Quantitative intravital microscopy of hepatic transport

Babbey, Clifford M.; Ryan, Jennifer; Gill, Erin M.; Ghabril, Marwan; Burch, Courtney R.; Paulman, April; Dunn, Kenneth W.

doi:10.4161/intv.21296

Cited by 25 publications

(33 citation statements)

References 61 publications

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“…Our overall approach for analyzing hepatic transport involves quantitative multiphoton microscopy of transport of sodium fluorescein in the intact liver of a living rat. Techniques of quantitative intravital microscopy follow those that we previously described (1). After 24 h in a metabolic cage, the rats were anesthetized with 130 mg/kg of Inactin given intraperitoneally.…”

Section: /6th Nephrectomy Rat Model Of Ckdmentioning

confidence: 99%

“…We have recently developed methods of quantitative intravital microscopy capable of dissecting hepatocyte transport in vivo (1). Time-lapse multiphoton fluorescence microscopy of the liver of living rats following intravenous injection of fluorescent transport substrates is combined with methods of quantitative digital image analysis to quantify the kinetics of transport into the cytosol of individual hepatocytes and into bile canaliculi.…”

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confidence: 99%

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Effects of chronic kidney disease on liver transport: quantitative intravital microscopy of fluorescein transport in the rat liver

Ryan

Dunn

Decker

2014

American Journal of Physiology-Regulatory, Integrative and Comp

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Ryan JC, Dunn KW, Decker BS. Effects of chronic kidney disease on liver transport: quantitative intravital microscopy of fluorescein transport in the rat liver. Am J Physiol Regul Integr Comp Physiol 307: R1488 -R1492, 2014. First published October 22, 2014 doi:10.1152/ajpregu.00371.2014.-Clinical studies indicate that hepatic drug transport may be altered in chronic kidney disease (CKD). Uremic solutes associated with CKD have been found to alter the expression and/or activity of hepatocyte transporters in experimental animals and in cultured cells. However, given the complexity and adaptability of hepatic transport, it is not clear whether these changes translate into significant alterations in hepatic transport in vivo. To directly measure the effect of CKD on hepatocyte transport in vivo, we conducted quantitative intravital microscopy of transport of the fluorescent organic anion fluorescein in the livers of rats following 5/6th nephrectomy, an established model of CKD. Our quantitative analysis of fluorescein transport showed that the rate of hepatocyte uptake was reduced by ϳ20% in 5/6th nephrectomized rats, consistent with previous observations of Oatp downregulation. However, the overall rate of transport into bile canaliculi was unaffected, suggesting compensatory changes in Mrp2-mediated secretion. Our study suggests that uremia resulting from 5/6th nephrectomy does not significantly impact the overall hepatic clearance of an Oatp substrate. chronic kidney disease; uremia; cytochrome P-450; hepatic transport; Mrp2; Oatp; sodium fluorescein PATIENTS WITH END-STAGE RENAL disease (ESRD) receiving dialysis take on average 12 medications to manage the complications of their renal failure and their comorbid disease, which places them at a substantial risk for adverse drug events (8,12,13). Currently, modifications in drug dosing to improve safety for ESRD patients have largely been confined to those medications excreted renally. This has likely improved the safety of these medications for the ESRD population, yet it remains an incomplete approach, since it does not include the hepatically metabolized medications. This oversight is significant because evidence increasingly indicates that hepatic drug metabolism and transport are reduced in patients with [14][15][16][17][18][19][20][21][22][25][26][27][28]30). Consequently, administering the full dose of a hepatically metabolized medication may place an ESRD patient at similar risk for an adverse drug event as administering the full dose of a renally eliminated medication.Experimental evidence suggests multiple mechanisms by which uremic solutes might influence hepatic drug disposition (20,25). Studies from the Pichette laboratory demonstrated that the activity and expression of cytochrome P-450 (CYP450) was reduced in the 5/6th nephrectomy (5/6N) rat model of chronic renal failure (6, 10, 11). Their work elucidated a direct role for uremic solutes with the observation that CYP450 activity and expression were reduced in primary rat hepatocytes incubated wit...

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Section: /6th Nephrectomy Rat Model Of Ckdmentioning

confidence: 99%

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confidence: 99%

Effects of chronic kidney disease on liver transport: quantitative intravital microscopy of fluorescein transport in the rat liver

Ryan

Dunn

Decker

2014

American Journal of Physiology-Regulatory, Integrative and Comp

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“…In our studies of kidney and liver [21][22][23][24] , an inverted microscope stand is used to image the surgically exposed organs that are secured to a glass-bottomed dish (see Figure 1). The anesthetized animal is placed on the microscope stage, and the animal's temperature is maintained using a heating pad and monitored using a rectal thermometer.…”

Section: ) Optimized Probes For Tplsmmentioning

confidence: 99%

A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy

2016

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“…Mice are housed in a controlled environment in the animal facility and are allowed to acclimate for one week prior to the experiment. This step is necessary for imaging the SGs since any distress results in enhanced secretory activity 12 . Water and food are provided ad libitum.…”

Section: Part 2: Animals and Anesthesiamentioning

confidence: 99%

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Masedunskas

Porat-Shliom

Tora

et al. 2013

JoVE

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Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton. Video LinkThe video component of this article can be found at

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Quantitative intravital microscopy of hepatic transport

Cited by 25 publications

References 61 publications

Effects of chronic kidney disease on liver transport: quantitative intravital microscopy of fluorescein transport in the rat liver

Effects of chronic kidney disease on liver transport: quantitative intravital microscopy of fluorescein transport in the rat liver

A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

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