(M0930-02-09) A 24 and 96 Well High-Throughput Renal Proximal Tubule MPS Model for Drug Development and Safety Studies

Purpose: The proximal tubule (PT) is the key nephron segment mediating renal drug elimination drug absorption and drug induced nephrotoxicity.  In vivo animal studies have proved to be  poorly predictive of human outcome. To address this roadblock, there has been a drive to develop physiological relevant microphysiological systems (MPS) of  PT to recapitulate differentiation and  function in vitro.  We have  recently developed  an  aProximateMPS™ human PT  platform, in which primary human PT cells are subject to  fluidic media flow and  a shear stress of between 0.1-2 dynes/cm²

Methods: Human primary PT cells were isolated from transplant grade kidney and seeded onto the underside of 24 or 96 well Transwell filter supports. After attachment cells were placed in the specially designed 24 or 96 well  filter holder such that the apical membrane of the cell monolayer was subject to media flow and a shear stress of 2 dyne/cm². Cells were grown for 7 days before being assessed.

Results: Under fluidic flow conditions, human primary PT cells formed confluent  monolayers  with a Transepithelial Electrical Resistance (TEER) of between 60-90Ωcm² by day 7 in culture, not significantly different from the 60-120Ωcm² of identical cells grown under static culture conditions.  Similarly measurement of barrier function using either lucifer yellow (P_app 15.6±06 cm/sec, n=6) or ¹⁴C-mannitol (P_app 14.4±0.3cm/sec, n=6) under flow conditions gave very similar results  (P_app 16.1±1.4 cm/sec (lucifer yellow) or 15.6±2.3 cm/sec (mannitol) to barrier function measured under static conditions. At the mRNA level for the key renal transporters, OAT1, OAT3, OCT2 MATE1, MDR1, megalin and cubulin. expression levels were elevated at least 5- fold compared to cell monolayers grown under static conditions. Immunohistochemistry revealed that flow induced a significant  increase  in the number  of  primary cilia by cells within the monolayer. To assess impact of flow upon transport function, the flux of ¹⁴C-creatinine was measured in the  absorptive (Jab)  and secretory (Jba) direction . In both cultures, a net secretion of creatinine was found. In monolayers exposed to flow the magnitude of net secretion was >3 fold that of static cultures (Jab 203.1±12.6 pmole/cm²/h, Jba 398.2±19.1,  Jnet  189.3±14.6, P< 0.001 , n=6 ). To assess nephrotoxicity, cells were exposed to a range of cisplatin  concentrations for 48 hours. The key biomarkers; NGAL, KIM1 and clusterin were all significantly elevated and the magnitude of release was significantly greater (P < 0.1, n=3) in monolayer under flow compared with static cultures.

Conclusion: This dataset, suggests that  growing human PT cells on Transwell filter supports with media flow across the apical membrane, significantly improves phenotype and function and has significant benefit to the utility and near-physiology  of the model