Method for measuring luminal efflux of fluorescent organic compounds in isolated, perfused renal tubules

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Abstract

To examine directly in real time the efflux of organic compounds [e.g., organic anions (OAs) such as fluorescein (FL)] across the luminal membrane of isolated, perfused renal tubules during net secretion, we devised an approach utilizing a recently developed epifluorescence microscopy system for continuous monitoring of fluorescence in the collected perfusate. To illustrate this approach, we measured the luminal efflux rate of FL in mineral oil-covered, isolated, perfused S2 segments of rabbit renal proximal tubules. The washout profile of FL showed a deviation from linearity at time 0 when plotted on a semilog scale, indicating that the luminal efflux of FL was a saturable process. We were able for the first time to determine the kinetic parameters of luminal efflux [FL concentration at one-half maximal FL efflux (K(t)/(lumen)) of ~560 μM and maximal rate of FL efflux across the luminal membrane (J(max)/(lumen)) of ~635 fmol·min-1·mm-1]. From the present study, we conclude that the transport step for OAs across the luminal membrane of OAs is a carrier-mediated process. This approach will work to measure luminal transport in real time for any secreted organic compound that is sufficiently fluorescent to be measured with commonly available, highly sensitive optical equipment.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Renal Physiology
Volume279
Issue number5 48-5
StatePublished - 2000

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Fluorescein
Kidney
Anions
Membranes
Mineral Oil
Proximal Kidney Tubule
Microscopy
Fluorescence
Rabbits
Equipment and Supplies

Keywords

  • Fluorescein
  • Kidney
  • P-aminohippurate
  • Transport

ASJC Scopus subject areas

  • Physiology
  • Physiology (medical)

Cite this

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title = "Method for measuring luminal efflux of fluorescent organic compounds in isolated, perfused renal tubules",
abstract = "To examine directly in real time the efflux of organic compounds [e.g., organic anions (OAs) such as fluorescein (FL)] across the luminal membrane of isolated, perfused renal tubules during net secretion, we devised an approach utilizing a recently developed epifluorescence microscopy system for continuous monitoring of fluorescence in the collected perfusate. To illustrate this approach, we measured the luminal efflux rate of FL in mineral oil-covered, isolated, perfused S2 segments of rabbit renal proximal tubules. The washout profile of FL showed a deviation from linearity at time 0 when plotted on a semilog scale, indicating that the luminal efflux of FL was a saturable process. We were able for the first time to determine the kinetic parameters of luminal efflux [FL concentration at one-half maximal FL efflux (K(t)/(lumen)) of ~560 μM and maximal rate of FL efflux across the luminal membrane (J(max)/(lumen)) of ~635 fmol·min-1·mm-1]. From the present study, we conclude that the transport step for OAs across the luminal membrane of OAs is a carrier-mediated process. This approach will work to measure luminal transport in real time for any secreted organic compound that is sufficiently fluorescent to be measured with commonly available, highly sensitive optical equipment.",
keywords = "Fluorescein, Kidney, P-aminohippurate, Transport",
author = "Apichai Shuprisha and Stephen Wright and Dantzler, {William H}",
year = "2000",
language = "English (US)",
volume = "279",
journal = "American Journal of Physiology",
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TY - JOUR

T1 - Method for measuring luminal efflux of fluorescent organic compounds in isolated, perfused renal tubules

AU - Shuprisha, Apichai

AU - Wright, Stephen

AU - Dantzler, William H

PY - 2000

Y1 - 2000

N2 - To examine directly in real time the efflux of organic compounds [e.g., organic anions (OAs) such as fluorescein (FL)] across the luminal membrane of isolated, perfused renal tubules during net secretion, we devised an approach utilizing a recently developed epifluorescence microscopy system for continuous monitoring of fluorescence in the collected perfusate. To illustrate this approach, we measured the luminal efflux rate of FL in mineral oil-covered, isolated, perfused S2 segments of rabbit renal proximal tubules. The washout profile of FL showed a deviation from linearity at time 0 when plotted on a semilog scale, indicating that the luminal efflux of FL was a saturable process. We were able for the first time to determine the kinetic parameters of luminal efflux [FL concentration at one-half maximal FL efflux (K(t)/(lumen)) of ~560 μM and maximal rate of FL efflux across the luminal membrane (J(max)/(lumen)) of ~635 fmol·min-1·mm-1]. From the present study, we conclude that the transport step for OAs across the luminal membrane of OAs is a carrier-mediated process. This approach will work to measure luminal transport in real time for any secreted organic compound that is sufficiently fluorescent to be measured with commonly available, highly sensitive optical equipment.

AB - To examine directly in real time the efflux of organic compounds [e.g., organic anions (OAs) such as fluorescein (FL)] across the luminal membrane of isolated, perfused renal tubules during net secretion, we devised an approach utilizing a recently developed epifluorescence microscopy system for continuous monitoring of fluorescence in the collected perfusate. To illustrate this approach, we measured the luminal efflux rate of FL in mineral oil-covered, isolated, perfused S2 segments of rabbit renal proximal tubules. The washout profile of FL showed a deviation from linearity at time 0 when plotted on a semilog scale, indicating that the luminal efflux of FL was a saturable process. We were able for the first time to determine the kinetic parameters of luminal efflux [FL concentration at one-half maximal FL efflux (K(t)/(lumen)) of ~560 μM and maximal rate of FL efflux across the luminal membrane (J(max)/(lumen)) of ~635 fmol·min-1·mm-1]. From the present study, we conclude that the transport step for OAs across the luminal membrane of OAs is a carrier-mediated process. This approach will work to measure luminal transport in real time for any secreted organic compound that is sufficiently fluorescent to be measured with commonly available, highly sensitive optical equipment.

KW - Fluorescein

KW - Kidney

KW - P-aminohippurate

KW - Transport

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