**4. Discussion**

OAT3 function is predominantly dependent on the amount located on the plasma membrane, which can be regulated by mitogen-activated protein kinase (MAPK), protein kinase A (PKA), PKC signaling pathways [43–45]. Ubiquitination is a significant posttranslational mechanism for OAT3 regulation. Our previous study had demonstrated the essential role of Nedd4-2 (a ubiquitin ligase) in the ubiquitination, surface expression, and transport activity of OAT3 [27]. Serum- and glucocorticoid-inducible kinases 1 (sgk1), PKC, janus tyrosine kinase 2 (JAK2) regulated OAT3 through Nedd4-2, which showed Nedd4-2 is molecular target for OAT3 regulation [27,37,38,46]. In this study, we further discovered proteasome was a novel target for regulation of OAT3 and stimulating OAT3 function can be achieved through inhibiting proteasomal activity.

COS-7 and HEK293 cells lacking in endogenous OATs were commonly utilized as heterologous expression systems for OATs. Both cell lines were broadly selected for study the regulation and mechanisms of the cloned OATs and other drug transporters in kidney with several advantages [13,47–49]. Expression of exogenous OAT3 will allow us to study the transport characteristics of OAT3 without being disturbed by other OATs. They are originated from the kidney and have the proteasome activity and signaling pathways involved in OAT3 regulation. COS-7 cells and HEK293 cells used in our studies will provide the research basis for the upcoming work focusing on validating whether primary epithelia possess the similar mechanisms.

Ixazomib is an FDA-approved anticancer drug, while oprozomib and delanzomib are in phases of clinical trials. All of them are administered orally, and preferentially bind reversibly (ixazomib and delanzomib) or irreversibly (oprozomib) and inhibit the chymotrypsin-like activity of the 20S proteasome in various tissues and organs. There were reports that ixazomib inhibited the proteasome activity in the whole blood and tumor; oprozomib could inhibit the proteasome activity in the blood, peripheral blood mononuclear cells, liver, kidney, and adrenal glands; and delanzomib inhibited the proteasome activity in blood mononuclear cells, kidney, and spleen [50–55]. Ixazomib prevented antibodymediated rejection in kidney transplantation and treated patients with metastatic kidney

cancer [56,57]. Delanzomib can ameliorate lupus nephritis in mice [55]. These results suggested that proteasomal inhibitors can be used to treat kidney diseases, through proteasome inhibition-mediated reduction in aberrant cytokines and antibodies, or downregulation of nuclear factor kappa B-dependent gene expression and resulted tumor growth [58,59].

Ixazomib, oprozomib, or delanzomib treatment substantially increased the accumulation of ubiquitinated OAT3 (Figure 1), which was consistent with decreased 20S proteasomal activity in cell lysate in OAT3-expressing cells (Figure 2), stimulated OAT3-mediated transport of estrone sulfate and p-aminohippuric acid (Figures 4–6), and increased OAT3 membrane expression (Figure 9). The enhanced transport activity of OAT3 following drug pretreatment resulted from an increase in maximum transport velocity without altering the binding affinity of the transporter (Figure 8). Ubiquitinated OAT3 exhibited the molecular mass above 180 kDa, ~100 kDa more than OAT3 (~80 kDa). As ubiquitin is an 8-kDa polypeptide, OAT3 may be modified by poly- or multiubiquitination (Figure 1).

The OAT3 function was chronically stimulated with 6 h of treatment with ixazomib, oprozomib, or delanzomib. As the alteration of trafficking processes, including internalization or recycling of OATs, can be reflected in function change during acute regulation (such as 0.5 h), we can exclude the reduced internalization and increased recycling that are the underlying mechanisms for those drugs [27–30,39]. With further exploring, the degradation of OAT3 was decelerated by ixazomib, oprozomib, or delanzomib (Figure 10). Our results showed they inhibited the 20S proteasome activity (Figure 2), and there was a negative association between proteasomal activity and transport activity at 10–40 nM ixazomib (Figure 7B). Together, ixazomib-, oprozomib-, and delanzomib-upregulated OAT3 function was mainly through suppression of proteasome activity and decelerated degradation of OAT3.

The concentrations of ixazomib (10–40 nM), oprozomib (50–400 nM), and delanzomib (10–50 nM) used in our study are in the clinically therapeutic range. After once-weekly oral dosing of 2.23 mg/m<sup>2</sup> for 3 weeks in combination therapy with lenalidomide and dexamethasone, the mean maximum plasma concentration (Cmax) of ixazomib in multiple myeloma patients at day 1 and day 15 was 22.3 ng/mL (61.7 nM) and 31.4 ng/mL (87.0 nM), respectively [60]. For oprozomib, after 2 consecutive days weekly oral dosing at 210 mg/day for 4 weeks plus pomalidomide and dexamethasone in relapsed/refractory multiple myeloma patients, the mean Cmax of oprozomib at day 1 and day 8 was 744 ng/mL (1.4 μM) and 1030 ng/mL (1.9 μM), respectively [61]. Until now, there were only reports about intravenous pharmacokinetic data of delanzomib in human. After 2 days weekly intravenous dosing 0.4–1.8 mg/m<sup>2</sup> for 2 weeks in patients with solid tumors and multiple myeloma, the mean Cmax of delanzomib on day 1 was 88.4–557.3 ng/mL (0.2–1.3 μM) [54]. Ixazomib and delanzomib have a long terminal plasma half-life of 3.6–11.3 days and 62.0 ± 43.5 h, respectively [54,62]. Though oprozomib has a short plasma half-life of about 1 h resulting from rapid systemic clearance, the recovery of proteasome activity in tissues needed a longer time of 24~72 h due to irreversible binding [63,64]. Therefore, the inductive effects of ixazomib, oprozomib, and delanzomib on drug elimination and DDIs potentially exist, though they are administered once or twice weekly. The in vitro regulation and related mechanisms in cell models were reported in this study, and further in vivo study in Sprague Dawley rats by oral ixazomib will be performed to further explore the roles of ixazomib in proteasome activity, OTA3 ubiquitination, drug uptake in kidney slices, membrane and total expression in kidney, and renal clearance of drugs by kidney in our lab.

Ixazomib, oprozomib, or delanzomib are all indicated in combination with dexamethasone, a synthetic glucocorticoid for the treatment of patients with multiple myeloma [61,65,66]. Our previous study showed dexamethasone stimulates OAT3 transport activity and membrane expression through the serum- and glucocorticoid-inducible kinases 1 signaling pathway, suggesting the stimulatory effect on OAT3 may be further magnified using ixazomib, oprozomib, and delanzomib in combination with dexamethasone [37].

Ixazomib is a low-affinity substrate of P-glycoprotein (P-gp); is not a substrate of breast cancer resistance protein (BCRP), multidrug resistance protein 2 (MRP2), or hepatic OATPs; and is not an inhibitor of P-gp, BCRP, MRP2, OATP1B1, OATP1B3, organic cation transporter 2 (OCT2), OAT1, OAT3, multidrug and toxin extruder 1 (MATE1), or MATE2-K. Therefore, the manufacturer claimed that ixazomib is not expected to cause transporter-mediated drug–drug interactions [67]. Consistent with this, our study found that ixazomib is not an inhibitor of OAT3 (Figure 3). However, although ixazomib did not cause DDIs through direct interaction (inhibiting or competing) with the transporters, our study showed that ixazomib can upregulate OAT3 activity through induced membrane expression, which may affect the disposition of other drugs in an indirect manner of transporter-mediated DDIs. Besides, potential DDIs may be occurred by direct OATs induction. There were reports that ursolic acid and ciprofloxacin stimulated OAT1-mediated p-aminohippuric acid uptake, and 1,5-dicaffeoylquinic acid and 18β-glycyrrhetinic acid stimulated hOAT4-mediated estrone sulfate uptake [68,69].

Proteasome inhibition drugs are now well utilized for cancer treatment. In contrast, impaired proteasome function and related elevation of toxic intracellular protein or aggregates are involved in neurodegenerative disorders (e.g., Parkinson's disease, Alzheimer's disease) and cardiac dysfunctions, and enhancement of proteasome activity may also be a promising therapeutic strategy for those diseases [70–72]. PD169316, pyrazolones and chlorpromazine as small molecules, were found to be proteasome activators [70,73,74]. It would be interesting to study whether proteasomal activators can regulate the OAT3 function.

Our findings that oral proteasome inhibitors ixazomib, oprozomib, and delanzomib can increase OAT3 transport activity have important physiological implications. First, it can accelerate the drugs clearance from body, resulting in reduced plasma concentration and therapeutic efficacy of drugs. We can also use this mechanism for noninvasive detoxification in the event of drug overdoses. Second, it may enhance the entering and distribution of drugs in proximal tubular cells, leading to potential nephrotoxicity. Those points should attract the attention of physicians and pharmacists for rational use of medicines and irrational drug combinations, and avoiding potential drug–drug interactions. Third, bilateral ureteral obstruction (BUO), a common clinical disease, impaired renal elimination of drugs partly resulted from reduced cell-surface expression of OAT3 [75]. Proteasome inhibition may provide a potential strategy to reverse BUO or other kidney-disease-induced downregulation of OAT3. Last, it also can promote renal clearance of toxins, metabolites, signaling molecules, nutrients, and other substances as OAT3 substrates, and maintain homeostasis within the body.
