Ferritin plays a central role in iron metabolism and is made of 24 subunits of 2 types: heavy chain and light chain. The ferritin heavy chain (FtH) has ferroxidase activity that is required for iron incorporation and limiting toxicity. The purpose of this study was to investigate the role of FtH in acute kidney injury (AKI) and renal iron handling by using proximal tubule-specific FtH-knockout mice (FtH PT-/-mice). FtH PT-/-mice had significant mortality, worse structural and functional renal injury, and increased levels of apoptosis in rhabdomyolysis and cisplatin-induced AKI, despite significantly higher expression of heme oxygenase-1, an antioxidant and cytoprotective enzyme. While expression of divalent metal transporter-1 was unaffected, expression of ferroportin (FPN) was significantly lower under both basal and rhabdomyolysis-induced AKI in FtH PT-/-mice. Apical localization of FPN was disrupted after AKI to a diffuse cytosolic and basolateral pattern. FtH, regardless of iron content and ferroxidase activity, induced FPN. Interestingly, urinary levels of the iron acceptor proteins neutrophil gelatinase-associated lipocalin, hemopexin, and transferrin were increased in FtH PT-/-mice after AKI. These results underscore the protective role of FtH and reveal the critical role of proximal tubule FtH in iron trafficking in AKI.

Collectively these data suggest the potential role of carbamylated LDL in accelerated atherosclerosis in patients with chronic renal disease and, possibly, in healthy individuals.

Melatonin is a potent antioxidant and direct radical scavenger. As keratinocytes represent the major population in the skin and UV light causes damage to these cells, the possible protective effects of melatonin against UV-induced cell damage in HaCaT keratinocytes were investigated in vitro. Cells were preincubated with melatonin at graded concentrations from 10(-9) to 10(-3) m for 30 min prior to UV irradiation at doses of 25 and 50 mJ/cm2. Biological markers of cellular viability such as DNA synthesis and colony-forming efficiency as well as molecular markers of apoptosis were measured. DNA synthesis was determined by [3H]-thymidine incorporation into insoluble cellular fraction, clonogenicity through plating efficiency experiments and apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. DNA synthesis experiments showed a strong protective effect by preincubation with melatonin at concentrations of 10(-4) m (P < 0.01) and 10(-3) m (P < 0.001). Additional postirradiation treatment with melatonin showed no increase in the pre-UV incubation protective effect. These results indicate that preincubation is a requirement for melatonin to exert its protective effects. The mechanism of melatonin's protective effect (10(-6) to 10(-3) m) includes inhibition of apoptosis as measured by TUNEL assay. Moreover, the biological significance of these effects is supported by clonogenic studies showing a significantly higher number of colonies in cultures treated with melatonin compared to controls. Thus, pretreatment with melatonin led to strong protection against UVB-induced damage in keratinocytes.

Cisplatin is commonly used for chemotherapy in a wide variety of tumors; however, its use is limited by kidney toxicity. Although the exact mechanism of cisplatin-induced nephrotoxicity is not understood, several studies showed that it is associated with DNA fragmentation induced by an unknown endonuclease. It was demonstrated previously that deoxyribonuclease I (DNase I) is a highly active renal endonuclease, and its silencing by antisense is cytoprotective against the in vitro hypoxia injury of kidney tubular epithelial cells. This study used recently developed DNase1 knockout (KO) mice to determine the role of this endonuclease in cisplatin-induced nephrotoxicity. The data showed that DNase I represents approximately 80% of the total endonuclease activity in the kidney and cultured primary renal tubular epithelial cells. In vitro, primary renal tubular epithelial cells isolated from KO animals were resistant to cisplatin (8 M) injury. DNase I KO mice were also markedly protected against the toxic injury induced by a single injection of cisplatin (20 mg/kg), by both functional (blood urea nitrogen and serum creatinine) and histologic criteria (tubular necrosis and in situ DNA fragmentation assessed by the terminal deoxynucleotidyl transferase nick end-labeling). These data provide direct evidence that DNase I is essential for kidney injury induced by cisplatin.

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Objective Carbamylated LDL (cLDL) has been recently shown to have robust pro-atherogenic effects upon human endothelial cells in vitro; suggesting cLDL may have a significant role in atherosclerosis in uremia. The current study was designed to determine, which receptors are used by cLDL and so may cause the pro-atherogenic effects. Methods and Results In ex vivo or in vitro models as well as in intact animals, administration of cLDL was associated with endothelial internalization of cLDL and subendothelial translocation (transcytosis). In vitro recombinant LOX-1 and SREC-1 receptors showed the greatest cLDL binding. However, pretreatment of the endothelial cells with specific inhibiting antibodies demonstrated that cLDL binds mainly to LOX-1 and CD36 receptors. The transcytosis was dependent on SR-A1, SREC-1 and CD36 receptors while LOX-1 receptor was not involved. The cytotoxicity was mediated by several studied scavenger receptors, but cLDL-induced monocyte adhesion depended only on LOX-1. The cLDL-induced synthesis of LOX-1 protein significantly contributed to both cytotoxicity and accelerated monocyte adhesion to endothelial cells. Conclusions Our data suggest that cLDL utilizes unique pattern of scavenger receptors. They show that LOX-1 receptor, and partially, CD36, SREC-1 and SR-A1 receptors are essential for the pro-atherogenic effects of cLDL on human endothelial cells.

Objective-Carbamylated low-density lipoprotein (LDL), the most abundant modified LDL isoform in human blood, has been recently implicated in causing the atherosclerosis-prone injuries to endothelial cells in vitro and atherosclerosis in humans. This study was aimed at testing the hypothesis that carbamylated LDL acts via inducing monocyte adhesion to endothelial cells and determining the adhesion molecules responsible for the recruitment of monocytes. Methods and Results-Exposure of human coronary artery endothelial cells with carbamylated LDL but not native LDL caused U937 monocyte adhesion and the induction of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 adhesion molecules as measured by cell enzyme-linked immunosorbent assay. Silencing of intercellular adhesion molecule-1 by siRNA or its inhibition using neutralizing antibody resulted in decreased monocyte adhesion to the endothelial cells. Similar silencing or neutralizing of vascular cell adhesion molecule-1 alone did not have an effect but was shown to contribute to intercellular adhesion molecule-1 when tested simultaneously. This process is caused by the upregulation of adhesion molecules on endothelial cells and an increased expression in the vascular wall of chemotactic factors to monocytes. Highly specific adhesive interactions between monocytes and endothelial cells are mediated by 3 main families of receptors: members of the immunoglobulin superfamily, selectins, and integrins. Recent studies indicate that intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), members of the immunoglobulin superfamily, are the most common participants in monocyte attraction induced by different stimuli. [1][2][3] The expression of adhesion molecules and monocyte adhesion can be triggered by a variety of plasma components, such as interferon-␥, 4 homocysteine, 5 and lipoproteins, like oxidized low-density lipoprotein (oxLDL), 6 or its derivates, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine 7 and lysophosphatidylcholine. 8 The identification of new plasma components that are responsible for monocyte adhesion is important in light of their potential application as pathogenic atherosclerosis markers, predictors or therapeutic targets. Conclusions-TakenCarbamylated low-density lipoprotein (cLDL) is a recently identified type of modified low-density lipoprotein (LDL) that seems to be important in atherosclerosis in humans. 9 -11 It is generated by irreversible chemical modification of the protein component of the LDL particle, apolipoprotein B, and by urea-derived cyanate present in human blood plasma. 9 Plasma levels of cLDL as determined by sandwich enzymelinked immunosorbent assay (ELISA) methods vary in a higher range of concentrations than oxLDL, which makes it the most abundant LDL isoform of human plasma. 11 The elevation of blood urea in uremic patients causes a proportional increase of plasma cLDL. 11 The LDL of chronic renal failure patients on dialysis was shown to induce g...

An overdose of acetaminophen (APAP) (N-acetyl-p-aminophenol) leads to hepatocellular necrosis induced by its metabolite N-acetyl-p-benzoquinone-imine, which is generated during the metabolic phase of liver intoxication. It has been reported that DNA damage occurs during the toxic phase; however, the nucleases responsible for this effect are unknown. In this study, we analyzed the participation of the hepatic endonuclease deoxyribonuclease 1 (DNASE1) during APAP-induced hepatotoxicity by employing a Dnase1 knockout (KO) mouse model. Male CD-1 Dnase1 wild-type (WT) (Dnase1 ؉/؉ ) and KO (Dnase1 ؊/؊ ) mice were treated with 2 different doses of APAP. Hepatic histopathology was performed, and biochemical parameters for APAP metabolism and necrosis were investigated, including depletion of glutathione/glutathione-disulfide (GSH؉GSSG), ␤-nicotinamide adenine dinucleotide (NADH؉NAD ؉ ), and adenosine triphosphate (ATP); release of aminotransferases and Dnase1; and occurrence of DNA fragmentation. As expected, an APAP overdose in WT mice led to massive hepatocellular necrosis characterized by the release of aminotransferases and depletion of hepatocellular GSH؉GSSG, NADH؉NAD ؉ , and ATP. These metabolic events were accompanied by extensive DNA degradation. In contrast, Dnase1 KO mice were considerably less affected. In conclusion, whereas the innermost pericentral hepatocytes of both mouse strains underwent necrosis to the same extent independent of DNA damage, the progression of necrosis to more outwardly located cells was dependent on DNA damage and only occurred in WT mice. Dnase1 aggravates APAP-induced liver necrosis. A cetaminophen, a widely used analgesic, is one of the most commonly overdosed pharmaceuticals. 1 Overdose leads to liver necrosis caused by the electrophilic metabolite N-acetyl-p-benzoquinoneimine (NAPQI), which is generated via oxidation of acetaminophen (APAP) by a microsomal cytochrome P450 containing mixed-function oxidase system. 2 Subsequent detoxication of NAPQI by cellular glutathione leads to depletion of glutathione/glutathione-disulfide (GSHϩGSSG) followed by NAPQI accumulation and induction of necrosis due to protein arylation and severe oxidative stress. 2,3 Nuclear accumulation of Ca 2ϩ and DNA damage have been described to occur during APAPinduced cytotoxicity of murine hepatocytes in vitro and in vivo. 4-6 DNA damage seems to be an important event, because APAP toxicity was prevented by known inhibitors of Ca 2ϩ -dependent endonucleases, including Ca 2ϩ -chelators such as aurintricarboxylic acid, glycol ether diamine tetra-acetic acid, and the Ca 2ϩ -calmodulin antagonist chlorpromazine. 5,7,8 However, the nucleases involved in APAP-induced DNA damage have not yet been identified.Deoxyribonuclease 1 (DNASE1), a Ca 2ϩ /Mg 2ϩ -dependent secreted endonuclease with a pH optimum of approximately 7.5, hydrolyzes double-stranded DNA generating 5Ј-phospho-tri-and/or tetra-oligonucleotides. 9,10 DNASE1 gene expression has been demonstrated in a number of different organs of the urogenital and ...