Delayed ischemic preconditioning effectively protects kidneys from ischemia-reperfusion injury but the mechanism underlying renal protection remains poorly understood. Here we examined the in vivo role of microRNA miR-21 in the renal protection conferred by delayed ischemic preconditioning in mice. A 15 minute renal ischemic preconditioning significantly increased the expression of miR-21 by 4 hours and substantially attenuated ischemia-reperfusion injury induced 4 days later. A locked nucleic acid-modified anti-miR-21 given at the time of ischemic preconditioning knocked down miR-21 and significantly exacerbated subsequent ischemia-reperfusion injury in the mouse kidney. Knockdown of miR-21 resulted in significant upregulation of programmed cell death protein 4, a pro-apoptotic target gene of miR-21, and substantially increased tubular cell apoptosis. Hypoxia inducible factor-1α in the kidney was activated after ischemic preconditioning and blockade of its activity with a decoy abolished the up-regulation of miR-21 in cultured human renal epithelial cells treated with the inducer cobalt chloride. In the absence of ischemic preconditioning, knockdown of miR-21 alone did not significantly affect ischemia-reperfusion injury in the mouse kidney. Thus, upregulation of miR-21 contributes to the protective effect of delayed ischemic preconditioning against subsequent renal ischemia-reperfusion injury.
We reported previously an approach for identifying microRNA (miRNA)-target pairs by combining miRNA and proteomic analyses. The approach was applied in the present study to examine human renal epithelial cells treated with transforming growth factor β1 (TGFβ1), a model of epithelial–mesenchymal transition important for the development of renal interstitial fibrosis. Treatment of human renal epithelial cells with TGFβ1 resulted in upregulation of 16 miRNAs and 18 proteins and downregulation of 17 miRNAs and 16 proteins. Of the miRNAs and proteins that exhibited reciprocal changes in expression, 77 pairs met the sequence criteria for miRNA–target interactions. Knockdown of miR-382, which was up-regulated by TGFβ1, attenuated TGFβ1-induced loss of the epithelial marker E-cadherin. miR-382 was confirmed by 3′-untranslated region reporter assay to target five genes that were downregulated at the protein level by TGFβ1, including superoxide dismutase 2 (SOD2). Knockdown of miR-382 attenuated TGFβ1-induced downregulation of SOD2. Overexpression of SOD2 ameliorated TGFβ1-induced loss of the epithelial marker. The study provided experimental evidence in the form of reciprocal expression at the protein level for a large number of predicted miRNA-target pairs and discovered a novel role of miR-382 and SOD2 in the loss of epithelial characteristics induced by TGFβ1.
Abstract-In a previous proteomic study, we found dramatic differences in fumarase in the kidney between Dahl salt-sensitive rats and salt-insensitive consomic SS-13 BN rats. Fumarase catalyzes the conversion between fumarate and L-malate in the tricarboxylic acid cycle. Little is known about the pathophysiological significance of fumarate metabolism in cardiovascular and renal functions, including salt-induced hypertension. The fumarase gene is located on the chromosome substituted in the SS-13 BN rat. Sequencing of fumarase cDNA indicated the presence of lysine at amino acid position 481 in Dahl salt-sensitive rats and glutamic acid in Brown Norway and SS-13 BN rats. Total fumarase activity was significantly lower in the kidneys of Dahl salt-sensitive rats compared with SS-13 BN rats, despite an apparent compensatory increase in fumarase abundance in Dahl salt-sensitive rats. Intravenous infusion of a fumarate precursor in SS-13 BN rats resulted in a fumarate excess in the renal medulla comparable to that seen in Dahl salt-sensitive rats. The infusion significantly exacerbated salt-induced hypertension in SS-13 BN rats (140Ϯ3 vs125Ϯ2 mm Hg in vehicle control at day 5 on a 4% NaCl diet; PϽ0.05). In addition, the fumarate infusion increased renal medullary tissue levels of H 2 O 2 . Treatment of cultured human renal epithelial cells with the fumarate precursor also increased cellular levels of H 2 O 2 . These data suggest a novel role for fumarate metabolism in salt-induced hypertension and renal medullary oxidative stress. BN rat has the same genomic makeup as the SS rat except for chromosome 13, which is introgressed from the Brown Norway (BN) rat and substantially attenuates salt-sensitive hypertension and renal injury. 3 A tree-like network of molecular, biochemical, and physiological mechanisms is likely involved in the development of Dahl salt-sensitive hypertension and renal injury. 4 Comparative analysis of SS and SS-13 BN rats has revealed several new components of this regulatory network. Examples include increased levels of superoxide and H 2 O 2 , 5,6 dysregulation of 11-hydroxysteroid dehydrogenase, and alterations of glucocorticoid metabolism 4 in the renal medulla of SS rats compared with SS-13 BN rats. Additional mechanisms and particularly sequence variations of specific genes involved in the SS phenotypes remain to be discovered or validated. 4,[7][8][9] Fumarase was one of the proteins exhibiting dramatic differences between SS and SS-13 BN rats according to a recent proteomic study. 10 The analysis indicated a consistent and substantial difference in the isoelectric point of fumarase in SS and SS-13 BN rats, as reflected by a significant shift of the protein spot on 2D gels. Fumarase catalyzes the reversible conversion between fumarate and L-malate in the tricarboxylic acid cycle in mitochondria. Rare loss-of-function mutations of fumarase in humans cause accumulation of fumarate and are associated with the development of hereditary leiomyomatosis, renal cell cancer, or encephalopathy. [11][1...
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