Hypoxia alters ocular drug transporter expression and activity in rat and calf models: implications for drug delivery

Abstract

Chronic hypoxia, a key stimulus for neovascularization, has been implicated in the pathology of proliferative diabetic retinopathy, retinopathy of prematurity, and wet age related macular degeneration. The aim of the present study was to determine the effect of chronic hypoxia on drug transporter mRNA expression and activity in ocular barriers. Sprague-Dawley rats were exposed to hypobaric hypoxia (PB = 380 mmHg) for 6 weeks, and neonatal calves were maintained under hypobaric hypoxia (PB = 445 mmHg) for 2 weeks. Age matched controls for rats, and calves were maintained at ambient altitude and normoxia. The effect of hypoxia on transporter expression was analyzed by qRT-PCR analysis of transporter mRNA expression in hypoxic and control rat choroid-retina. The effect of hypoxia on the activity of PEPT, OCT, ATB(0+), and MCT transporters was evaluated using in vitro transport studies of model transporter substrates across calf cornea and sclera-choroid-RPE (SCRPE). Quantitative gene expression analysis of 84 transporters in rat choroid-retina showed that 29 transporter genes were up regulated or down regulated by ≥1.5-fold in hypoxia. Nine ATP binding cassette (ABC) families of efflux transporters including MRP3, MRP4, MRP5, MRP6, MRP7, Abca17, Abc2, Abc3, and RGD1562128 were up-regulated. For solute carrier family transporters, 11 transporters including SLC10a1, SLC16a3, SLC22a7, SLC22a8, SLC29a1, SLC29a2, SLC2a1, SLC3a2, SLC5a4, SLC7a11, and SLC7a4 were up regulated, while 4 transporters including SLC22a2, SLC22a9, SLC28a1, and SLC7a9 were down-regulated in hypoxia. Of the three aquaporin (Aqp) water channels, Aqp-9 was down-regulated, and Aqp-1 was up-regulated during hypoxia. Gene expression analysis showed down regulation of OCT-1, OCT-2, and ATB(0+) and up regulation of MCT-3 in hypoxic rat choroid-retina, without any effect on the expression of PEPT-1 and PEPT-2. Functional activity assays of PEPT, OCT, ATB(0+), and MCT transporters in calf ocular tissues showed that PEPT, OCT, and ATB(0+) functional activity was down-regulated, whereas MCT functional activity was up-regulated in hypoxic cornea and SCRPE. Gene expression analysis of these transporters in rat tissues was consistent with the functional transport assays except for PEPT transporters. Chronic hypoxia results in significant alterations in the mRNA expression and functional activity of solute transporters in ocular tissues.

Figure 1

Fold change in ATP-binding cassette (ABC) transporters expression in hypoxic rat choroid-retina when compared to normoxic rat choroid-retina. Values above +1 indicate the up regulation and values below −1 indicates the down regulation of transporters in hypoxic condition. Thick black lines at ± 1.5 are cutoff lines for 50 % up regulation and down regulation. Data are expressed as mean for three biological replicates.

Figure 2

Fold change in solute carrier transporters (SLC) expression in hypoxic rat choroid-retina when compared to normoxic rat choroid-retina. Values above +1 indicate the up regulation and values below −1 indicates the down regulation of transporters in hypoxic condition. Thick black lines at ± 1.5 are cutoff lines for 50 % up regulation and down regulation. Data are expressed as mean for three biological replicates.

Figure 3

Fold change in miscellaneous transporter expression in hypoxic rat choroid-retina when compared to normoxic rat choroid-retina. Values above +1 indicate the up regulation and values below −1 indicates the down regulation of transporters in hypoxic condition. Thick black lines at ± 1.5 are cutoff lines for 50 % up regulation and down regulation. Data are expressed as mean for three biological replicates.

Figure 4

Transport of Gly-Sar, MPP+, and valacyclovir is significantly higher across normoxic calf SCRPE than hypoxic calf SCRPE. On the other hand, transport of phenylacetic acid is significantly higher across hypoxic SCRPE than normoxic SCRPE. Transport of all four transporter substrates was significantly inhibited in the presence of inhibitor cocktail. A) Gly-Sar; B) MPP+; C) Valacyclovir; and D) Phenylacetic acid. Data are expressed as mean ± SD for n =4.

Figure 5

Apparent permeability (Papp) of Gly-Sar, MPP+, and valacyclovir is significantly higher across normoxic SCRPE than hypoxic SCRPE. For phenylacetic acid, Papp is significantly higher across hypoxic SCRPE than normoxic SCRPE. Apparent permeability of all four transporter substrates was significantly inhibited in the presence of inhibitor cocktail. Effect of hypoxia and transporter inhibitors on apparent permeability of A) Gly-Sar, B) MPP +, C) Valacyclovir, and D) Phenylacetic acid across normoxic and hypoxic calf SCRPE. Data are expressed as mean ± SD for n =4. * Significantly different from normoxic at P ≤ 0.05. + Significantly different from hypoxic at P ≤ 0.05

Figure 6

Transport of Gly-Sar, MPP+, and valacyclovir is significantly higher across normoxic calf cornea than hypoxic calf cornea. For phenylacetic acid, transport across hypoxic cornea is significantly higher than normoxic cornea. A) Gly-Sar; B) MPP+; C) Valacyclovir; and D) Phenylacetic acid. Data are expressed as mean ± SD for n =4.

Figure 7

Apparent permeability (Papp) of Gly-Sar, MPP+, and valacyclovir is significantly higher across normoxic cornea than hypoxic cornea. For phenylacetic acid, Papp is significantly higher across hypoxic cornea than normoxic cornea. Effect of hypoxia on apparent permeability of A) Gly-Sar, B) MPP +, C) Valacyclovir, and D) Phenylacetic acid across calf cornea. Data are expressed as mean ± SD for n =4. *Significantly different from normoxic at P ≤ 0.05.

Figure 8

Relative gene expression of PEPT, ATB⁰⁺, OCT, and MCT transporters in hypoxic rat choroid-retina, normalized to normoxic rat choroid-retina. Data are expressed as mean for n =3. Gene expression in normoxic animal was set to 100 % and relative change in hypoxic animal was expressed in % up regulation