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and Slc22a8 becoming detectable in late embryogenesis [32, 35, 64]. Employing organ culture models of nephrogenesis, the patterns of transporter expression and function were found to closely resemble those observed in vivo [64]. The approximately co‐temporal expression of these transporters in the developing proximal tubule of the kidney suggests the existence of a common transcriptional regulatory pathway for expression of the Oats [35].

      Analyses of the maturation of these transporters after birth showed that Oat expression in the postnatal kidney increased postnatally in rodents with the highest level detectable in the adult [63]. A comprehensive analysis of the expression of the Slc and Abc transporters through all stages of rodent kidney development (i.e., embryonic, postnatal, and mature) from existing transcriptomic data sets revealed increases between the postnatal and mature stages of renal development [65]. Importantly, Slc22a6 and Slc22a8 were significantly upregulated in the maturing proximal tubule. In vivo studies also revealed a 4‐fold increase in the clearance of para‐aminohippurate (PAH, a classic Oat1 substrate), during postnatal development. A recent human study of OAT function reveals a roughly similar pattern of postnatal PAH handling [66].

      The findings described above not only indicate that the OATs are functionally expressed during early proximal tubule differentiation and development; they also highlight the importance of studying postnatal expression and functional maturation. Given the increasingly lower gestational ages at which infants are being delivered, these types of studies likely have clinical implications for the dosing of drugs to premature infants and full‐term newborns, as well as children [63]. Furthermore, given that the gut–liver–kidney axis needs to become functionally efficient after birth, it has been argued that there must be coordinated regulation of these transporters [67].

      4.3.3 Transcriptional Regulation

      Although increases in both the mRNA levels and the function of the Oats are seen in the kidney in the postnatal to mature periods, it remains unclear what triggers these increases. Postnatal maturation of organic anion transport has been found to be influenced by a number of factors, including substrate availability, age, and gender [63]. For example, increased organic anion uptake into renal cortical slices can be induced by prior exposure to organic anions. This substrate exposure may induce the synthesis of either the Oats themselves or, conceivably, of other proteins required for Oat‐mediated uptake. Numerous studies have also shown that Oat expression and function are influenced by sex steroids, while the corticosteroid dexamethasone also increases Oat functional activity [68]. Interestingly, a recent study found that the sex‐dependent differences in the expression of some of the Oats appear to be regulated by Bcl6 which transactivates the promoters for Oat1 and Oat3 [69]. Thyroid hormones have also been implicated in the regulation of Oat activity in the postnatal kidney [65]. The impetus for up‐regulation in expression may also be linked to the feeding and fasting behavior that is newly established after birth, as may be occurring with the regulation of liver transporters [70].

      Detailed studies have implicated the hepatocyte nuclear factors 1α (Hnf1α) and Hnf4α in regulating the expression of many of the OATs [23, 24]. Bioinformatics analysis of time‐series microarray data yielded a network of genes (including Hnf1α, hyaluronic acid‐CD44, and notch pathways) with connectivity biased toward Hnf4α [71]. When taken with ChIP‐qPCR data which showed that Hnf4α occupies the proximal promoters of Slc22a6, Slc22a8, and Slc22a1 in the in vivo differentiating rat kidney, the data suggested that a network of genes centered around Hnf4α regulates the terminal differentiation of the nascent proximal tubule. The importance of Hnf4a in proximal tubule differentiation has recently been confirmed in kidney organ culture, stem cell models of renal differentiation, and in knockout mice [72–74].

      Oats are not only regulated during development, but their expression and function can be altered in response to various stimuli (e.g., injury, ischemia, inflammation, drugs toxins, growth factors, and various hormones). For instance, chronic renal failure has been shown to alter the expression of a variety of transporters, including the Oats, in multiple organs (e.g., intestine, liver, kidney and brain) [75, 76], while acute kidney injury induced by either ischemia, drugs, or toxicants (e.g., cisplatin, inorganic mercury) alter the expression and function of both Oat1 and Oat3 [77, 78]. In addition, Chinese herbal medicines, which are widely used as prescription drugs, complementary alternative medicines and dietary supplements, have been found to alter the expression and function of renal Oats [79].

      4.3.4 Post‐translational Regulation

As described above, the OATs are characterized by two large interconnecting loops, one between TMD 1/2 and another between TMD 6/7 (Fig. 4.1). The first large loop is extracellular and contains multiple consensus N‐glycosylation sites [32], the role of which remains unclear, although glycosylation of OAT1 was shown to be required for protein trafficking to the membrane [57]. The second large loop is intracellular and together with the intracellular carboxy terminus contains several potential phosphorylation sites for PKC, PKA [53], casein kinase II and tyrosine kinase [13], as well as for ubiquitination, another type of post‐translational modification (PTM). Once considered to be static at the cell membrane, OATs have recently been shown to undergo internalization and recycling to and from the cell surface constitutively or in response to stimuli [80–82]. As the levels of OATs at the cell surface are critical to their transport activity, the dynamic expression of OATs at the cell surface would improve the transporter’s ability to initiate trafficking in response to stimuli, thus providing efficient and fast fine‐tuning of OAT activity.

      While most of the information discussed below refers to OAT1 and OAT3, the term OAT is used generically to emphasize concepts applicable to other OATs as well. Several PTMs are involved in the regulation of OAT trafficking. PTMs, the modifications on the target protein following synthesis, refer to the covalent addition of functional group(s) to the target proteins and include glycosylation, ubiquitination, phosphorylation, and many others [83, 84]. Most of the PTMs are catalyzed by specific enzymes that attach or remove functional group(s) and are dynamic and reversible. Through promoting and demoting the modifications, PTMs add complexity to the functional diversity of the proteome. Since different PTMs can modify target proteins individually or simultaneously through various mechanisms, the functional diversity of the target proteins exceeds their molecular diversity [85–87].

      Ubiquitination, the addition of ubiquitin molecule(s) to the lysine residue(s) on the substrate protein, is a type of PTM that can occur in different conjugations, including monoubiquitination, multiubiquitination, and polyubiquitination. Monoubiquitination refers to the addition of a single ubiquitin molecule to a single lysine residue on the target protein, while multiubiquitination is the conjugation of several monoubiquitin molecules to multiple lysine residues on the target protein. Polyubiquitination refers to the conjugation of a polyubiquitin chain to the substrate. The polyubiquitin chain is formed between a lysine residue of one ubiquitin molecule and a glycine residue of another ubiquitin molecule [88, 89]. Ubiquitin itself has seven lysine residues, which includes K6, K11, K27, K29, K33, K48, and K63. Ubiquitination regulates the target proteins through altering their cellular location, stability, activity, and protein–protein interactions.

      OAT ubiquitination is catalyzed by the ubiquitin ligases Nedd4‐1 or Nedd4‐2 and facilitates the internalization of OATs from the cell surface to the intracellular early endosomes. Once in the endosomes, OATs move to the proteolytic system for degradation or becomes deubiquitinated, resulting in OATs recycling back to the cell membrane. Nedd4‐2 is mainly involved in protein‐kinase‐regulated OAT ubiquitination, whereas Nedd4‐1 is largely involved in the constitutive OAT ubiquitination [80–82,90–92]. Several protein kinases have been reported to exert their regulation on OATs through phosphorylating Nedd4‐2 at different sites, which either weaken or strengthen the protein–protein interaction between OATs and Nedd4‐2. The strength of this interaction leads to a decreased or enhanced OAT ubiquitination and, ultimately, to stimulated or inhibited OAT transport activity. For example, the short‐term activation of PKC enhances Nedd4‐2 phosphorylation, and therefore OAT ubiquitination, which leads to accelerated

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