Chloroquine and Hydroxychloroquine, as Proteasome Inhibitors, Upregulate the Expression and Activity of Organic Anion Transporter 3.

Organic anion transporter 3 (OAT3), at the basolateral membrane of kidney proximal tubule cells, facilitates the elimination of numerous widely used drugs. Earlier investigation from our laboratory revealed that ubiquitin conjugation to OAT3 leads to OAT3 internalization from the cell surface, followed by degradation in the proteasome. In the current study, we examined the roles of chloroquine (CQ) and hydroxychloroquine (HCQ), two well-known anti-malarial drugs, in their action as proteasome inhibitors and their effects on OAT3 ubiquitination, expression, and function. We showed that in cells treated with CQ and HCQ, the ubiquitinated OAT3 was considerably enhanced, which correlated well with a decrease in 20S proteasome activity. Furthermore, in CQ- and HCQ-treated cells, OAT3 expression and OAT3-mediated transport of estrone sulfate, a prototypical substrate, were significantly increased. Such increases in OAT3 expression and transport activity were accompanied by an increase in the maximum transport velocity and a decrease in the degradation rate of the transporter. In conclusion, this study unveiled a novel role of CQ and HCQ in enhancing OAT3 expression and transport activity by preventing the degradation of ubiquitinated OAT3 in proteasomes.

Pancreatic Hormone Insulin Modulates Organic Anion Transporter 1 in the Kidney: Regulation via Remote Sensing and Signaling Network.

Organic anion transporter 1 (OAT1) expressed in the kidney plays an important role in the elimination of numerous anionic drugs used in the clinic. We report here that insulin, a pancreas-secreted hormone, regulated the expression and activity of kidney-specific OAT1 both in cultured cells and in rats. We showed that treatment of OAT1-expressing cells with insulin led to an increase in OAT1 expression, transport activity, and SUMOylation. Such insulin-induced increase was blocked by afuresertib, a specific inhibitor for protein kinase B (PKB), suggesting insulin regulates OAT1 through PKB signaling pathway. Furthermore, insulin stimulated transport activity and SUMOylation of endogenously expressed OAT1 in rat kidneys. In conclusion, our data support a remote sensing and signaling model, in which OAT1 plays an essential role in intercellular and inter-organ communication and in maintaining local and whole-body homeostasis. Such complex and dedicated communication is carried out by insulin, and PKB signaling and membrane sorting.

Inhibition of proteasome, but not lysosome, upregulates organic anion transporter 3 in vitro and in vivo.

Organic anion transporter 3 (OAT3), an indispensable basolateral membrane transporter predominantly distributed in the kidney proximal tubules, mediated the systemic clearance of substrates including clinical drugs, nutrients, endogenous and exogenous metabolites, toxins, and critically sustains body homeostasis. Preliminary data in this study showed that classical proteasome inhibitors (e.g., MG132), but not lysosome inhibitors, significantly increased the OAT3 ubiquitination and OAT3-mediated transport of estrone sulfate (ES) in OAT3 stable expressing cells, indicating that proteasome rather than lysosome is involved in the intracellular fate of OAT3. Next, bortezomib and carfilzomib, two FDA-approved and widely applied anticancer agents through selective targeting proteasome, were further used to define the role of inhibiting proteasome in OAT3 regulation and related molecular mechanisms. The results showed that 20S proteasome activity in cell lysates was suppressed with bortezomib and carfilzomib treatment, leading to the increased OAT3 ubiquitination, stimulated transport activity of ES, enhanced OAT3 surface and total expression. The upregulated OAT3 function by proteasome inhibition was attributed to the augment in maximum transport velocity and stability of membrane OAT3. Lastly, in vivo study using Sprague Dawley rats validated that proteasome inhibition using bortezomib induced enhancement of OAT3 ubiquitination and membrane expression in kidney. These data suggest that activity of proteasome but not lysosome could have an impact on the physiological function of OAT3, and proteasome displayed a promising target for OAT3 regulation in vitro and in vivo, and could be used in restoring OAT3 impairment under pathological conditions, avoiding OAT3-associated toxicity and diseases, ensuring drug efficacy and safety.

Oral Proteasomal Inhibitors Ixazomib, Oprozomib, and Delanzomib Upregulate the Function of Organic Anion Transporter 3 (OAT3): Implications in OAT3-Mediated Drug-Drug Interactions.

Organic anion transporter 3 (OAT3) is mainly expressed at the basolateral membrane of kidney proximal tubules, and is involved in the renal elimination of various kinds of important drugs, potentially affecting drug efficacy or toxicity. Our laboratory previously reported that ubiquitin modification of OAT3 triggers the endocytosis of OAT3 from the plasma membrane to intracellular endosomes, followed by degradation. Oral anticancer drugs ixazomib, oprozomib, and delanzomib, as proteasomal inhibitors, target the ubiquitin-proteasome system in clinics. Therefore, this study investigated the effects of ixazomib, oprozomib, and delanzomib on the expression and transport activity of OAT3 and elucidated the underlying mechanisms. We showed that all three drugs significantly increased the accumulation of ubiquitinated OAT3, which was consistent with decreased intracellular 20S proteasomal activity; stimulated OAT3-mediated transport of estrone sulfate and p-aminohippuric acid; and increased OAT3 surface expression. The enhanced transport activity and OAT3 expression following drug treatment resulted from an increase in maximum transport velocity of OAT3 without altering the substrate binding affinity, and from a decreased OAT3 degradation. Together, our study discovered a novel role of anticancer agents ixazomib, oprozomib, and delanzomib in upregulating OAT3 function, unveiled the proteasome as a promising target for OAT3 regulation, and provided implication of OAT3-mediated drug-drug interactions, which should be warned against during combination therapies with proteasome inhibitor drugs.

Protein kinase C regulates organic anion transporter 1 through phosphorylating ubiquitin ligase Nedd4-2.

BACKGROUND: Organic anion transporter 1 (OAT1) is a drug transporter expressed on the basolateral membrane of the proximal tubule cells in kidneys. It plays an essential role in the disposition of numerous clinical therapeutics, impacting their pharmacological and toxicological properties. The activation of protein kinase C (PKC) is shown to facilitate OAT1 internalization from cell surface to intracellular compartments and thereby reducing cell surface expression and transport activity of the transporter. The PKC-regulated OAT1 internalization occurs through ubiquitination, a process catalyzed by a E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2). Nedd4-2 directly interacts with OAT1 and affects ubiquitination, expression and stability of the transporter. However, whether Nedd4-2 is a direct substrate for PKC-induced phosphorylation is unknown. RESULTS: In this study, we investigated the role of Nedd4-2 phosphorylation in the PKC regulation of OAT1. The results showed that PKC activation enhanced the phosphorylation of Nedd4-2 and increased the OAT1 ubiquitination, which was accompanied by a decreased OAT1 cell surface expression and transport function. And the effects of PKC could be reversed by PKC-specific inhibitor staurosporine. We further discovered that the quadruple mutant (T197A/S221A/S354A/S420A) of Nedd4-2 partially blocked the effects of PKC on Nedd4-2 phosphorylation and on OAT1 transport activity. CONCLUSIONS: Our investigation demonstrates that PKC regulates OAT1 likely through direct phosphorylation of Nedd4-2. And four phosphorylation sites (T197, S221, S354, and S420) of Nedd4-2 in combination play an important role in this regulatory process.

Research Progress of Coronavirus Based on Bibliometric Analysis.

BACKGROUND: COVID-19 has become one of the most serious global epidemics in the 21st Century. This study aims to explore the distribution of research capabilities of countries, institutions, and researchers, and the hotspots and frontiers of coronavirus research in the past two decades. In it, references for funding support of urgent projects and international cooperation among research institutions are provided. METHOD: the Web of Science core collection database was used to retrieve the documents related to coronavirus published from 2003 to 2020. Citespace.5.6.R2, VOSviewer1.6.12, and Excel 2016 were used for bibliometric analysis. RESULTS: 11,036 documents were retrieved, of which China and the United States have contributed the most coronavirus studies, Hong Kong University being the top contributor. Regarding journals, the JournalofVirology has contributed the most, while in terms of researchers, Yuen Kwok Yung has made the most contributions. The proportion of documents published by international cooperation has been rising for decades. Vaccines for SARS-CoV-2 are under development, and clinical trials of several drugs are ongoing. CONCLUSIONS: international cooperation is an important way to accelerate research progress and achieve success. Developing corresponding vaccines and drugs are the current hotspots and research directions.