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Kinetic Measurements of Di- and Tripeptide and Peptidomimetic Drug Transport in Different Kidney Regions Using the Fluorescent Membrane Potential-Sensitive Dye, DiS-C3-(3)

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Abstract

Tri- and dipeptides are transported in the kidney by PEPT1 and PEPT2 isoforms. The aim of this study was to investigate differences in transport kinetics between renal brush border (BBMV) and outer medulla (OMMV) membrane vesicles (where PEPT1 and PEPT2 are sequentially available) for a range of di- and tripeptides and peptidomimetic drugs. This was accomplished through the use of the potential-sensitive fluorescent dye 3,3′-dipropylthiacarbocyanine iodide [DiS–C3-(3)]. BBMV and OMMV were prepared from the rat kidney using standard techniques. The presence of PEPT1 in BBMV and PEPT2 in OMMV was confirmed using Western blotting. Fluorescence changes were measured when extravesicular medium at pH 6.6 containing 0–1 mM substrates was added to a cuvette containing vesicles pre-equilibrated at pH 7.4 and 2.71 μM DiS-C3-(3). An increase in fluorescence intensity occurred upon substrate addition reflecting the expected positive change in membrane potential difference. Of the range of substrates studied, OMMV manifested the highest affinity to cefadroxil and valacyclovir (K m 4.3 ± 1.2 and 11.7 ± 3.2 µM, respectively) compared to other substrates, whilst the BBMV showed a higher affinity to Gly-His (K m 15.4 ± 3.1 µM) compared to other substrates. In addition, OMMV showed higher affinity and capacity to Gly-Gln (K m 47.1 ± 9.8 µM, 55.5 ± 2.8 ΔF/s/mg protein) than BBMV (K m 78.1 ± 13.3 µM and 35.5 ± 1.7 ΔF/s/mg protein, respectively). In conclusion, this study successfully separated the expression of PEPT1 and PEPT2 into different vesicle preparations inferring their activity in different regions of the renal proximal tubule.

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References

  • Alghamdi OA, King N (2012) Novel application of the dye, DiS-C3-(3) as a biosensor of the renal membrane potential difference. Int J Biosci Biochem Bioinform 9(4):282–286

    Google Scholar 

  • Amasheh S, Wenzel U, Boll M, Dorn D, Weber WM, Clauss W, Daniel H (1997) Transport of charged dipeptides by the intestinal H+/peptide symporter PepT1 expressed in Xenopus laevis oocytes. J Membr Biol 155(3):247–256

    Article  CAS  PubMed  Google Scholar 

  • Anderson CM, Thwaites DT (2010) Hijacking solute carriers for proton-coupled drug transport. Physiology 25(6):364–377. doi:10.1152/physiol.00027.2010 (Bethesda, MD)

    Article  CAS  PubMed  Google Scholar 

  • Beck JC, Sacktor B (1978) Membrane potential-sensitive fluorescence changes during Na+-dependent d-glucose transport in renal brush border membrane vesicles. J Biol Chem 253(20):7158

    CAS  PubMed  Google Scholar 

  • Biber J, Stieger B, Haase W, Murer H (1981) A high yield preparation for rat kidney brush border membranes. Different behaviour of lysosomal markers. Biochem Biophys Acta 647(2):169–176

    Article  CAS  PubMed  Google Scholar 

  • Blostein R (1999) Structure-function studies of the sodium pump. Biochem Cell Biol 77(1):1–10. doi:10.1139/o99-018

    Article  CAS  PubMed  Google Scholar 

  • Brandsch M, Knutter I, Bosse-Doenecke E (2008) Pharmaceutical and pharmacological importance of peptide transporters. J Pharm Pharmacol 60(5):543–585. doi:10.1211/jpp.60.5.0002

    Article  CAS  PubMed  Google Scholar 

  • Brown CD, King N, Simmons NL (1993) Co-expression of an anion conductance pathway with Na(+)-glucose cotransport in rat renal brush-border membrane vesicles. Pflugers Arch 423(5–6):406–410

    Article  CAS  PubMed  Google Scholar 

  • Cassano G, Maffia M, Vilella Storelli C (1988) Effects of membrane potential on Na Cotransporters in eel intestinal brush-border membrane vesicles: studies with fluorescent dye. J Membr Biol 101:225–236

    Article  CAS  Google Scholar 

  • Chen XZ, Zhu T, Smith DE, Hediger MA (1999) Stoichiometry and kinetics of the high-affinity H+-coupled peptide transporter PepT2. J Biol Chem 274(5):2773–2779

    Article  CAS  PubMed  Google Scholar 

  • Daniel H, Kottra G (2004) The proton oligopeptide cotransporter family SLC15 in physiology and pharmacology. Pflugers Arch 447(5):610–618. doi:10.1007/s00424-003-1101-4

    Article  CAS  PubMed  Google Scholar 

  • Daniel H, Rubio-Aliaga I (2003) An update on renal peptide transporters. Am J Physiol Ren Physiol 284(5):F885–F892. doi:10.1152/ajprenal.00123.2002

    Article  CAS  Google Scholar 

  • Daniel H, Morse EL, Adibi SA (1991) The high and low affinity transport systems for dipeptides in kidney brush border membrane respond differently to alterations in pH gradient and membrane potential. J Biol Chem 266(30):19917–19924

    CAS  PubMed  Google Scholar 

  • Donowitz M, Emmer E, McCullen J, Reinlib L, Cohen ME, Rood RP, Madara J, Sharp GW, Murer H, Malmstrom K (1987) Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles. Am J Physiol 252(6 Pt 1):G723–G735

    CAS  PubMed  Google Scholar 

  • Faller LD (2008) Mechanistic studies of sodium pump. Arch Biochem Biophys 476(1):12–21. doi:10.1016/j.abb.2008.05.017

    Article  CAS  PubMed  Google Scholar 

  • Inui K, Terada T, Masuda S, Saito H (2000) Physiological and pharmacological implications of peptide transporters, PEPT1 and PEPT2. Nephrol Dial Transplant 15(Suppl 6):11–13

    Article  CAS  PubMed  Google Scholar 

  • Jorgensen P (1986) Structure, function and regulation of Na, K-ATPase in the kidney. Kidney Int 29(1):10–20

    Article  CAS  PubMed  Google Scholar 

  • Kaur J, Bachhawat AK (2009) A modified Western blot protocol for enhanced sensitivity in the detection of a membrane protein. Anal Biochem 384(2):348–349. doi:10.1016/j.ab.2008.10.005

    Article  CAS  PubMed  Google Scholar 

  • Kottra G, Spanier B, Verri T, Daniel H (2013) Peptide transporter isoforms are discriminated by the fluorophore-conjugated dipeptides beta-Ala- and d-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid. Physiol Rep 1(7):e00165. doi:10.1002/phy2.165

    Article  PubMed  PubMed Central  Google Scholar 

  • Kragh-Hansen U, Sheikh MI (1984) Serine uptake by luminal and basolateral membrane vesicles from rabbit kidney. J Physiol 354:55–67

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kragh-Hansen U, Jorgensen KE, Sheikh MI (1982) The use of potential-sensitive cyanine dye for studying ion-dependent electrogenic renal transport of organic solutes. Spectrophotometric measurements. Biochem J 208(2):359–368

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Leibach FH, Ganapathy V (1996) Peptide transporters in the intestine and the kidney. Annu Rev Nutr 16:99–119. doi:10.1146/annurev.nu.16.070196.000531

    Article  CAS  PubMed  Google Scholar 

  • Ocheltree SM, Shen H, Hu Y, Keep RF, Smith DE (2005) Role and relevance of peptide transporter 2 (PEPT2) in the kidney and choroid plexus: in vivo studies with glycylsarcosine in wild-type and PEPT2 knockout mice. J Pharmacol Exp Ther 315(1):240–247. doi:10.1124/jpet.105.089359

    Article  CAS  PubMed  Google Scholar 

  • Plášek J, Dale RE, Sigler K, Laskay G (1994) Transmembrane potentials in cells: a diS-C3(3) assay for relative potentials as an indicator of real changes. Biochem Biophys Acta 1196:181–190

    Article  PubMed  Google Scholar 

  • Ries M, Wenzel U, Daniel H (1994) Transport of cefadroxil in rat kidney brush-border membranes is mediated by two electrogenic H+-coupled systems. J Pharmacol Exp Ther 271(3):1327–1333

    CAS  PubMed  Google Scholar 

  • Rubio-Aliaga I, Daniel H (2008) Peptide transporters and their roles in physiological processes and drug disposition. Xenobiotica 38(7–8):1022–1042. doi:10.1080/00498250701875254

    Article  CAS  PubMed  Google Scholar 

  • Shen H, Smith DE, Yang T, Huang YG, Schnermann JB, Brosius FC III (1999) Localization of PEPT1 and PEPT2 proton-coupled oligopeptide transporter mRNA and protein in rat kidney. Am J Physiol 276(5):F658–F665

    CAS  PubMed  Google Scholar 

  • Shen H, Ocheltree SM, Hu Y, Keep RF, Smith DE (2007) Impact of genetic knockout of PEPT2 on cefadroxil pharmacokinetics, renal tubular reabsorption, and brain penetration in mice. Drug Metab Dispos 35(7):1209–1216. doi:10.1124/dmd.107.015263

    Article  PubMed  Google Scholar 

  • Smith DE, Pavlova A, Berger UV, Hediger MA, Yang T, Huang YG, Schnermann JB (1998) Tubular localization and tissue distribution of peptide transporters in rat kidney. Pharm Res 15(8):1244–1249

    Article  CAS  PubMed  Google Scholar 

  • Steel A, Nussberger S, Romero M, Boron W, Boyd C, Hediger M (1997) Stoichiometry and pH dependence of the rabbit proton-dependent oligopeptide transporter PepT1. J Physiol 498(Pt 3):563

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Takahashi K, Nakamura N, Terada T, Okano T, Futami T, Saito H, Inui KI (1998) Interaction of beta-lactam antibiotics with H+/peptide cotransporters in rat renal brush-border membranes. J Pharmacol Exp Ther 286(2):1037–1042

    CAS  PubMed  Google Scholar 

  • Terada T, Saito H, Mukai M, Inui K (1997) Recognition of beta-lactam antibiotics by rat peptide transporters, PEPT1 and PEPT2, in LLC-PK1 cells. Am J Physiol 273(5 Pt 2):F706–F711

    CAS  PubMed  Google Scholar 

  • Vayro S, Simmons NL (1996) An effect of Ca2+ on the Intrinsic Cl(-)-conductance of rat kidney cortex brush border membrane vesicles. J Membr Biol 150(2):163–173

    Article  CAS  PubMed  Google Scholar 

  • Vieira F, Malnic G (1968) Hydrogen ion secretion by rat renal cortical tubules as studied by an antimony microelectrode. Am J Physiol 214(4):710–718

    CAS  PubMed  Google Scholar 

  • Wright SH, Krasne S, Kippen I, Wright EM (1981) Na+-dependent transport of tricarboxylic acid cycle intermediates by renal brush border membranes. Effects on fluorescence of a potential-sensitive cyanine dye. Biochem Biophys Acta 640(3):767–778

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the Saudi Ministry of Higher Education and University of New England. We would like to thank Mr. Brian Cross and Mr. Jonathon Clay for their excellent technical assistance.

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Authors and Affiliations

  1. School of Science and Technology, University of New England, Armidale, NSW, 2351, Australia

    Othman A. Alghamdi, Graham L. Jones & Pierre D. J. Moens

  2. School of Biomedical and Healthcare Sciences, Plymouth University Peninsula Schools of Medicine and Dentistry, Plymouth University, Plymouth, PL4 8AA, UK

    Nicola King

  3. Department of Biochemistry, Faculty of Science, Centre for Science and Medical Research, University of Jeddah, Jeddah, Kingdom of Saudi Arabia

    Othman A. Alghamdi

Authors
  1. Othman A. Alghamdi
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  2. Nicola King
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  4. Pierre D. J. Moens
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Correspondence to Nicola King.

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Apart from the funding sources (listed on the title page), the authors declare that they have no other conflicts of interest.

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Alghamdi, O.A., King, N., Jones, G.L. et al. Kinetic Measurements of Di- and Tripeptide and Peptidomimetic Drug Transport in Different Kidney Regions Using the Fluorescent Membrane Potential-Sensitive Dye, DiS-C3-(3). J Membrane Biol 250, 641–649 (2017). https://doi.org/10.1007/s00232-017-9990-x

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