2012-07-441824 ◽

2012 ◽

Vol 120 (16) ◽

pp. 3336-3344 ◽

Cited By ~ 36

Author(s):

Anu Laitala ◽

Ellinoora Aro ◽

Gail Walkinshaw ◽

Joni M. Mäki ◽

Maarit Rossi ◽

...

Keyword(s):

Cultured Cells ◽

Mrna Level ◽

Hypoxia Inducible Factor ◽

Mrna Levels ◽

Wild Type ◽

Α Subunit ◽

Hepcidin Expression ◽

In The Wild ◽

Hepcidin Mrna

AbstractAn endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm−/− mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm−/− mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497–treated Hif-p4h-2 hypomorphic (Hif-p4h-2gt/gt) and Hif-p4h-3−/− mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm−/− and wild-type mice, but caused higher increases in both values in the Hif-p4h-2gt/gt mice and in hematocrit value in the Hif-p4h-3−/− mice than in the wild-type. Hif-p4h-2gt/gt/P4h-tm−/− double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2gt/gt or P4h-tm−/− mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

Download Full-text

Upregulations of Clcn3 and P-Gp Provoked by Lens Osmotic Expansion in Rat Galactosemic Cataract

Journal of Diabetes Research ◽

10.1155/2017/3472735 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8

Author(s):

Lixia Ji ◽

Lixia Cheng ◽

Zhihong Yang

Keyword(s):

Osmotic Stress ◽

Early Stage ◽

Lens Epithelial Cells ◽

Anterior Capsule ◽

Glycoprotein P ◽

Protein Levels ◽

P Glycoprotein ◽

Sugar Cataract

Objective.Lens osmotic expansion, provoked by overactivated aldose reductase (AR), is the most essential event of sugar cataract. Chloride channel 3 (Clcn3) is a volume-sensitive channel, mainly participating in the regulation of cell fundamental volume, and P-glycoprotein (P-gp) acts as its modulator. We aim to study whether P-gp and Clcn3 are involved in lens osmotic expansion of galactosemic cataract.Methods and Results.In vitro, lens epithelial cells (LECs) were primarily cultured in gradient galactose medium (10–60 mM), more and more vacuoles appeared in LEC cytoplasm, and mRNA and protein levels of AR, P-gp, and Clcn3 were synchronously upregulated along with the increase of galactose concentration. In vivo, we focused on the early stage of rat galactosemic cataract, amount of vacuoles arose from equatorial area and scattered to the whole anterior capsule of lenses from the 3rd day to the 9th day, and mRNA and protein levels of P-gp and Clcn3 reached the peak around the 9th or 12th day.Conclusion. Galactosemia caused the osmotic stress in lenses; it also markedly leads to the upregulations of AR, P-gp, and Clcn3 in LECs, together resulting in obvious osmotic expansion in vitro and in vivo.

Download Full-text

Abstract 564: Differential Expression and Regulation of Angiotensinogen in Renal Proximal Tubule Cells From S1, S2 and S3 Segments

Hypertension ◽

10.1161/hyp.62.suppl_1.a564 ◽

2013 ◽

Vol 62 (suppl_1) ◽

Author(s):

Ryousuke Satou ◽

Kathleen S Hering-Smith ◽

L G Navar

Keyword(s):

Proximal Tubule ◽

Kidney Injury ◽

Specific Cell ◽

S2 Cells ◽

Ang Ii ◽

Proximal Tubule Cells ◽

Protein Levels ◽

Pathogenic Factors ◽

Renal Proximal Tubule Cells

In angiotensin II (Ang II)-dependent hypertension, intrarenal angiotensinogen (AGT) augmentation induced by Ang II and associated pathogenic factors including interleukin 6 (IL-6) cause further elevation of intratubular Ang II production, leading to the progression of hypertension and kidney injury. Recent studies have suggested that renal proximal straight tubules (S3 segment) are the main source of intrarenal AGT and that S1 and S2 segments do not express AGT mRNA under normal conditions. However, AGT expression and its regulation by Ang II and/or IL-6 in each proximal tubule segment have not been demonstrated an in vitro setting. The availability of specific cell lines derived from mouse S1, S2 and S3 segments provided an opportunity to decisively determine each segments’ capability to express AGT and respond to stimuli. Thus, this study was performed to determine AGT expression and its response to stimulation with Ang II and IL-6 in S1, S2 and S3 cell line. Basal AGT mRNA and protein levels were detected by RT-PCR and western blot analysis. Basal levels of Ang II type 1 receptor (AT1R) and STAT3, which is a transcription factor in IL-6 signaling pathway, were also measured. In addition, the cells were incubated with 100 nM Ang II and/or 400 nM IL-6 for 24 h. Basal AGT levels in S1 and S3 cells were lower than in mouse whole kidney (0.09-fold and 0.33-fold compared with mouse whole kidney). S2 cells exhibited the highest basal AGT levels (4.15-fold) among these cells. In S1 cells, AGT expression was stimulated by IL-6 (1.89 ± 0.32, ratio to control) and co-stimulation with Ang II and IL-6 (1.85 ± 0.28) although Ang II alone did not alter AGT levels. In S2 cells, only the co-stimulation increased AGT expression (1.35 ± 0.01). No changes were observed by similar treatments in S3 cells. Basal AT1R levels were lower in S3 than in S1 and S2 cells (0.97 ± 0.09 in S2, 0.32 ± 0.07 in S3, ratio to S1). S1 cells showed the highest basal levels of STAT3. Basal STAT3 levels in S3 cells were lower than that in S1 and S2 cells. These results indicate that S2 cells are main source of intrarenal AGT which can be augmented by Ang II and IL-6 during the development of Ang II-dependent hypertension. Furthermore, low basal levels of AT1R and STAT3 in S3 cells explain why these cells do not respond to Ang II and IL-6.

Download Full-text

Immuno-oncology agent IPI-549 is a modulator of P-glycoprotein (P-gp, MDR1, ABCB1)-mediated multidrug resistance (MDR) in cancer: In vitro and in vivo

Cancer Letters ◽

10.1016/j.canlet.2018.10.020 ◽

2019 ◽

Vol 442 ◽

pp. 91-103 ◽

Cited By ~ 9

Author(s):

Albert A. De Vera ◽

Pranav Gupta ◽

Zining Lei ◽

Dan Liao ◽

Silpa Narayanan ◽

...

Keyword(s):

Multidrug Resistance ◽

Glycoprotein P ◽

P Glycoprotein

Download Full-text

Transport of Alzheimer’s associated amyloid-β catalyzed by P-glycoprotein

PLoS ONE ◽

10.1371/journal.pone.0250371 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0250371

Author(s):

James W. McCormick ◽

Lauren Ammerman ◽

Gang Chen ◽

Pia D. Vogel ◽

John G. Wise

Keyword(s):

Cell Culture ◽

Atp Hydrolysis ◽

Amyloid Β ◽

Md Simulations ◽

Membrane Transporter ◽

Biochemical Activity ◽

Glycoprotein P ◽

P Glycoprotein ◽

Hydrolysis Activity

P-glycoprotein (P-gp) is a critical membrane transporter in the blood brain barrier (BBB) and is implicated in Alzheimer’s disease (AD). However, previous studies on the ability of P-gp to directly transport the Alzheimer’s associated amyloid-β (Aβ) protein have produced contradictory results. Here we use molecular dynamics (MD) simulations, transport substrate accumulation studies in cell culture, and biochemical activity assays to show that P-gp actively transports Aβ. We observed transport of Aβ40 and Aβ42 monomers by P-gp in explicit MD simulations of a putative catalytic cycle. In in vitro assays with P-gp overexpressing cells, we observed enhanced accumulation of fluorescently labeled Aβ42 in the presence of Tariquidar, a potent P-gp inhibitor. We also showed that Aβ42 stimulated the ATP hydrolysis activity of isolated P-gp in nanodiscs. Our findings expand the substrate profile of P-gp, and suggest that P-gp may contribute to the onset and progression of AD.

Download Full-text

Expression of hamster P-glycoprotein and multidrug resistance in DNA-mediated transformants of mouse LTA cells

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.718-724.1987 ◽

1987 ◽

Vol 7 (2) ◽

pp. 718-724

Author(s):

K L Deuchars ◽

R P Du ◽

M Naik ◽

D Evernden-Porelle ◽

N Kartner ◽

...

Keyword(s):

Multidrug Resistance ◽

Dna Sequences ◽

Multidrug Resistant ◽

Cross Resistance ◽

Recipient Mouse ◽

Strongly Correlated ◽

Wild Type ◽

Glycoprotein P ◽

Single Drug ◽

P Glycoprotein

The overexpression of a plasma membrane glycoprotein, P-glycoprotein, is strongly correlated with the expression of multidrug resistance. This phenotype (frequently observed in cell lines selected for resistance to a single drug) is characterized by cross resistance to many drugs, some of which are used in cancer chemotherapy. In the present study we showed that DNA-mediated transformants of mouse LTA cells with DNA from multidrug-resistant hamster cells acquired the multidrug resistance phenotype, that the transformants contained hamster P-glycoprotein DNA sequences, that these sequences were amplified whereas the recipient mouse P-glycoprotein sequences remained at wild-type levels, and that the overexpressed P-glycoprotein in these cells was of hamster origin. Furthermore, we showed that the hamster P-glycoprotein sequences were transfected independently of a group of genes that were originally coamplified and linked within a 1-megabase-pair region in the donor hamster genome. These data indicate that the high expression of P-glycoprotein is the only alteration required to mediate multidrug resistance.

Download Full-text