Protein-bound uremic toxins (<500 Da) are difficult to eliminate by dialysis because of their protein-binding capacity. These toxins, which are mostly produced from intestinal bacterial fermentation, include phenolic compounds from tyrosine and phenylalanine, as well as indolic compounds from tryptophan, such as indoxyl sulfate (IS, C8H7NO4S) [1]. Uremic toxins induce inflammation by increasing the production of reactive oxygen species (ROS) and proinflammatory cytokine levels [2]. Inflammation plays a key role in renal fibrosis and mitochondrial dysfunction contributes significantly to the development of chronic kidney disease (CKD) development [3].

Inflammasomes comprise large multimeric protein complexes with receptor proteins including Nod-like receptor (NLR) family pyrin domain-containing (NLRP) 1, NLRP3, NLR family CARD domain-containing 4 (NLRC4), and absent in melanoma 2 (AIM2) from the HIN–200 family. Inflammasomes activate caspase-1, which promotes interleukin (IL)-1β and IL-18 maturation and secretion, triggering inflammation and cell injury [4]. ROS play a crucial role in inflammasome activation, particularly the formation and activation of NLRP3 [5]. NLRP3 and caspase-1 expression levels are elevated in the kidneys of patients with CKD or fibrosis [6], [7], suggesting that the NLRP3 inflammasome is involved in renal pathology. Additionally, transforming growth factor beta receptor type I/II (TGF-βRI/II) phosphorylates suppressor of Smad2 and Smad3, which form a complex with Smad4 that translocates to the nucleus to upregulate the expression of fibrotic genes. The activation of the TGF-β1–Smad2/3 signaling axis drives fibrosis in most CKD forms [8].

Natural plant compounds are active substances that modulate multiple pathways, including epithelial-to-mesenchymal transition (EMT), through antioxidant, anti-inflammatory, and antifibrotic mechanisms [9], [10]. Rosmarinic acid (RA, C18H16O8), an ester of caffeic acid and 3, 4-dihydroxyphenylacetic acid, is found in over 160 plant species, particularly Lamiaceae and is known for its wide range of desirable biological activities [11]. Previous studies, including our study, demonstrated effectiveness of RA against kidney injury induced by cisplatin [12], or unilateral ureteral obstruction (UUO) [13]. Specifically, RA administration ameliorated kidney damage, by reducing fibrosis-related markers, such as TGF-β1, vimentin, and phosphorylated protein kinase B (p-AKT), while increasing E-cadherin expression in both UUO kidneys and IS-treated NRK-52E cells [13].

MCC950 (N-[[(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)amino] carbonyl]-4-(1-hydroxy-1-methylethyl)-2-furansulfonamide) is a potent selective inhibitor of both canonical and non-canonical NLRP3 inflammasome activation. It disrupts ASC oligomerization [14] and has shown promising outcomes in the treatment of inflammatory diseases in both human and animal models [15], [16]. In this study, we examined how IS affected the expression of the inflammasome components NLRP3, NLRC4, and AIM2, caspase-1 activity, IL-1β secretion, inflammation, and oxidative stress in vitro, using NRK-52E cells. We also investigated the efficacy of RA alone and in combination with MCC950 against renal fibrosis in a murine model of UUO.