Insights into the structure and function of the human organic anion transporter 1 in lipid bilayer membranes

The human SLC22A6/OAT1 plays an important role in the disposition of a broad range of endogenous substances and xenobiotics. This is particularly important from the pharmacological point of view since OAT1 is involved in drug elimination events. Furthermore, OAT1 is also involved in key physiological events such as the remote inter-organ communication. Despite its significance, the knowledge about OAT1 structure and the transport mechanism at the atomic level remains fragmented owing to the lack of resolved structures. By means of protein-threading modeling refined by μs-scaled Molecular Dynamics simulations, the present study provides the first robust model of hOAT1 in outward-facing conformation. Taking advantage of the AlphaFold 2 predicted structure of hOAT1 in inward-facing conformation, we here provide the essential structural and functional features comparing both states. The intracellular motifs conserved among Major Facilitator Superfamily members create a so-called "charge-relay system" that works as molecular switches modulating the conformation. The principal element of the event points at interactions charged residues that appear crucial for the transporter dynamics and function. Besides, hOAT1 model was embedded in different lipid bilayer membranes highlighting the crucial structural dependence on lipid-protein. MD simulations supported the pivotal role of phosphatidylethanolamine (PE) components on the protein conformation stability. The present model is made available to decipher the impact of any observed polymorphism and mutation on drug transport as well as to understand substrate binding modes.

Single particle cryo-EM structure of the outer hair cell motor protein prestin

The mammalian outer hair cell (OHC) protein prestin (Slc26a5) differs from other Slc26 family members due to its unique piezoelectric-like property that drives OHC electromotility, the putative mechanism for cochlear amplification. Here, we use cryo-electron microscopy to determine prestin’s structure at 3.6 Å resolution. Prestin is structurally similar to the anion transporter Slc26a9. It is captured in an inward-open state which may reflect prestin’s contracted state. Two well-separated transmembrane (TM) domains and two cytoplasmic sulfate transporter and anti-sigma factor antagonist (STAS) domains form a swapped dimer. The transmembrane domains consist of 14 transmembrane segments organized in two 7+7 inverted repeats, an architecture first observed in the bacterial symporter UraA. Mutation of prestin’s chloride binding site removes salicylate competition with anions while retaining the prestin characteristic displacement currents (Nonlinear Capacitance), undermining the extrinsic voltage sensor hypothesis for prestin function.

Indoxyl Sulfate Contributes to mTORC1-Induced Renal Fibrosis via The OAT/NADPH Oxidase/ROS Pathway

Activation of mTORC1 (mechanistic target of rapamycin complex 1) in renal tissue has been reported in chronic kidney disease (CKD)-induced renal fibrosis. However, the molecular mechanisms responsible for activating mTORC1 in CKD pathology are not well understood. The purpose of this study was to identify the uremic toxin involved in mTORC1-induced renal fibrosis. Among the seven protein-bound uremic toxins, only indoxyl sulfate (IS) caused significant activation of mTORC1 in human kidney 2 cells (HK-2 cells). This IS-induced mTORC1 activation was inhibited in the presence of an organic anion transporter inhibitor, a NADPH oxidase inhibitor, and an antioxidant. IS also induced epithelial–mesenchymal transition of tubular epithelial cells (HK-2 cells), differentiation of fibroblasts into myofibroblasts (NRK-49F cells), and inflammatory response of macrophages (THP-1 cells), which are associated with renal fibrosis, and these effects were inhibited in the presence of rapamycin (mTORC1 inhibitor). In in vivo experiments, IS overload was found to activate mTORC1 in the mouse kidney. The administration of AST-120 or rapamycin targeted to IS or mTORC1 ameliorated renal fibrosis in Adenine-induced CKD mice. The findings reported herein indicate that IS activates mTORC1, which then contributes to renal fibrosis. Therapeutic interventions targeting IS and mTORC1 could be effective against renal fibrosis in CKD.

SLC22 Transporters in the Fly Renal System Regulate Response to Oxidative Stress In Vivo

Several SLC22 transporters in the human kidney and other tissues are thought to regulate endogenous small antioxidant molecules such as uric acid, ergothioneine, carnitine, and carnitine derivatives. These transporters include those from the organic anion transporter (OAT), OCTN/OCTN-related, and organic cation transporter (OCT) subgroups. In mammals, it has been difficult to show a clear in vivo role for these transporters during oxidative stress. Ubiquitous knockdowns of related Drosophila SLC22s—including transporters homologous to those previously identified by us in mammals such as the “Fly-Like Putative Transporters” FLIPT1 (SLC22A15) and FLIPT2 (SLC22A16)—have shown modest protection against oxidative stress. However, these fly transporters tend to be broadly expressed, and it is unclear if there is an organ in which their expression is critical. Using two tissue-selective knockdown strategies, we were able to demonstrate much greater and longer protection from oxidative stress compared to previous whole fly knockdowns as well as both parent and WT strains (CG6126: p < 0.001, CG4630: p < 0.01, CG16727: p < 0.0001 and CG6006: p < 0.01). Expression in the Malpighian tubule and likely other tissues as well (e.g., gut, fat body, nervous system) appear critical for managing oxidative stress. These four Drosophila SLC22 genes are similar to human SLC22 transporters (CG6126: SLC22A16, CG16727: SLC22A7, CG4630: SLC22A3, and CG6006: SLC22A1, SLC22A2, SLC22A3, SLC22A6, SLC22A7, SLC22A8, SLC22A11, SLC22A12 (URAT1), SLC22A13, SLC22A14)—many of which are highly expressed in the kidney. Consistent with the Remote Sensing and Signaling Theory, this indicates an important in vivo role in the oxidative stress response for multiple SLC22 transporters within the fly renal system, perhaps through interaction with SLC22 counterparts in non-renal tissues. We also note that many of the human relatives are well-known drug transporters. Our work not only indicates the importance of SLC22 transporters in the fly renal system but also sets the stage for in vivo studies by examining their role in mammalian oxidative stress and organ crosstalk.

Early Elimination of Uremic Toxin Ameliorates AKI to CKD Transition

Acute kidney injury (AKI)-related fibrosis is a major driver of chronic kidney disease (CKD) development. Aberrant kidney recovery after AKI is multifactorial and still unclear. The accumulation of indoxyl sulfate (IS), a protein-bound uremic toxin, has been identified as a detrimental factor of renal fibrosis. However, the mechanisms underlying IS-related aberrant kidney recovery after AKI is still unknown. The study aims to elucidate the effects of IS in the pathogenesis of AKI to CKD transition. Our results showed that serum IS started to accumulate associated with the downregulation of tubular organic anion transporter, but not observed in the small-molecule uremic toxins of the unilateral ischemia-reperfusion injury without a contralateral nephrectomy model(UIRI). Serum IS is positively correlated with renal fibrosis and ER stress-related protein expression induction in the UIRI with a contralateral nephrectomy model (UIRI+Nx). To evaluate the effects of IS in the AKI to CKD transition, we administered indole, a precursor of IS, at the early stage of UIRI. Our results demonstrated IS potentiates renal fibrosis, senescence-associated secretory phenotype (SASP), and activation of ER, which is attenuated by synergistic AST-120 administration. Furthermore, we clearly demonstrated that IS exposure potentiated hypoxia-reperfusion (H/R) induced G2/M cell cycle arrest, epithelial-mesenchymal transition, and aggravated ER stress induction in vitro. Finally, the ER chemical chaperon, 4-PBA, successfully reversed the above-mentioned AKI to CKD transition. Taken together, early IS elimination in the early stage of AKI is likely to be a useful strategy in the prevention or treatment of the AKI to CKD transition.

Enhancing the Therapeutic Effect of 2-211At-astato-α-methyl-L-phenylalanine with Probenecid Loading

L-type amino acid transporter 1 (LAT1) might be a useful target for tumor therapy since it is highly expressed in various types of cancers. We previously developed an astatine-211 (211At)-labeled amino acid derivative, 2-211At-astato-α-methyl-L-phenylalanine (2-211At-AAMP), and demonstrated its therapeutic potential for LAT1-positive cancers. However, the therapeutic effect of 2-211At-AAMP was insufficient, probably due to its low tumor retention. The preloading of probenecid, an organic anion transporter inhibitor, can delay the clearance of some amino acid tracers from the blood and consequently increase their accumulation in tumors. In this study, we evaluated the effect of probenecid preloading on the biodistribution and therapeutic effect of 2-211At-AAMP in mice. In biodistribution studies, the blood radioactivity of 2-211At-AAMP significantly increased with probenecid preloading. Consequently, the accumulation of 2-211At-AAMP in tumors was significantly higher with probenecid than without probenecid loading. In a therapeutic study, tumor growth was suppressed by 2-211At-AAMP with probenecid, and the tumor volume was significantly lower in the treatment group than in the untreated control group from day 2 to day 30 (end of the follow-up period) after treatment. These results indicate that probenecid loading could improve the therapeutic effect of 2-211At-AAMP by increasing its accumulation in tumors.

Nephroprotective Effect of Functional Beverage of Dolichous Biflorus Seeds on Ethylene Glycol Induced Urolithiasis: A Mechanistic Approach

Calcium oxalate is the most common type of urolithiasis and is suggested due to membrane oxalate transporters dysfunction. This study has used Dolichos biflorus seeds as a functional beverage to explore its bioactive substances’ role on membrane transporters for managing urolithiasis. Urolithiasis model of ethylene glycol induced calcium oxalate crystals in albino Wistar rats was used in this study. Treatment of functional beverage of Dolichos biflorus seeds restored normal blood and urinary oxalate and citrate level and showed anti-urolithiasis activity. Probenecid, a membrane organic anion transporter (Slc26) inhibitor, treatment has abolished anti-urolithiasis activity of functional beverage and suggested Dolichos biflorus produce effect through the restoration of membrane oxalate transporters activity. An oxalate-less diet did not significantly reduce urinary, and plasma oxalate in probenecid treated animals and suggested that dietary oxalate is not primarily involved in calcium oxalate urolithiasis and conditions like hyperoxalemia and hyperoxaluria. Therefore, our findings suggested that endogenous oxalate in conjunction with idiopathic or chronic reduced activity or defective Slc26 transporters due to aging results in calcium oxalate kidney stones. The functional beverage of Dolichos biflorus seeds exhibited anti-urolithiasis activity through improving the activity of Slc26 transporters.