P345 Primary Human Renal Proximal Tubule Cell Model for Study of Transepithelial Transport

Caitlin Brown , Celsis In Vitro Technologies, Baltimore, MD
Yuko Kosaka , 3-V Biosciences, Menlo Park, CA
Marc J. Evanchik , 3-V Biosciences, Menlo Park, CA
Scott Heyward , Celsis In Vitro Technologies, Baltimore, MD
Ji Young Lee , Celsis In Vitro Technologies, Baltimore, MD
Renal elimination is a major clearance route for many drugs and their metabolites.  This includes active tubular secretion, which takes place primarily in the proximal tubule (PT) cells, and is mediated by uptake and efflux transporters.  Currently, most in vitro kidney transporter data are obtained from renal slices, isolated tubule fragments, or animal-derived cell lines that can exhibit differing transport characteristics or altered transporter behavior compared to human.  Primary cells cultured on transwells have been shown to be an effective in vitro model and may represent a more physiologically relevant system.1,2  To that end, freshly isolated primary kidney cells were plated onto semipermeable membrane filters and assessed for monolayer integrity and transporter functionality.  A gamma glutamyltransferase (GGT) assay was used to determine percentage of PT cells with respect to distal tubule (DT) cells.  An approximate 3:1 ratio of PT to DT cells has been determined to be optimal for appropriate tight junction formation.2   In addition, transepithelial electrical resistance (TEER) measurements and mannitol permeability verified monolayer integrity.  Prototypical substrates, para-aminohippuric acid (PAH) and 1-methyl-4-phenylpyridinium (MPP+), were used to evaluate expression of the key transporter pathways OATs to MRP2/4 and OCT2 to MATE1, respectively.  A flux ratio of secretory to absorptive (B→A/A→B) greater than 1.5 was defined as significant and corresponded well to TEER measurements starting at about 110 ohms/cm2.  Transporter function diminished or was abolished at much higher TEER values.  Shipment of these transwell cultures on agarose media caused a drop in TEER values and a loss of net flux after transfer to liquid media.  However, after some days in culture, transepithelial transport was re-established as measured by PAH and MPP+ movement.  Thus, a primary kidney cell model has been shown to present appropriate transporter functioning and retention of these transporters upon shipment in an agarose system.

1.  Lash LH, Putt DA, Hongliang C. 2006 Aug 24. Membrane transport function in primary cultures of human proximal tubular cells. Toxicology 228: 200-218.

2.  Brown CDA, Sayer R, Windass AS, Haslam IS, De Broe ME. 2008 Sept 18. Characterisation of human tubular cell monolayers as a model of proximal tubular xenobiotic handling. Toxicol Appl Pharm 223: 428-438.