The Microbiome Product Urolithin A Abolishes TGFβ‐Dependent Stimulation of PAI‐1 in Renal Epithelial Cells

Abstract

Introduction and HypothesisEllagitannins are abundant in various foods including fruits, nuts and wines, as well as plant‐based supplements. Urolithin A is derived from ellagitannins metabolism by the microbiome, is absorbed by the gut epithelium and may significantly contribute to the biological actions of ellagic acid. Previous studies demonstrate renal protective effects of both ellagic acid and urolithin A; however, the cellular mechanisms that underlie the benefits of urolithin A are unclear. Activation of the renal TGFβ‐PAI‐1 pathway is a key cellular event that contributes to fibrosis, oxidative stress and progressive kidney injury. Therefore, the current study assessed the effects of urolithin A on TGFβ‐induced stimulation of PAI‐1 in renal proximal tubule (PT) cells. We hypothesized that urolithin A would present protective effects to attenuate PAI‐1 expression in PT cells and exhibit greater potency than the parent compound ellagic acid.ResultsWe initially determined an optimal dose of TGFβ (0.1 to 10 ng/ml) to stimulate PAI‐1 release in the NRK‐52e cell line. Cells were maintained in serum free conditions for 48 hours prior to TGFβ treatment. At a concentration of 5 ng/ml, TGFβ increased PAI‐1 approximately 5‐fold (480 ± 35%, n=4; p<0.01) above unstimulated or basal levels following a 24 hour exposure. As expected, pretreatment with the TGFβ receptor (TGFβR) kinase inhibitor SB431542 (1 uM) abolished the increase in PAI‐1. Moreover, the EGFR kinase inhibitor AG1478 (1 μM) completely abrogated the PAI‐1 release in response to TGFβ and confirms earlier reports that TGFβ stimulation of PAI‐1 reflects the complete dependence on activation of the EGFR. In contrast, the cSRC kinase inhibitor SU6056 failed to block PAI‐1 release suggesting that TGFβ stimulates the ligand‐dependent transactivation of EGFR rather than direct cSRC activation of the EGFR. Similar to TGR and EGFR inhibition, pretreatment of the cells with urolithin A (40 μM) abolished the increase in PAI‐1 to TGFβ (5 ± 4% of basal release, n=4). Dose response studies of urolithin A revealed an ED50 of 22 ± 3 μM (n=3) to inhibit PAI‐1. Ellagic acid also inhibited TGFβ stimulation of PAI‐1 release, but was less potent (ED50 >50 μM) than Urolithin A.ConclusionWe conclude that the protective effects of urolithin A may involve inhibition of the TGFβ‐EGFR‐PAI‐1 pathway in renal epithelial cells. Although the precise inhibitory mechanisms are presently unknown, urolithin A may potentially target either TGFβR‐ or EGFR‐dependent kinases to abrogate PAI‐1 expression.Support or Funding InformationAHA Transformative Grant 18TPA34170522; Anonymous Contribution 15‐01401This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.