Digital Imaging Analysis for Quantitative Subcellular Localization of MRP2 during Hepatic Ischemia–Reperfusion Injury in a Rat Model: Can You Capture Time?

Abstract

Introduction: The mechanism of cholestasis in hepatic ischemia–reperfusion injury (IRI) remains to be elucidated. Short-term changes in bile formation are regulated by translocating transporter proteins between the cell membrane and the cytosolic compartment. We sought to develop a method that quantifies the degree of translocation of multidrug resistance-associated protein 2 (MRP2) along the hepatocyte canalicular membrane. Method: Fifteen male Sprague Dawley rats were divided into three groups based on warm ischemia time (WIT): 0 min, 10 min, and 20 min. IRI was induced by temporarily clamping pedicles to the median and lateral lobes, followed by 60 min of reperfusion. Liver tissue was analyzed for subcellular protein fractionation and immunofluorescence staining. CD13 served as a reference protein for the canalicular membrane. Digital imaging analyses measured the area of MRP2 and CD13 staining. The size of the MRP2 area without overlap with CD13 (i.e., MRP2 in the cytosolic compartment) in proportion to the total MRP2 area was defined as the MRP2 translocation index (MTI). Results: The proportion of CD13 along the canalicular membrane was larger than that of MRP2 (median 93.3% versus 43.4%, respectively; P<0.0001, Figure A). The subcellular distribution of CD13 was consistent among the three WIT groups. Immunofluorescence colocalization analysis for CD13 and MRP2 demonstrated a negative linear correlation between MTI and WIT (P=0.0183, Y=-1.052X+25.00, Figure B). Conclusions: The degree of MRP2 translocation relative to CD13 was quantified, which correlated with WIT. This novel approach can be used to determine the pathophysiology of cholestasis in hepatic IRI.