Alena Savenka

Chronic Uremia Stimulates LDL Carbamylation and Atherosclerosis

Carbamylated LDL (cLDL) is a potential atherogenic factor in chronic kidney disease (CKD). However, whether elevated plasma cLDL associates with atherosclerosis in vivo is unknown. Here, we induced CKD surgically in apolipoprotein E-deficient (ApoE(-/-)) mice fed a high-fat diet to promote the development of atherosclerosis. These mice had two- to threefold higher plasma levels of both oxidized LDL (oxLDL) and cLDL compared with control mice. Oral administration of urea increased cLDL approximately eightfold in ApoE(-/-) mice subjected to unilateral nephrectomy and a high-fat diet, but oxLDL did not rise. Regardless of the model, the uremic mice with high plasma cLDL had more severe atherosclerosis as measured by intravital ultrasound echography and en face aortic staining of lipid deposits. Furthermore, cLDL accumulated in the aortic wall and colocalized with ICAM-1 and macrophage infiltration. In summary, these data demonstrate that elevated plasma cLDL may represent an independent risk factor for uremia-induced atherosclerosis.

Interferon Regulatory Transcription Factor 3 Protects Mice from Uterine Horn Pathology during Chlamydia muridarum Genital Infection

ABSTRACT Mice with the type I interferon (IFN) receptor gene knocked out (IFNAR KO mice) or deficient for alpha/beta IFN (IFN-α/β) signaling clear chlamydial infection earlier than control mice and develop less oviduct pathology. Initiation of host IFN-β transcription during an in vitro chlamydial infection requires interferon regulatory transcription factor 3 (IRF3). The goal of the present study was to characterize the influence of IRF3 on chlamydial genital infection and its relationship to IFN-β expression in the mouse model. IRF3 KO mice were able to resolve infection as well as control mice, overcoming increased chlamydial colonization and tissue burden early during infection. As previously observed for IFNAR KO mice, IRF3 KO mice generated a potent antigen-specific T cell response. However, in contrast to IFNAR KO mice, IRF3 KO mice exhibited unusually severe dilatation and pathology in the uterine horns but normal oviduct pathology after infection. Although IFN-β expression in vivo was dependent on the presence of IRF3 early in infection (before day 4), the IFN-independent function of IRF3 was likely driving this phenotype. Specifically, early during infection, the number of apoptotic cells and the number of inflammatory cells were significantly less in uterine horns from IRF3 KO mice than in those from control mice, despite an increased chlamydial burden. To delineate the effects of IFN-β versus IRF3, neutralizing IFN-β antibody was administered to wild-type (WT) mice during chlamydial infection. IFN-β depletion in WT mice mimicked that in IFNΑR KO mice but not that in IRF3 KO mice with respect to both chlamydial clearance and reduced oviduct pathology. These data suggest that IRF3 has a role in protection from uterine horn pathology that is independent of its function in IFN-β expression.

Deoxyribonuclease I is Essential for DNA Fragmentation Induced by Gamma Radiation in Mice

Abstract Gamma radiation is known to induce cell death in several organs. This damage is associated with endonuclease-mediated DNA fragmentation; however, the enzyme that produces the latter and is likely to cause cell death is unknown. To determine whether the most abundant cytotoxic endonuclease DNase I mediates γ-radiation-induced tissue injury, we used DNase I knockout mice and zinc chelate of 3,5-diisopropylsalicylic acid (Zn-DIPS), which, as we show, has DNase I inhibiting activity in vitro. The study demonstrated for the first time that inactivation or inhibition of DNase I ameliorates radiation injury to the white pulp of spleen, intestine villi and bone marrow as measured using a quantitative TUNEL assay. The spleen and intestine of DNase I knockout mice were additionally protected from radiation by Zn-DIPS, perhaps due to the broad radioprotective effect of the zinc ions. Surprisingly, the main DNase I-producing tissues such as the salivary glands, pancreas and kidney showed no effect of DNase I inactivation. Another unexpected observation was that even without irradiation, DNA fragmentation and cell death were significantly lower in the intestine of DNase I knockout mice than in wild-type mice. This points to the physiological role of DNase I in normal cell death in the intestinal epithelium. In conclusion, our results suggested that DNase I-mediated mechanism of DNA damage and subsequent tissue injury are essential in γ-radiation-induced cell death in radiosensitive organs.

Mechanism of graphene-induced cytotoxicity: Role of endonucleases

Graphene, a crystalline allotrope or carbon, presents numerous useful properties; however, its toxicity is yet to be determined. One of the most dramatic and irreversible toxic abilities of carbon nanomaterials is the induction of DNA fragmentation produced by endogenous cellular endonucleases. This study demonstrated that pristine graphene exposed to cultured kidney tubular epithelial cells is capable of inducing DNA fragmentation measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which is usually associated with cell death. TUNEL (cell death) and endonuclease activity measured using a near infrared fluorescence probe was significantly higher in cells containing graphene aggregates detected by Raman spectroscopy. The elevation of TUNEL coincided with the increased abundance of heme oxygenase 1 (HO‐1), heat shock protein 90 (HSP90), active caspase‐3 and endonucleases (deoxyribonuclease I [DNase I] and endonuclease G [EndoG]), as measured by quantitative immunocytochemistry. Specific inhibitors for HO‐1, HSP90, caspase‐3, DNase I and EndoG almost completely blocked the DNA fragmentation induced by graphene exposure. Therefore, graphene induces cell death through oxidative injury, caspase‐mediated and caspase‐independent pathways; and endonucleases DNase I and EndoG are important for graphene toxicity. Inhibition of these pathways may ameliorate cell injury produced by graphene. Copyright

Serotonin transporter protects the placental cells against apoptosis in caspase 3-independent pathway

Serotonin (5‐HT) and its specific transporter, SERT play important roles in pregnancy. Using placentas dissected from 18d gestational SERT‐knock out (KO), peripheral 5‐HT (TPH1)‐KO, and wild‐type (WT) mice, we explored the role of 5‐HT and SERT in placental functions in detail. An abnormal thick band of fibrosis and necrosis under the giant cell layer in SERT‐KO placentas appeared only moderately in TPH1‐KO and minimally present in WT placentas. The majority of the changes were located at the junctional zone of the placentas in SERT. The etiology of these findings was tested with TUNEL assays. The placentas from SERT‐KO and TPH1‐KO showed 49‐ and 8‐fold increase in TUNEL‐positive cells without a concurrent change in the DNA repair or cell proliferation compared to WT placentas. While the proliferation rate in the embryos of TPH1‐KO mice was 16‐fold lower than the rate in gestational age matched embryos of WT or SERT‐KO mice. These findings highlight an important role of continuous 5‐HT signaling on trophoblast cell viability. SERT may contribute to protecting trophoblast cells against cell death via terminating the 5‐HT signaling which changes cell death ratio in trophoblast as well as proliferation rate in embryos. However, the cell death in SERT‐KO placentas is in caspase 3‐independent pathway.

Protective effect of zinc-N-acetylcysteine on the rat kidney during cold storage

Cold storage of kidneys before transplantation is problematic because of the limited survival time of the allografts. In this study, zinc-N-acetylcysteine (ZnNAC) was shown to be a potent endonuclease inhibitor and antioxidant, and it was tested as a potential additive to a cold storage solution for kidney preservation. Exposure of normal rat kidney NRK-52E cells to ZnNAC resulted in zinc delivery to the cells as determined by TFL-Zn fluorophore and partial protection of the cells against injury by cold storage in University of Wisconsin solution (UWS) as measured by propidium iodide assay. Ex vivo, rat kidneys demonstrated time- and temperature-dependent DNA fragmentation as assessed by TUNEL assay, indicating irreversible cell death. DNA fragmentation was faster in the medulla than in the cortex, and tubules were affected more than glomeruli. Perfusion of rat kidneys with cold ZnNAC solution in UWS significantly inhibited cell death both in the cortex and medulla at concentrations of 0.3-30 mM compared with UWS alone, with a maximum effect at 1-10 mM ZnNAC. Cold storage of the kidney significantly increased quantities of cleaved caspase-3 and endonuclease G (EndoG) in the tissue, which were abolished by 10 mM ZnNAC, indicating its ability to suppress both caspase-dependent and -independent cell death. Therefore, supplementation of UWS with ZnNAC can decrease DNA fragmentation and protect kidney allografts from cell death due to cold storage.