No studies which have elucidated the fibrotic mechanism of TMAO in the molecular level in human renal fibroblasts. Our findings show that the Akt/mTOR pathway mediates the signaling by which TMAO exerts its collagenproducing and proliferative effect on renal fibroblasts. Our findings show that TMAO enhanced the phosphorylation of Akt and mTOR but didn’t have an effect on their total protein level. At the functional level, the Akt (MK-2206) and mTOR (ridaforolimus) inhibitors significantly inhibited TMAO-induced proliferation and collagen production. Nonetheless, the PI3K inhibitor (wortmannin) did not minimize TMAO-induced proliferation. Looking at the gene expression of collagens, TMAO did not induce an improved gene expression of collagen 1, three, or four, which have previously been related with renal fibrosis [37,38]. This suggests that the improve of total collagen may possibly be an effect on the increased proliferation of renal fibroblasts induced by TMAO. The PI3K/Akt/mTOR pathway features a variety of (S)-Mephenytoin Purity & Documentation biological effects on cells both in the physiological and pathological levels. In the physiological level, it promotes cell viability, prevents apoptosis, and induces autophagy in erythropoiesis [39,40]. Furthermore, it’s involved in cell proliferation and cell fate determination [415]. At the pathological level, its role is established in neurodegenerative disease, tumor growth, tumor cells proliferation, and metabolism [39,46]. There’s a selection of current studies on biological agents targeting PI3K, Akt, and mTOR to treat hematological malignancies and solid tumors [475]. Substantially research exists on the newly identified plant derivatives that use the PI3K/Akt/mTOR pathway as a mediator to influence fibroblast apoptosis [56,57] or proliferation [58]. Taken with each other, our findings indicate that only Akt and mTOR, but not PI3K, mediates the effect of TMAO on collagen production and human renal fibroblast proliferation. Lately TMAO was discovered to directly bind to and activate protein kinase R-like endoplasmic reticulum kinase (PERK), an ER stress kinase in hepatocytes. The study suggested that PERK was a TMAO receptor [59]. In our findings, we observed that inhibition of PERK reduced the TMAO-mediated collagen production and proliferation of renal fibroblasts. It has been shown, in agreement with our findings, that activated PERK can mediate the activation of the PI3K/Akt/mTOR pathway via its lipid kinase activity. PERKs lipid kinase activity converts diacylglycerol to phosphatidic acid (PA), and PA is very important for mTOR complex formation and Akt activation [603]. This shows that there is a hyperlink amongst PERK and mTOR/Akt in collagen production and renal fibroblast proliferation. We also investigated whether or not NLRP3 inflammasome activation may be involved in TMAO-induced fibroblast proliferation. Several different studies help the association of the NLRP3 inflammasome with fibrosis, TMAO, Akt and mTOR [22,23,647]. Applying NLRP3 and caspase-1 knockout cell lines, we located that the proliferative effect of TMAO on human renal fibroblasts is NLRP3 and caspase-1 dependent. We also found increased protein levels of NLRP3 and caspase-1 after TMAO therapy. Nevertheless, TMAO stimulation of renal fibroblasts didn’t induce the release of IL-1, indicating that theInt. J. Mol. Sci. 2021, 22,9 ofrole of NLRP3 and Mifamurtide supplier capsase-1 in TMAO-mediated fibroblast proliferation is independent of NLRP3 inflammasome activation. It has previously been shown that NLRP3 via an inflammasome-independent ro.