Kidney Ischemia/Reperfusion Injury Induces Changes in the Drug Transporter Expression at the Blood–Brain Barrier in vivo and in vitro

Abstract

Ischemia/reperfusion injury is a major cause of acute kidney injury (AKI). AKI is characterized by a sudden decrease in kidney function, systemic inflammation, oxidative stress, and dysregulation of the sodium, potassium, and water channels. While AKI leads to uremic encephalopathy, epidemiological studies have shown that AKI is associated with a subsequent risk for developing stroke and dementia. To get more insights into kidney–brain crosstalk, we have created an in vitro co-culture model based on human kidney cells of the proximal tubule (HK-2) and brain microvascular endothelial cells (BMEC). The HK-2 cell line was grown to confluence on 6-well plates and exposed to oxygen/glucose deprivation (OGD) for 4 h. Control HK-2 cells were grown under normal conditions. The BMEC cell line cerebED was grown to confluence on transwells with 0.4 μm pores. The transwell filters seeded and grown to confluence with cereEND were inserted into the plates with HK-2 cells with or without OGD treatment. In addition, cerebEND were left untreated or treated with uremic toxins, indole-3-acetic acid (IAA) and indoxyl sulfate (IS). The protein and mRNA expression of selected BBB-typical influx transporters, efflux transporters, cellular receptors, and tight junction proteins was measured in BMECs. To validate this in vitro model of kidney–brain interaction, we isolated brain capillaries from mice exposed to bilateral renal ischemia (30 min)/reperfusion injury (24 h) and measured mRNA and protein expression as described above. Both in vitro and in vivo systems showed similar changes in the expression of drug transporters, cellular receptors, and tight junction proteins. Efflux pumps, in particular Abcb1b, Abcc1, and Abcg2, have shown increased expression in our model. Thus, our in vitro co-culture system can be used to study the cellular mechanism of kidney and brain crosstalk in renal ischemia/reperfusion injury.

Role of microRNAs in the regulation of blood-brain barrier function in ischemic stroke and under hypoxic conditions in vitro

“…An alternated expression and functional changes of ABC transporters were observed after hypoxia or ischemia (Tornabene and Brodin, 2016;Burek et al, 2020). In a model of focal cerebral ischemia, the activity and expression of the brain luminal transporter P-gp was increased in mice (Spudich et al, 2006) as well in rats (DeMars et al, 2017) being consistent with in vitro studies showing that OGD treatment was able to upregulate P-gp both at mRNA and protein level (Neuhaus et al, 2014;DeMars et al, 2017).…”

Analysis of microRNAs in Exosomes of Breast Cancer Patients in Search of Molecular Prognostic Factors in Brain Metastases

“…Protein concentration was estimated using BCA Protein Assay (Thermo Fisher Scientific) following the manufacturer’s instructions. Western blot was performed as previously described [ 57 , 58 , 59 ]. Briefly, 120 µg of protein extract was mixed with 2× Tris-Glycine Sample Buffer and Reducing Agent (Thermo Fisher Scientific).…”

Protocadherin Gamma C3 (PCDHGC3) Is Strongly Expressed in Glioblastoma and Its High Expression Is Associated with Longer Progression-Free Survival of Patients

“…Transfected clones were selected with 3 µg/mL puromycin, and knockout was verified by Western blot as described above. RNA was isolated from WT and PCDHGC3 KO U343 cells as previously described [ 35 , 36 ] using the RNA isolation kit NucleoSpin ® RNA (Machery-Nagel, Düren, Germany) according to manufacturer’s instructions. Total RNA (500 ng) was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA).…”