Meprin, a brush-border enzyme, plays an important role in hypoxic/ischemic acute renal tubular injury in rats

Abstract

It has been shown that non-congenic mice strains with lower levels of renal meprin develop less renal injury following renal ischemia and reperfusion. We have demonstrated that following ischemia-reperfusion renal injury, there is a rapid shift of meprin localization and intensity from the brush border to the cytoplasmic compartment, tubular lumens and the tubular basement membranes. Radical shifts in the localization of an activated enzyme to potentially sensitive areas of the tubule suggest a toxic role for meprin in ischemia-reperfusion injury. Though meprin degrades extracellular matrix components and other substrates, to our knowledge meprin cytotoxicity has never been examined. Therefore, the first objective of this study was to determine if meprin is directly cytotoxic to renal cells in vitro. The second objective was to determine if inhibition of meprin is protective against hypoxia-reoxygenation injury in vitro and ischemia-reperfusion injury in vivo. The immortalized porcine epithelial cell line (LLC-PK1) and Madin-Darby canine kidney (MDCK) cells in culture were exposed to meprin in various concentrations and for various times. Cell death was determined by Trypan Blue exclusion, lactate dehydrogenase (LDH) release and the 3-[4,5] dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) assay. Renal slices were used to examine the effect of the meprin inhibitor, actinonin, on hypoxic injury in vitro. Male Sprague-Dawley rats were used in ischemia-reperfusion injury studies to determine the effect of actinonin on renal function as measured by plasma urea nitrogen, creatinine and renal histology. Meprin is cytotoxic to LLC-PK1 and MDCK cells in a concentration and time dependent manner. The meprin inhibitor 1,10-phenanthroline completely abolished the cytotoxic effect. Renal slices exposed to hypoxia and hypoxia followed by reoxygenation showed marked cell death. Pre-treatment with the actinonin was markedly protective while not interfering with the hypoxia-induced fall in adenosine 5'-triphosphate (ATP) levels. In in vivo studies, rats exposed to ischemia/reperfusion injury were markedly protected against acute renal failure by IP treatment with actinonin. Meprin is cytotoxic to cultured renal tubular epithelial cells in vitro. Renal slices are protected from hypoxia-reoxygenation injury in vitro by the meprin inhibitor actinonin. Meprin inhibition is protective against rat renal hypoxia-reoxygenation injury. These data strongly support the concept that meprin is cytotoxic and may play a key role in renal ischemia-reperfusion induced renal injury.