actinonin and Ischemia

We investigated the effects of actinonin, an inhibitor of a matrix-degrading enzyme meprin, on ischemic acute kidney injury in male and female rats, and these were compared with the effects of verapamil, a Ca(2+) channel blocker. Ischemic acute kidney injury was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. At 24 h after reperfusion, renal function and histology of both males and females showed significant deterioration. The degrees of renal dysfunction and histological damage were much more severe in males than in females. Pre-ischemic treatment with actinonin (10 or 30 mg/kg, i.v.) dose-dependently attenuated the ischemia/reperfusion-induced renal injury in male rats, but failed to improve the renal injury in female rats. On the other hand, verapamil (1 mg/kg, i.v.) could efficiently prevent the ischemic acute kidney injury in female rats, as well as male rats. These results indicate that the renoprotective effect of actinonin is male-specific, thereby suggesting that meprin is involved in exacerbation of ischemia/reperfusion-induced renal injury in male rats. The possibility that meprin is a key factor involved in the sex difference in the pathogenesis of ischemic acute kidney injury, warrants further attention.

Topics: Acute Disease; Animals; Female; Hydroxamic Acids; Ischemia; Kidney; Male; Metalloendopeptidases; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sex Characteristics; Verapamil

It is known that female rats are resistant to ischemic acute renal failure (ARF), compared with male rats. To elucidate sex differences in ischemic ARF, we searched global protein expression in post-ischemic kidneys using proteomic techniques. Ischemic ARF was induced by 45-min ischemia followed by reperfusion. By proteomic analysis, many male- or female-dominant proteins were detected in sham-operated rat kidneys, and significantly increased or decreased proteins were found in post-ischemic kidneys 2 h after reperfusion, at which there were no significant deterioration in renal function of both sexes. We detected 86 proteins showing more than 1.5-fold significant alterations (p<0.01) in both sexes by ischemia/reperfusion (I/R) treatment. Among the altered proteins, we identified a significantly up-regulated protein in male rat kidneys, meprin alpha, a subunit of meprin which had been reported to play a role in the pathophysiology of I/R-induced ARF. In addition, it is known that a potent meprin alpha inhibitor, actinonin, can protect against I/R-induced renal injury when administered to male rats. We therefore compared the effect of actinonin on I/R-induced renal dysfunction between male and female rats. Renal function of both males and females showed significant deterioration when measured at 24 h after the reperfusion, although the degree of renal dysfunction was much less in females than in males. Pre-ischemic treatment with actinonin (30 mg/kg, i.v.) prevented the I/R-induced renal dysfunction in males but not in females. Our results provide information on differences in protein expression at an early phase after the reperfusion between male and female rats. Moreover, the present study suggests that up-regulation of meprin alpha in the post-ischemic kidney is at least partly involved in aggravation of I/R-induced renal injury in male rats. The possibility that meprin alpha is a key component of the sex difference in ischemic ARF, warrants further attention.

Topics: Acute Kidney Injury; Animals; Blood Urea Nitrogen; Creatinine; Electrophoresis, Gel, Two-Dimensional; Enzyme Inhibitors; Female; Hydroxamic Acids; Ischemia; Kidney Function Tests; Male; Metalloendopeptidases; Myocardial Reperfusion Injury; Proteins; Proteomics; Rats; Rats, Sprague-Dawley; Sex Characteristics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

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.

Topics: Animals; Cell Death; Cell Line; Dogs; Hydroxamic Acids; Hypoxia; In Vitro Techniques; Ischemia; Kidney; Kidney Tubules; LLC-PK1 Cells; Male; Metalloendopeptidases; Microvilli; Oxygen; Rats; Rats, Sprague-Dawley; Swine