Spatiotemporal Changes in P-glycoprotein Levels in Brain and Peripheral Tissues Following Ischemic Stroke in Rats

Abstract

P-glycoprotein (P-gp) is known to transport a diverse array of xenobiotics, including therapeutic drugs. A member of the ATP-binding cassette (ABC) transporter family, P-gp is a protein encoded by the gene Mdr1 in humans and Abcb1 in rodents (represented by 2 isoforms Abcb1a and Abcb1b). Lining the luminal and abluminal membrane of brain capillary endothelial cells, P-gp is a promiscuous efflux pump extruding a variety of exogenous toxins and drugs. In this study, we measured dynamic changes in Abcb1a and Abcb1b transcripts and P-gp protein in the brain, liver, and kidney after experimental stroke. P-glycoprotein has been shown to increase in brain endothelial cells following hypoxia in vitro or after exposure to proinflammatory cytokines. Using a rat model of ischemic stroke, we hypothesized that P-gp expression will be increased in the brain, liver, and kidney in response to neuroinflammation following ischemic stroke. Adult Sprague Dawley rats underwent middle cerebral artery occlusion (MCAO) for 90 minutes and were killed at 4, 14, 24, and 48 hours postreperfusion onset to determine the time course of P-gp expression. To mimic ischemia occurring at the blood-brain barrier, rat brain endothelial (RBE4) cells were subjected to hypoxia and low glucose (HLG) for 16 hours. Immunoblotting analyses showed P-gp increases in brain and liver following 90-minute MCAO, as well as in cultured RBE4 cells after 16-hour HLG treatment, but fluctuated in the kidney depending on the time point. The relative roles of each isoform in the protein expression were analyzed with quantitative reverse transcriptase polymerase chain reaction. Ischemic stroke leads to significant increases in P-gp levels not only in the brain but also in the liver. The increase in P-gp could dramatically reduce the bioavailability and efficacy of neuroprotective drugs. Therefore, P-gp represents a big hurdle to drug delivery to the ischemic brain.

Keywords: Abcb1/Mdr1; P-glycoprotein; ischemia; kidney; liver; stroke.

Figure 1.

(A) and (C) Western blot data of cortical tissue and representative image showing P-glycoprotein (P-gp) levels normalized to actin in sham (n = 6), 4-hour (n = 5), 14-hour (n = 6), 24-hour (n = 7), and 48-hour (n = 6) groups in the ipsilateral (i) and contralateral (c) hemispheres. Data are presented as a function of total reperfusion time until being killed. P-gp is significantly increased by 48 hours in the ipsilateral cortex (*P < 0.05). (B) and (D) Western blot in subcortical tissue showing P-gp levels normalized to actin in sham (n = 6), 4-hour (n = 5), 14-hour (n = 6), 24-hour (n = 7), and 48-hour (n = 6) groups. There are nonsignificant increases in P-gp at 24 and 48 hours in the ipsilateral hemisphere. (E) Abcb1a expression normalized to Ywhaz in cortical samples from sham (n = 6), 4-hour (n = 5), 14-hour (n = 6), 24-hour (n = 7), and 48-hour (n = 6) groups; *P < 0.05, **P < 0.01, ***P < 0.001. (F) Abcb1b expression normalized to Ywhaz in cortical samples from sham (n = 6), 4-hour (n = 5), 14-hour (n = 6), 24-hour (n = 7), and 48-hour (n = 6) groups; *P < 0.05, ***P < 0.001. (G) Abcb1a expression normalized to Ywhaz in subcortical samples from sham (n = 6), 4-hour (n = 5), 14-hour (n = 6), 24-hour (n = 7), and 48-hour (n = 6) groups; ***P < 0.001. (H) Abcb1b expression normalized to Ywhaz in subcortical samples from sham (n = 6), 4-hour (n = 5), 14-hour (n = 6), 24-hour (n = 7), and 48-hour (n = 6) groups; **P < 0.01, ***P < 0.001.

Figure 2.

Immunohistochemistry (IHC) of the brain. Rat brain diagram with rectangle depicts region where images were taken within the ipsilateral cortex near the border between infarcted and “healthy” tissue. IHC at ×40 with naïve, sham, and middle cerebral artery occlusion 48 hours post-reperfusion onset showing co-localization of P-glycoprotein (P-gp) and endothelial cells with RECA-1. DAPI indicates 4′,6-diamidino-2-phenylindole.

Figure 3.

(A) Western blot of RBE4 cell lysate measuring HIF-1α in response to 16 hours of HLG compared with control and representative image (n = 4, ***P < 0.001). (B) Western blot of RBE4 cell lysate showing increased P-gp with 16 hours of hypoxia/low glucose compared with control and representative image (n = 5, ** P < 0.01). HIF-1α indicates hypoxia-inducible factor 1 alpha; HLG, hypoxia and low glucose; P-gp, P-glycoprotein; RBE4, rat brain endothelial cells.

Figure 4.

(A) and (B) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) changes in Abcb1a and Abcb1b expression in the liver of sham (n = 6), 4-hour (n = 5), 14-hour (n = 6), 24-hour (n = 7), and 48-hour (n = 6) groups normalized to Ywhaz. Data are presented as a function of total reperfusion time until being killed. Abcb1b is increased in a time-dependent manner (***P < 0.001). (C) and (D) qRT-PCR changes in Abcb1a (*P < 0.05, *** P < 0.001) and Abcb1b (**P < 0.01) expression in the kidney of sham (n = 6), 4-hour (n = 5), 14-hour (n = 6), 24-hour (n = 7), and 48-hour (n = 6) groups normalized to Ywhaz. (E) and (G) Western blot data of kidney tissue and representative image showing P-gp levels normalized to actin in sham (n = 6), 4-hour (n = 5), 14-hour (n = 6), 24-hour (n = 7), and 48-hour (n = 6) groups. P-gp is significantly decreased by 4 hours (*P < 0.05) and 14 hours (*** P < 0.001). (F) and (H) Western blot data and representative image of P-gp normalized to actin in the liver showing sham (n = 6), 4-hour (n = 5), 14-hour (n = 6), 24-hour (n = 7), and 48-hour (n = 6) groups. P-gp is significantly increased by 48 hours (**P < 0.01). P-gp indicates P-glycoprotein.