Digital Fluorescence Imaging of Organic Cation Transport in Freshly Isolated Rat Proximal Tubules

Pietruck, Frank; Hörbelt, Markus; Feldkamp, Thorsten; Engeln, Katrin; Herget–Rosenthal, Stefan; Philipp, Thomas; Kribben, Andreas

doi:10.1124/dmd.105.006403

Cited by 6 publications

(5 citation statements)

References 15 publications

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“…Nevertheless, ASP þ uptake appeared to be highly effective, which is consistent with earlier in vitro reports. 10,14,15 However, we observed differences in the way intracellular ASP þ fluorescence decreased in proximal and distal tubules. In proximal tubules, intracellular ASP þ fluorescence decreased over time.…”

Section: Discussionmentioning

confidence: 58%

“…Additionally, ASP þ was used for OC transport studies in other cell culture models [11][12][13] and in isolated proximal tubules. 14,15 Various experimental models have previously been applied for studies of OC transport including cellular substructures, 16,17 cell expression systems, 5 and cultured cells. 18,19 However, these experimental models possess particular limitations.…”

mentioning

confidence: 99%

“…20 In contrast, freshly isolated proximal tubules retain a high degree of structural integrity as well as biochemical and functional properties of the in vivo state and they represent a good model to exclude vascular or hormonal effects. 15,21 Structural and functional properties may even be better retained in the context of the surrounding tissue using the model of in situ perfusion of individual tubules and their associated capillaries in living animals. 22,23 However, in vivo studies of intact kidneys most likely approach original renal physiology, since the context of the whole organism is retained.…”

mentioning

confidence: 99%

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Organic cation transport in the rat kidney in vivo visualized by time-resolved two-photon microscopy

Hörbelt

Wotzlaw

Sutton

et al. 2007

Kidney International

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Secretion of cationic drugs and endogenous metabolites is a major function of the kidney accomplished by tubular organic cation transport systems. A cationic styryl dye (ASP(+)) was developed as a fluorescent substrate for renal organic cation transporters. The dye was injected intravenously and continuously monitored in externalized rat kidneys by time-resolved two-photon laser scanning microscopy. To investigate changes in transport activity, cimetidine, a competitive inhibitor of organic cation transport was co-injected with ASP(+). Shortly after injection, fluorescence increased in peritubular capillaries. Simultaneously, fluorescence was transiently found at the basolateral membrane of the proximal and distal tubules at a higher intensity and shorter wavelength indicating membrane association of ASP(+). Subsequently, intracellular fluorescence increased steeply within 10 s. In the proximal tubules, intracellular fluorescence decreased by 50% within 5 min, while in the distal tubules the fluorescence decreased by only 5% within the same time frame. Intracellular uptake of ASP(+) into proximal tubules was significantly reduced by cimetidine. Our studies show that organic cation transport of the kidney can be visualized in vivo by two-photon laser scanning microscopy.

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Section: Discussionmentioning

confidence: 58%

mentioning

confidence: 99%

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confidence: 99%

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Organic cation transport in the rat kidney in vivo visualized by time-resolved two-photon microscopy

Hörbelt

Wotzlaw

Sutton

et al. 2007

Kidney International

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“…Investigating the mechanisms underlying the renal secretion of organic ions is fundamental to gather new knowledge on the renal secretion of some molecules acting as uremic toxins and some drugs. To this aim, the ASP+ fluorescent molecule has been previously used [9][10][11][12][13]. Besides the usage of ASP+ in kidney research, ASP+ has been widely used, e.g., to identify dopamine transporter-positive neurons in vitro [20] and serotonin transporter-(SERT-) expressing cells and to evaluate the activity and regulation of SERT in vitro [21].…”

Section: Discussionmentioning

confidence: 99%

“…The organic cation 4-dimethylaminostyryl)-N-methylpyridinium (ASP+) was first defined as a probe for renal OC transport in experiments using the stopped flow technique in intact rat kidneys [9]. Since then, ASP+ has been proposed to be used for drug transport analysis [10] and widely used in various experimental settings [9][10][11][12][13]. Prior to the use in renal research, ASP+ was described as a fluorescent probe for living cells, which showed variable emission spectra depending on its interaction with different cellular structures [14] including mitochondria [15].…”

Section: Introductionmentioning

confidence: 99%

The Probe for Renal Organic Cation Secretion (4-Dimethylaminostyryl)-N-Methylpyridinium (ASP+)) Shows Amplified Fluorescence by Binding to Albumin and Is Accumulated In Vivo

et al. 2022

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Accumulation of uremic toxins may lead to the life-threatening condition “uremic syndrome” in patients with advanced chronic kidney disease (CKD) requiring renal replacement therapy. Clinical evaluation of proximal tubular secretion of organic cations (OC), of which some are uremic toxins, is desired, but difficult. The biomedical knowledge on OC secretion and cellular transport partly relies on studies using the fluorescent tracer 4-dimethylaminostyryl)-N-methylpyridinium (ASP+), which has been used in many studies of renal excretion mechanisms of organic ions and which could be a candidate as a PET tracer. This study is aimed at expanding the knowledge of the tracer characteristics of ASP+ by recording the distribution and intensity of ASP+ signals in vivo both by fluorescence and by positron emission tomography (PET) imaging and at investigating if the fluorescence signal of ASP+ is influenced by the presence of albumin. Two-photon in vivo microscopy of male Münich Wistar Frömter rats showed that a bolus injection of ASP+ conferred a fluorescence signal to the blood plasma lasting for about 30 minutes. In the renal proximal tubule, the bolus resulted in a complex pattern of fluorescence including a rapid and strong transient signal at the brush border, a very low signal in the luminal fluid, and a slow transient intracellular signal. PET imaging using 11C-labelled ASP+ showed accumulation in the liver, heart, and kidney. Fluorescence emission spectra recorded in vitro of ASP+ alone and in the presence of albumin using both 1-photon excitation and two-photon excitation showed that albumin strongly enhance the emission from ASP+ and induce a shift of the emission maximum from 600 to 570 nm. Conclusion. The renal pattern of fluorescence observed from ASP+ in vivo is likely affected by the local concentration of albumin, and quantification of ASP+ fluorescent signals in vivo cannot be directly translated to ASP+ concentrations.

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Organic Cation Transport Measurements Using Fluorescence Techniques

Ciarimboli¹,

Schlatter²

2016

Neuromethods

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Digital Fluorescence Imaging of Organic Cation Transport in Freshly Isolated Rat Proximal Tubules

Abstract: ABSTRACT:The secretion of cationic drugs and endogenous metabolites is a major function of the kidney. This is accomplished by organic cation transport systems, mainly located in the proximal tubules.

Cited by 6 publications

References 15 publications

Organic cation transport in the rat kidney in vivo visualized by time-resolved two-photon microscopy

Organic cation transport in the rat kidney in vivo visualized by time-resolved two-photon microscopy

The Probe for Renal Organic Cation Secretion (4-Dimethylaminostyryl)-N-Methylpyridinium (ASP+)) Shows Amplified Fluorescence by Binding to Albumin and Is Accumulated In Vivo

Organic Cation Transport Measurements Using Fluorescence Techniques

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