Abstract 129: The Microbiome Product Urolithin a Abrogates the Tgf-β Egfr Pai-1 Pathway by Inhibiting Egfr Activation and Expression

Abstract

Introduction: Ellagitanins are highly abundant in various foods including fruits, nuts and wines, and particularly high in commercial muscadine grape-derived supplements. The microbiome metabolizes ellagitanins to urolithins. Urolithin A (UA), the final product of the microbiome processing pathway may contribute to the cardioprotective effects of ellagitanins; however, the cellular signaling mechanisms of UA are unclear. Since the TGFβ-PAI-1 pathway contributes to fibrosis, cellular senescence and progressive kidney injury, we evaluated the effects of UA on TGFβ-induced stimulation of PAI-1 in rat renal proximal tubule cells (rPTCs - NRK52E). We hypothesize that UA may be an endogenous anti-fibrotic compound that attenuates the TGFβ-PAI-1 response. Results: TGFβ (5 ng/ml) treatment markedly increased PAI-1 levels 10-fold [43.9 ± 0.7 vs 3.5 ± 0.3 ng/ml; P<0.001, n=3] in the rPTCs at 24 hrs. The TGFβ receptor (TGFβR) kinase (ALK5) inhibitor SB525334 or UA abolished the increase in PAI-1 to TGFβ [SB: 1.3 ± 0.3; UA: 2.0 ± 0.4 ng/ml; P<0.001, n=3]; UA exhibits an IC50 of 3.1 μM to abrogate the TGFβ-PAI-1 response. Since the TGFβ pathway may transactivate the EGFR, treatment with the EGFR kinase inhibitors AG1478 (AG, 1 μM) and Lapatinib (LAP, 1 μM) also abrogated the PAI-1 response to TGFβ [AG: 3.3 ± 0.4; LAP: 6.2 ± 1.4 ng/ml; P<0.001, n=3]. Addition of EGF alone increased PAI-1 to a similar extent as TGFβ [46.6 ± 2.7 ng/ml; P<0.001, n=3], and treatment with UA or AG abolished the EGF-PAI-1 response [2.4 ± 0.1 and 1.3 ± 0.1 ng/ml, respectively; P<0.001, n=3]. Given the dependence on EGFR signaling for PAI-1, we determined whether UA directly attenuates EGFR stimulation. Indeed, UA treatment inhibited peak EGFR phosphorylation to EGF at 5 mins [10 μM: 67 ± 4%; 50 μM: 71 ± 5%; P<0.05, n=3], and reduced total EGFR expression at 24 hrs [10 μM: 27 ± 11%; 50 μM: 66 ± 7%; P<0.05, n=6]. UA was devoid of cytotoxic effects (LDH release). Conclusion: The present study is the first to establish that UA abrogates the TGFβ-EGFR-PAI-1 pathway via the inhibition of EGFR stimulation and expression. Moreover, the potency of UA to attenuate PAI-1 is comparable to its physiological levels. The microbiome product UA may convey anti-fibrotic actions within the kidney by targeting the EGFR system.