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Cationic uremic toxins affect human renal proximal tubule cell functioning through interaction with the organic cation transporter

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Abstract

Several organic cations, such as guanidino compounds and polyamines, have been found to accumulate in plasma of patients with kidney failure due to inadequate renal clearance. Here, we studied the interaction of cationic uremic toxins with renal organic cation transport in a conditionally immortalized human proximal tubule epithelial cell line (ciPTEC). Transporter activity was measured and validated in cell suspensions by studying uptake of the fluorescent substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium-iodide (ASP+). Subsequently, the inhibitory potencies of the cationic uremic toxins, cadaverine, putrescine, spermine and spermidine (polyamines), acrolein (polyamine breakdown product), guanidine, and methylguanidine (guanidino compounds) were determined. Concentration-dependent inhibition of ASP+ uptake by TPA, cimetidine, quinidine, and metformin confirmed functional endogenous organic cation transporter 2 (OCT2) expression in ciPTEC. All uremic toxins tested inhibited ASP+ uptake, of which acrolein required the lowest concentration to provoke a half-maximal inhibition (IC50 = 44 ± 2 μM). A Dixon plot was constructed for acrolein using three independent inhibition curves with 10, 20, or 30 μM ASP+, which demonstrated competitive or mixed type of interaction (K i = 93 ± 16 μM). Exposing the cells to a mixture of cationic uremic toxins resulted in a more potent and biphasic inhibitory response curve, indicating complex interactions between the toxins and ASP+ uptake. In conclusion, ciPTEC proves a suitable model to study cationic xenobiotic interactions. Inhibition of cellular uptake transport was demonstrated for several uremic toxins, which might indicate a possible role in kidney disease progression during uremia.

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Abbreviations

ASP+ :

4-(4-(Dimethylamino)styryl)-N-methylpyridinium-iodide

ciPTEC:

Conditionally immortalized proximal tubule epithelial cells

CKD:

Chronic kidney disease

DMEM:

Dulbecco's modified eagle medium

ESRD:

End-stage renal disease

FCS:

Fetal calf serum

IC50 :

Half-maximal inhibitory concentration

MATE:

Multidrug and toxic compound extrusion protein

MTT:

(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

OCT:

Organic cation transporter

RLU:

Relative light units

TPA:

Tetrapentylammonium

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Acknowledgment

This work was supported by the BioMedical Materials Institute (BMM, Project P3.01 BioKid); the Dutch Ministry of Economic Affairs, Agriculture and Innovation; the Nierstichting Nederland; and the Netherlands Institute for Regenerative Medicine (NIRM, grant no. FES0908). M.J.G. Wilmer and R. Masereeuw were supported by a collaboration research grant from AstraZeneca (grant no. A10-0324). J. Hoenderop was supported by an EURYI award.

Ethical standards

The experiments described in this article comply with the current laws of the country in which they were performed (the Netherlands).

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The authors declare that they have no conflict of interest.

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Correspondence to Rosalinde Masereeuw.

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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Carolien M.S. Schophuizen, Martijn J. Wilmer, Lambert P. van den Heuvel, and Rosalinde Masereeuw contributed equally to this work.

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Fig9

Online Resource 1 OCT-mediated ASP+ uptake by mature ciPTEC analyzed by measuring the fluorescence of transported ASP+ in the absence (white bar) or presence (gray bar) of OCT2 inhibitor tetrapentylammonium (TPA). Additionally, uptake was performed at 4 °C (black bar). Uptake was significantly decreased in ciPTEC in the presence of TPA or at 4 °C (**P < 0.001). Results are shown as mean values ± SEM of three experiments (GIF 7 kb)

High resolution image (TIFF 285 kb)

Fig10

Online Resource 2 Time dependence of ASP+ uptake. OCT-mediated ASP+ uptake by ciPTEC analyzed by measuring the fluorescence of transported ASP+ for 60 min. Uptake was found to be linear for at least 60 min. Results are shown as mean values ± SEM of three experiments (GIF 10 kb)

High resolution image (TIFF 327 kb)

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Schophuizen, C.M.S., Wilmer, M.J., Jansen, J. et al. Cationic uremic toxins affect human renal proximal tubule cell functioning through interaction with the organic cation transporter. Pflugers Arch - Eur J Physiol 465, 1701–1714 (2013). https://doi.org/10.1007/s00424-013-1307-z

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