A Comprehensive Analysis of Ontogeny of Renal Drug Transporters: mRNA Analyses, Quantitative Proteomics, and Localization

Abstract

Human renal membrane transporters play key roles in the disposition of renally cleared drugs and endogenous substrates, but their ontogeny is largely unknown. Using 184 human postmortem frozen renal cortical tissues (preterm newborns to adults) and a subset of 62 tissue samples, we measured the mRNA levels of 11 renal transporters and the transcription factor pregnane X receptor (PXR) with quantitative real-time polymerase chain reaction, and protein abundance of nine transporters using liquid chromatography tandem mass spectrometry selective reaction monitoring, respectively. Expression levels of p-glycoprotein, urate transporter 1, organic anion transporter 1, organic anion transporter 3, and organic cation transporter 2 increased with age. Protein levels of multidrug and toxin extrusion transporter 2-K and breast cancer resistance protein showed no difference from newborns to adults, despite age-related changes in mRNA expression. Multidrug and toxin extrusion transporter 1, glucose transporter 2, multidrug resistance-associated protein 2, multidrug resistance-associated protein 4 (MRP4), and PXR expression levels were stable. Using immunohistochemistry, we found that MRP4 localization in pediatric samples was similar to that in adult samples. Collectively, our study revealed that renal drug transporters exhibited different rates and patterns of maturation, suggesting that renal handling of substrates may change with age.

Figure 1

Sample analysis scheme. The subset of 62 samples from sample set 2 for quantitative proteomics consisted of the 57 African American samples and 5 adult white samples (see Table 1 ). BCRP, breast cancer resistance protein; GLUT, glucose transporter; MATE, multidrug and toxin extrusion protein; MDR, multidrug resistance protein; MRP, multidrug resistance‐associated protein; OAT, organic anion transporter; OCT, organic cation transporter; PXR, pregnane X receptor; URAT, urate transporter.

Figure 2

Relative mRNA expression of 11 renal membrane transporters and pregnane X receptor in different age groups. Transporters are grouped according to their primary localization in the kidney (basolateral or apical). The bar represents the median for each age group. *P < 0.05, **P < 0.01, ***P < 0.001. BCRP, breast cancer resistance protein; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; GLUT, glucose transporter; MATE, multidrug and toxin extrusion protein; MDR, multidrug resistance protein; MRP, multidrug resistance‐associated protein; OAT, organic anion transporter; OCT, organic cation transporter; PXR, pregnane X receptor; URAT, urate transporter.

Figure 3

Protein abundance levels of nine renal membrane transporters in different age groups. The bar represents the median for each age group. Term newborn and infants were combined here for analysis because there was only one term newborn included for this part of the study. *P < 0.05, **P < 0.01, ***P < 0.001. GLUT, glucose transporter; MATE, multidrug and toxin extrusion protein; MDR, multidrug resistance protein; MRP, multidrug resistance‐associated protein; OAT, organic anion transporter; OCT, organic cation transporter; PXR, pregnane X receptor; URAT, urate transporter.

Figure 4

Ontogeny of protein abundance of P‐gp, OAT1, OAT3, and OCT2 as described by sigmoidal maximum effect (E_max) model. Individual sigmoidal curves are presented in (a) (solid lines). Dashed lines represent the 95% confidence bands. Superimposing the sigmoidal curves as presented in (b) showed that the pair transporters, P‐gp/OCT2 and OAT1/OAT3, shared similar maturation patterns. OAT, organic anion transporter; OCT, organic cation transporter; P‐gp, p‐glycoprotein; PNA, postnatal age; TM₅₀, half of the adult expression was reached.

Figure 5

Apical proximal tubule localization of multidrug resistance‐associated protein (MRP4) (arrow) by immunohistochemically staining in postmortem tissue of samples with (a) gestational age (GA) of 27.7 weeks; postnatal age (PNA) age 3.3 weeks, and (b) GA of 40.0 weeks; PNA 3.1 years (c) represents the negative control, and (d) the semiquantification of MRP4 staining in various age groups: negative (0), low staining (+1), or high staining (+2).