Stabilizing Activated Rac1 in a Long-Term Primary Renal Tubular Culture Model Improves Transport Without Causing Monolayer Leakiness

Background: Inhibition of Transforming Growth Factor-β is known to improve primary renal tubule cell differentiation in Long-Term Culture but strategies to enhance this mechanism have not been developed. The small Rho GTPase, Rac1, is a key regulator of epithelial cell integrity, that functions by promoting actin polymerization and organization. Using gene-targeting approaches Rac1 was identified as an important modulator of kidney tubular function. Whether exogenous activation of Rac1 is a feasible non-toxic approach to enhance tubular transport and differentiation is not known. A screen of small-molecule agonists recently identified the specific Rac1-activating compound 3744270 which reversibly stabilizes the activated GTP-bound state. Methods: Primary human renal proximal tubule cells (RPTEC, Lonza, Basel) were seeded onto permeable supports and cultured on orbital shakers. Apicobasal leak was assessed using TRITC-labelled dextrans and apicobasal transport was measured by media mass change. Media was supplemented with 3744270 (different concentrations) and 10µM SB431542 (selective inhibitor of the TGF-β type I receptor). Results: There was no difference in apicobasal transport or leak at any concentration of 3744270 at 6 weeks, but by 8 weeks there was a nonsignificant trend towards increased transport in the 10 µM 3744270 group which persisted after 3744270 supplementation was changed to 0. Leak rates remained unchanged. Addition of SB431542 increased transport. The difference in transport between 3744270-treated cells and controls was more pronounced at week 30 even though the Rac1-activating compound had been withdrawn from media for 52 days. Well-to-well variation in transport was much lower in 3744270-treated wells than in control wells (standard error/mean 3% vs 38%). Conclusion: In primary renal tubular cells in long-term culture that were induced to differentiate using TGF-β inhibition, a Rac1 activating compound potentially improves apicobasal tubular transport without disrupting the epithelial monolayer. This effect appeared to persist after the Rac1 activator was removed. Follow-up studies will determine the mechanism whereby temporary Rac1 stimulation permanently alters tubular differentiation and activity. Funding: NIDDK Support