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Updated: Jun 2, 2026
Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells
Published on: June 20, 2018
Properties and regulation of organic cation transport in freshly isolated mouse proximal tubules analyzed with a
Svenja K Holle1, Giuliano Ciarimboli, Bayram Edemir
1Medizinische Klinik und Poliklinik D, Experimentelle Nephrologie, Universität Münster, Domagkstrasse 3a, Münster, Germany.
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.
Insights
Researchers studied organic cation (OC) transporters in mouse kidney tubules using a new fluorescence method. They identified key regulators of OC transport, revealing complex control mechanisms in the renal proximal tubule.
Area of Science:
- Pharmacology
- Nephrology
- Molecular Biology
Background:
- The renal proximal tubule (PT) is the primary site for eliminating organic cations (OCs).
- Organic cation transporters (OCTs) handle endogenous and exogenous substances, including xenobiotics.
- Transgenic mouse models are crucial for translational medicine research.
Purpose of the Study:
- To investigate the functional properties and regulation of OCTs in isolated mouse PTs.
- To establish a novel fluorescence reader-based method for analyzing OC transport in larger numbers of tubules.
- To characterize substrate specificity and regulatory pathways of OC transport in mouse PTs.
Main Methods:
- Isolated mouse PTs were used to measure the initial uptake of the fluorescent dye 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP) as a model OC.
- A microtiter plate fluorescence reader enabled semi-automated analysis of OC transport.
- Relative mRNA expression of OCT1, OCT2, and OCT3 was quantified in different PT segments.
- ASP uptake was assessed in wild-type and OCT1/2(-/-) mouse PTs.
Main Results:
- ASP was identified as a substrate for mouse PTs with a K(m) of 6 μM, inhibited by TEA, TPA, and cimetidine.
- Angiotensin II significantly stimulated ASP uptake (+63%), while protein kinase C (PKC) stimulation reduced it (-37%).
- Inhibition of p56(lck) tyrosine kinase, PI3K, Ca(2+)/calmodulin, and PKA pathways reduced OC transport.
- ASP uptake was significantly decreased (~20%) in PTs from OCT1/2(-/-) mice, indicating their crucial role.
Conclusions:
- A novel fluorescence reader-based method allows efficient study of OC transport in isolated mouse PTs.
- Mouse PT OC transport is subject to complex acute regulation by various signaling pathways.
- Mouse PT OC transport exhibits both similarities and specific differences compared to human and rat counterparts.
