Organic Anion Transport in the Developing Kidney

Introduction

Rapid and complex changes in physiology occur when the fetus transitions from an intrauterine to an extrauterine environment. From dependency on the maternal circulation via the placenta for nutrition, oxygen, and waste excretion, the infant shifts to reliance on its own organ systems for vital functions. These organ systems include the pulmonary system for oxygen and carbon dioxide exchange, the cardiovascular system for blood circulation, the gastrointestinal system for food absorption, and the renal system for waste excretion, fluid, and acid-base balance.

The kidneys and the tubular transporters, in particular, play an important role in maintaining the chemical homeostasis within the body as the infant adapts to its rapidly changing environment. The transporters involved include the solute carrier (SLC) and ATP-binding cassette (ABC) family of transporters. A major subfamily of the SLC family of transporters is the organic anion transporters (OATs), multispecific transporters located in the proximal tubule that mediate the secretion and absorption of a wide array of endogenous solutes and drugs including diuretics, methotrexate, β-lactam and sulfonamide antibiotics, nonsteroidal antiinflammatory drugs (NSAIDs), angiotensin-converting enzyme (ACE) inhibitors, and antiviral drugs. The OATs, especially OAT3, also transport glucuronides and sulfate esters, phase II reaction products, and therefore may play an essential role in elimination of toxic organic anions in the infant. Members of the OAT family share structural similarities, including a putative 12 transmembrane helical domain, with an extracellular loop containing N-linked glycosylation sites between helices 1 and 2, and an intracellular loop containing protein kinase C (PKC) phosphorylation sites between helices 6 and 7. ^, Probably the most studied OATs are OAT1, the prototypical OAT originally identified as NKT, and OAT3, which are located on the basolateral membrane of the proximal tubule where they function via a tertiary transport system. ^, This transport system begins with the Na ⁺ gradient generated by the Na ⁺ /K ⁺ -ATPase, which drives the sodium-dicarboxylate co-transporter (NaDC3) to transport Na ⁺ and dicarboxylates, including α-ketoglutarate, into the cell. The resulting high intracellular concentration of dicarboxylates then drives the outward movement of dicarboxylates and the inward movement of organic anions, an exchange mediated by the OATs ( Fig. 101.1 ).

Several studies have shown time-dependent expression patterns in the OATs and ABC transporters with kidney development, and these expression patterns appear to be affected by gender and transcriptional and posttranscriptional regulation. The difference in expression patterns of OATs in the infant likely contributes to the differences in drug pharmacokinetics and therapeutic efficiency observed in children compared to adults, and emphasizes the importance of special consideration in drug dosing for pediatric patients. Furthermore, the immaturity of the transport system can render the newborn more vulnerable to drug overdosing and to environmental stressors such as ischemia.

Age-Dependent Expression and Activity of Renal Oats in Rodent and Mammalian Models

Several studies have described the temporal expression patterns of the SLC and ABC transporter families in the mammalian kidney. Initial studies investigated the transport of p-aminohippurate (PAH), the prototypical substrate for OAT1, in mammalian models, and reported rapid increases in renal PAH transport activity within the first few weeks of life in rabbit kidney slices and within the first few days after birth in newborn sheep. Transcript analysis and live in vitro influx/efflux assays showed that OAT1 and OAT3 expression begins as early as embryonic(e) day 10.5 in the murine mesonephros. These findings suggest that the mammalian mesonephros, a transient renal structure, can play an important role in the excretion of products in conjunction with the placenta in the developing embryo. The expression of Oat1 mRNA levels was detected on embryonic day 18 in the fetal rat kidney, with levels remaining low prenatally and dramatically increasing on the day of birth until postnatal day 2. The expression levels from postnatal days 1 and 2 were equivalent to adult rat expression levels. Other studies have shown that Oat1 mRNA levels increased gradually until 30 days of age, at which point they reached adult levels.

Rat Oat2 and Oat3 mRNA levels were also shown to be low at birth. Oat2 expression remained low until 30 days of age and increased from day 35 to 45 in only female rats and not male rats. An increase in Oat3 expression occurred earlier compared to the other Oats , with mRNA levels increasing significantly during the first 10 days of life. In the mouse, Oat1 mRNA transcripts were first detected by in situ hybridization in the proximal tubules at e14, with expression levels increasing throughout the rest of gestation and into adulthood. Organic cation transporter 1 ( Oct1 ) expression was first detected in the mouse kidney between e15 and e16, and was found to increase postnatally until adult levels. Oat2 mRNA levels were detected by in situ hybridization at e14 in the murine kidney, and these levels also increased postnatally. Sweeney and colleagues performed time-series microarray experiments and found a twofold or greater increase in mRNA expression between two temporally consecutive stages of kidney development in Slc22 transporters, including Oat1 , 2 , 3 , and 5 ; Oct1 , 2 ; Octn1 ; Octn2 ; and Rst (the murine URAT1 ortholog). Interestingly, the largest increase in expression occurred between the postnatal stage (birth to week 1) and the mature stage (week 4 to adult) ( Fig. 101.2 ). Furthermore, an increase in the expression of ABC transporters with renal development was also observed, again with the largest increase occurring between the postnatal and mature stage of kidney development ( Fig. 101.3 ). Using the GUDMAP consortium microarray datasets, the expression of six transporters including Oat1 , Oat3 , Oct 1 Oct 2 Mate 1 and Pept 2 were found to be increased at least twofold compared to other structures in the developing proximal tubules at e15.5. These findings suggest that drug transport may occur as early as e15.5 and that the transporters already have a distinct localization in the nephron at this early stage.

The up-regulation of the transporters studied correlated with an increase in PAH clearance observed in vivo during 1, 2, and 3 weeks of age and in the mature stages, supporting the finding that although the transporters are present and functional at an early age, they do not possess full activity until later in age. Clearance of PAH was also investigated in Oat1 and Oat3 knockouts at 2 weeks of age, and confirmed that, as in the adult kidney, OAT1 is the main transporter of PAH in the postnatal kidney.

Further localization of OAT1 and OAT3 expression was also described by immunohistochemical analysis of murine kidneys. OAT1 immunoreactivity was noted at e15 in the proximal tubules of mice and in the outer cortex 7 days postnatally. OAT3 was found to be expressed in the distal tubule of e14 mice and in the S2 segment of the proximal tubule of e16 mice. At the time of birth, expression of OAT3 shifted to the S1 and S3 segments.