Intracellular targets in heme protein-induced renal injury

Intracellular targets in heme protein-induced renal injury. We examined two potential intracellular targets in the glycerol model of acute renal failure, namely, the mitochondrion and the nucleus. Within three hours, alterations in mitochondrial function are already apparent. With either glutamate/malate or succinate/rotenone, state 3 and uncoupled respirations were decreased at three hours, and at 24 hours, such decrements were quite pronounced; in the presence of glutamate/malate, state 2 respiration was also depressed at 24 hours, while with succinate/rotenone state 2 was increased. Marked ultrastructural changes were observed in mitochondria studied at three hours, including the novel finding of degenerate mitochondria in autophagic vacuoles. Since the heme content in mitochondria was increased some tenfold within three hours, mitochondrial function was studied after exposure to concentrations of heme that reproduced such contents of heme: mitochondria initially displayed increased respiration, and subsequently, a persistent decline in oxygen consumption until oxygen consumption was virtually undetectable. With higher concentrations of heme, the early increase in oxygen consumption was blunted and the progressive decline in oxygen consumption was hastened. The antioxidant iron chelator, deferoxamine, prevented the early rise in oxygen consumption but did not prevent or delay the subsequent decline. We also assessed nuclear damage as a potential lesion in the glycerol model. DNA laddering was not observed at any time point. At 3 and 24 hours there was DNA injury by the TUNEL technique in the distal nephron but not in the proximal nephron. The 8-hydroxydeoxyguanosine/deoxyguanosine content was increased in the glycerol kidneys at 24 hours but not at three hours. At neither time point was evidence of apoptosis observed by light or electron microscopy. In studies undertaken in cell culture models, heme, at concentrations of 10 μM, failed to evince any such changes in LLC-PK1 cells, a cell line from the proximal tubule, or in MDCK cells, a cell line derived from the distal tubule. At concentrations of 50 μM, heme induced approximately 20% positivity in MDCK cells but none in LLC-PK1 cells by the TUNEL technique. We conclude that mitochondria and nuclei are prominent targets for injury in the glycerol model of acute renal failure. The presence of TUNEL-positive cells in the distal nephron but not at proximal sites in vivo underscores the increasing appreciation of the distinct responses of these nephron sites to nephrotoxic insults

Disruption of an SP2/KLF6 repression complex by SHP is required for farnesoid X receptor-induced endothelial cell migration

The farnesoid X receptor (FXR) signaling pathway regulates bile acid and cholesterol homeostasis. Here, we demonstrate, using a variety of gain- and loss-of-function approaches, a role of FXR in the process of cell motility, which involves the small heterodimeric partner (SHP)-dependent up-regulation of matrix metalloproteinase-9. We use this observation to reveal a transcriptional regulatory mechanism involving the SP/KLF transcription factors, SP2 and KLF6. Small interference RNA-based silencing studies in combination with promoter, gel shift, and chromatin immunoprecipitation assays indicate that SP2 and KLF6 bind to the matrix metalloproteinase-9 promoter and together function to maintain this gene in a silenced state. However, upon activation of FXR, SHP interacts with SP2 and KLF6, disrupting the SP2/KLF6 repressor complex. Thus, together, these studies identify a mechanism for antagonizing Sp/KLF protein repression function via SHP, with this process regulating endothelial cell motility

A monocyte-TNF-endothelial activation axis in sickle transgenic mice: Therapeutic benefit from TNF blockade

Elaboration of tumor necrosis factor (TNF) is a very early event in development of ischemia/reperfusion injury pathophysiology. Therefore, TNF may be a prominent mediator of endothelial cell and vascular wall dysfunction in sickle cell anemia, a hypothesis we addressed using NY1DD, S+SAntilles, and SS‐BERK sickle transgenic mice. Transfusion experiments revealed participation of abnormally activated blood monocytes exerting an endothelial activating effect, dependent upon Egr‐1 in both vessel wall and blood cells, and upon NFκB(p50) in a blood cell only. Involvement of TNF was identified by beneficial impact from TNF blockers, etanercept and infliximab, with less benefit from an IL‐1 blocker, anakinra. In therapeutic studies, etanercept ameliorated multiple disturbances of the murine sickle condition: monocyte activation, blood biomarkers of inflammation, low platelet count and Hb, vascular stasis triggered by hypoxia/reoxygenation (but not if triggered by hemin infusion), tissue production of neuro‐inflammatory mediators, endothelial activation (monitored by tissue factor and VCAM‐1 expression), histopathologic liver injury, and three surrogate markers of pulmonary hypertension (perivascular inflammatory aggregates, arteriolar muscularization, and right ventricular mean systolic pressure). In aggregate, these studies identify a prominent—and possibly dominant—role for an abnormal monocyte‐TNF‐endothelial activation axis in the sickle context. Its presence, plus the many benefits of etanercept observed here, argue that pilot testing of TNF blockade should be considered for human sickle cell anemia, a challenging but achievable translational research goal

H-ferritin ferroxidase induces cytoprotective pathways and inhibits microvascular stasis in transgenic sickle mice

Hemolysis, oxidative stress, inflammation, vaso-occlusion and organ infarction are hallmarks of sickle cell disease (SCD). We have previously shown that increases in heme oxygenase-1 (HO-1) activity detoxify heme and inhibit vaso-occlusion in transgenic mouse models of SCD. HO-1 releases Fe2+ from heme, and the ferritin heavy chain (FHC) ferroxidase oxidizes iron to catalytically-inactive Fe3+ inside ferritin. FHC overexpression has been shown to be cytoprotective. In this study, we hypothesized that overexpression of FHC and its ferroxidase activity will inhibit inflammation and microvascular stasis in transgenic sickle mice in response to stroma-free hemoglobin. We utilized a Sleeping Beauty transposase plasmid to deliver a human wild-type-ferritin heavy chain (wt-hFHC) transposable element by hydrodynamic tail vein injections to NY1DD SCD mice. Control mice were infused with the same volume of lactated Ringer's solution (LRS) or a triple missense human FHC (ms-hFHC) plasmid with no ferroxidase activity. Eight weeks later, LRS-injected mice had ~40% microvascular stasis (% non-flowing venules) when infused with stroma-free hemoglobin at 1 h, while mice overexpressing wt-hFHC had only 5% stasis (p< 0.05), and ms-hFHC mice had 33% stasis suggesting vascular protection by ferroxidase active wt-hFHC. The wt-hFHC SCD mice had marked increases in splenic hFHC mRNA and hepatic hFHC protein, light chain ferritin, 5-aminolevulinic acid synthase (5-ALA-synthase), heme content, ferroportin, nuclear factor erythroid 2-related factor 2 (Nrf2), nuclear hFHC, and microsomal HO-1 activity and protein, and a decrease in activated nuclear phosho-nuclear factor-kappa B (NF-κB) p65. HO-1 activity was not essential for the protection by FHC. We conclude that wt-hFHC ferroxidase activity enhances cytoprotective Nrf2-regulated proteins including HO-1, thereby resulting in decreased NF-κB-activation, inflammation and microvascular stasis in transgenic SCD mice

Blood outgrowth endothelial cell migration and trapping in vivo: a window into gene therapy

Human blood outgrowth endothelial cells (hBOEC) may be useful delivery-cells for gene therapy. hBOEC have high expansion capacity and stable phenotype. If incorporated into blood vessels, hBOEC could release therapeutic agents directly into the blood stream. However, little is known about lodging and homing of hBOEC in vivo. We examined the homing patterns of hBOEC in mice, and explored extending cell-based FVIII gene therapy from mice to larger animals. hBOEC were injected into NOD/SCID mice to determine where they localize, how localization changes over time and if there were toxic effects on host organs. The presence of hBOEC in mouse organs was determined by qPCR and immunofluorescence microscopy. hBOEC lodged most notably in mouse lungs at 3 h, but by 24 h there were no differences between 9 organs. hBOEC longevity was assessed up to 7 months in vivo. hBOEC expanded well and then plateaued in vivo. hBOEC from older cultures expanded equally well in vivo as younger. hBOEC caused no noticeable organ toxicity up to 3 days post-injection. When mice were pretreated with antibodies to E-selectin, P-selectin or anti-α4 integrin prior to hBOEC injection, the number of hBOEC in lungs at 3h was inhibited. Preliminary studies infusing hemophilic dogs with autologous canine BOEC over-expressing FVIII (B-domain deleted) showed improvement in whole blood clotting times (WBCT). In conclusion, the survivability, expandability and lack of toxicity of BOEC in vivo indicate that they may be valuable host cells for gene therapy

Erythroid-Specific Expression of β-globin from Sleeping Beauty-Transduced Human Hematopoietic Progenitor Cells

Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34+ cells with DsRed and a hybrid IHK–β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p = 0.05), indicating expression of β-globin from the integrated SB transgene. IHK–β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK–β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK–β-globin transgene for gene therapy of sickle cell disease