title: Properties and regulation of organic cation transport in freshly isolated mouse proximal tubules analyzed with a fluorescence reader-based method.
abstract: The main elimination site of organic cations (OCs) is the renal proximal tubule (PT). OC transporters (OCT) accept endogenous and exogenous substances and xenobiotics. As transgenic mouse models are increasingly used in translational medicine, functional properties with special focus on regulation of OCT of isolated mouse PTs were studied with a new fluorescence reader-based method, which allows studying larger numbers of tubules per kidney. OC transport across the basolateral membrane of PTs from male mice was measured as initial uptake of the fluorescent dye 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP). A microtiter plate fluorescence reader was used to semi-automatically analyze OC transport in freshly isolated tubules. Relative mRNA expression of OCT1/OCT2/OCT3 in PTs was 1/0.3/0.01 and did not vary from S1 to S3 segments. ASP was transported by PTs with a K (m) of 6 μM. It was inhibited by TEA, TPA, or cimetidine (IC(50)=5, 19, or 53 μM, respectively). Angiotensin II stimulated ASP uptake (+63%), while stimulation of PKC reduced (-37%) OC transport. Inhibition of p56(lck) tyrosine kinase (-60%), of PI3K (-36%), of Ca(2+)/calmodulin (-25%), or of PKA (-33%) reduced OC transport. In PTs from OCT1/2(-/-) mice ASP uptake was reduced to ~20%. Using this fluorescence reader-based method, we report substrate specificities and a complex pattern of acute regulation of OC transport in isolated mouse PTs. Compared to isolated human PTs or rat and human OCT isoforms expressed in HEK293-cells, OC transport across the basolateral membrane of freshly isolated mouse PTs shows similarities but also specific differences.
authors.affiliation: Medizinische Klinik und Poliklinik D, Experimentelle Nephrologie, Universität Münster, Domagkstrasse 3a, Münster, Germany.
authors.forename: Svenja K
authors.initials: SK
authors.lastname: Holle
authors.forename: Giuliano
authors.initials: G
authors.lastname: Ciarimboli
authors.forename: Bayram
authors.initials: B
authors.lastname: Edemir
authors.forename: Ute
authors.initials: U
authors.lastname: Neugebauer
authors.forename: Hermann
authors.initials: H
authors.lastname: Pavenstädt
authors.forename: Eberhard
authors.initials: E
authors.lastname: Schlatter
mesh_terms: D000818:Animals; D002412:Cations / Q000378:metabolism*; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D017136:Ion Transport / Q000502:physiology*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D027702:Organic Cation Transporter 1 / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
chemical_list: D002412:Cations; D005456:Fluorescent Dyes; D027702:Organic Cation Transporter 1; D020033:Protein Isoforms; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C