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.

Abstract-MicroRNAs are endogenous repressors of gene expression. We examined microRNAs in the renal medulla of Dahl salt-sensitive rats and consomic SS-13 BN rats. Salt-induced hypertension and renal injury in Dahl salt-sensitive rats, particularly medullary interstitial fibrosis, have been shown previously to be substantially attenuated in SS-13 BN rats. Of 377 microRNAs examined, 5 were found to be differentially expressed between Dahl salt-sensitive rats and consomic SS-13 BN rats receiving a high-salt diet. Real-time PCR analysis demonstrated that high-salt diets induced substantial upregulation of miR-29b in the renal medulla of SS-13 BN rats but not in SS rats. miR-29b was predicted to regulate 20 collagen genes, matrix metalloproteinase 2 (Mmp2), integrin ␤1 (Itgb1), and other genes related to the extracellular matrix. Expression of 9 collagen genes and Mmp2 was upregulated by a high-salt diet in the renal medulla of SS rats, but not in SS-13 BN rats, an expression pattern opposite to miR-29b. Knockdown of miR-29b in the kidneys of SS-13 BN rats resulted in upregulation of several collagen genes. miR-29b reduced expression levels of several collagen genes and Itgb1 in cultured rat renal medullary epithelial cells. Moreover, miR-29b suppressed the activity of luciferase when the reporter gene was linked to a 3Ј-untranslated segment of collagen genes Col1a1, Col3a1, Col4a1, Col5a1, Col5a2, Col5a3, Col7a1, Col8a1, Mmp2, or Itgb1 but not Col12a1. The result demonstrated broad effects of miR-29b on a large number of collagens and genes related to the extracellular matrix and suggested involvement of miR-29b in the protection from renal medullary injury in SS-13 BN rats. (Hypertension. 2010;55:974-982.)

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...