2013 rice university regional undergraduate symposium presentation

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  • Minyi Chen

    Dr. David Sheikh-Hamads LabStanniocalcin-1 protects ischemia/reperfusion kidney injury

    *

  • BACKGROUNDIn humans, acute kidney injury is highly prevalent; and the associated mortality rate remains high (up to 50%).Ischemia/reperfusion (I/R) kidney injury, is a major cause of renal failure in both native and transplanted organs.Mouse/rat experimental I/R kidney injury is a model for human I/R kidney injury. It is associated with phenomenon:decreased urine output and Glomerular Filtration Rate(GFR);inflammation;tubular cell injury, characterized by vacuolization, cell necrosis, and hyaline cast formation; full recovery is complete within 8 days.

    *

  • Mechanism of free radical generation in ischemia reperfusion injuryBulkey, G. B. Br. J. cancer, 1987

    The mechanism of free radical generation in ischemia reperfusion injury shows in this figure.With the onset of ischemia, there is rapid proteolytic conversion ofxanthine dehydrogenase to xanthine oxidase which induce oxygen to generate superoxide free radical and hydrogen peroxide, all of these species can cause cellular and tissues injury*

  • STANNIOCALCIN-1 (STC1) EFFECTS ON: ROS; ENDOTHELIAL AND MACROPHAGE FUNCTIONSTC1 decreases superoxide generation in macrophages, via UCP2-depenent pathway (Wang et al., J Leukoc Biol. 2009 86:981-8).STC1 inhibits macrophage migration and response to chemoattractants (Kanellis, J. et al. Am J Physiol Renal Physiol, 2004, 286: F356-F362).STC1 blocks Tumour Necrosis Factor(TNF)--induced rise in endothelial monolayer permeability and decreases TNF--induced activation of Jun N-terminal Kinase(JNK), NF- B, and superoxide in endothelial cells (Chen, C. et al. Arterioscler Thromb Vasc Biol 2008; 28:906-912).STC1 inhibits T-cell and macrophage transmigration across IL-1-treated endothelial monolayer (Chakraborty et al., Am J Physiol Renal Physiol. 2007;292:F895-904).

    In our previous research we found that (read the slide)*

  • WT STC1 Superoxide generation in STC1 Tg and WT kidney under normal condition without clamping Labeling with 5 uM MitoSOX Red

    This slide showed freshly isolated kidneys were sectioned andincubated in PBS containing 5 M MitoSOX Red reagent for 10 min. MitoSOXpermeates live cells and selectively targets the mitochondria. It is rapidly oxidized bysuperoxide and emits red fluorescence. kidney sliceswere rinsed in PBS, fixed in 4% paraformaldehyde, imbedded in OCT and 5 M sectionswere viewed under fluorescence microscope. The panel showed less superoxide generaation in STC1 Tg mice compare with Wild Type mice.*

  • HYPOTHESIS Stanniocalcin-1 protects from I/R kidney injury by modulating key factors in the pathogenesis of I/R. These include:Decreased Reactive Oxygen Species(ROS) generation;Endothelial protection preservation of normal endothelial permeability - decreasing trans-endothelial migration of macromolecules and inflammatory cells (macrophages and T-cells), into the kidney;Direct inhibition of macrophages.

    *Base on our previous research and reports, we hypothesis (read the slide)

  • Decline in Creatinine clearance (CrCl) after I/R kidney injury in WT mice; but preservation of normal CrCl in STC1 Tg miceReduce of kidney function indicated by Glomerular Filtration Rate(GFR);

    First, we established mouse model of kidney I/R injury induced by clamping of bilateral renal pedicles for 30 min, Micewere sacrificed at 24h, 48h, 72h or 8d after surgical procedure, blood samples were obtainedfor creatinine measurement and kidneys were harvested for analysisof: histology; immunohistochemistry; Western blotting to assess the activities of MAPKsand STC1 protein expression; superoxide and H2O2. As this figure showed decreased creatinine clearence after kidney injury in wild type mice but not in STC1 transgenic mice.*

  • Kidney morphology in WT and STC1 Tg mice after I/R kidney injuryloss of brush border membrane (arrowheads), cellular vacuolization (v),tubular dilatation (dt) with cast formation (c)

    Kidney tissues were harvested at 24h, 48h, 72h or 8d following I/R and kidneys, and PAS staining was performed for morphology, as the up panel showed WT micedisplayed loss of brush border membrane (arrowheads), severe cellular vacuolization (v) andtubular dilatation (dt) with cast formation (c), starting 24h after I/R, persisting through the72h time point. Recovery of morphology in WT kidneys is observed by day 8, but low panel showed no evidence of cellular vacuolization, tubular dilatation or castFormation in STC1 Tg mice.*

  • Preservation of normal vascular permeability in kidneys of STC1 Tg mice after I/R injury compared to WT mice

    As the morphology showing, inflammatory cells were infiltrated in kidney, therefore we determine whether vascular permeability increased after ischemia reperfusion. Mice were given an injection of 2% Evans blue dye through the tail vein after Five hours after I/R, and then mice were anesthetized five min after dye injection, and kidneys were perfusedwith PBS for 20 minutes. Kidneys were then homogenized and dye retention was measured fluorometrically. As the figure showed preservation of normal vascular permeability in kidneys of STC1 Tg mice after I/R injury compared to WT mice. *

  • Increased T-cells and macrophages in kidney of WT mice after I/R injury; but no change is observed in kidneys of STC1 Tg mice

    The tissues were stained with anti-CD3 which is T-cells marker and anti-F4/80 which is macrophages marker. CD3 or F4/80 positively-stainedcells infiltrating 10 grids were counted. This figure showed increased T-cells and macrophages in kidney of WT mice after I/R injury; but no change is observed in kidneys of STC1 Tg mice. *

  • Increased superoxide and hydroperoxide in WT kidneys 24h after I/R injury; but not in kidneys of STC1 Tg mice

    Because reactive oxygen species play very important role in ischemia reperfusion injury, therefore we analyzed superoxide and H2O2 after ischemia reperfusion injury using semi-quantitative methods. The figure showed marked elevation of both superoxide andH2O2 in WT kidneys after I/R, but not in STC1 Tg kidneys.*

  • STC1 protects from I/R kidney injury through suppression of oxidant stressSTC1 Tg mice were given a single i.p. injection of saline or paraquat (12.5 mg/kg). qaraquat is the generator of superoxide. I/R mice were clamped 2h later than injection, and killed 24h, or 72h. (A) Superoxide, (B) hydroperoxide, (C) CrCl expressed , (D) PAS staining for morphology.

    To test whether STC1 protects from I/R kidney injury through suppression of oxidant stress, STC1 Tg mice were given a single i.p. injection of saline or paraquat (12.5 mg/kg) which is the generation of superoxide 2h beforeclamping, and killed 24h, or 72h following I/R. Superoxide (A), H2O2 (B), CrCl expressed(C) and PAS staining for morphology (D) were determined. In paraquat-pretreated STC1 Tg mice, I/R results in: increasedsuperoxide and H2O2 levels at the 24h through the 72h time points post I/R; drop in CrCl atthe 72h post I/R, and morphological changes similar to those observed in WT mice after I/R.*

  • WT mice displayDecline in CrCl (Glomerular Filtration Rate) at the 72h to 40% of sham-treated controls.Tubular injury (vacuolization, cell necrosis and cast formation).Increased vascular permeabilityInfiltration with macrophages and T-cells starting from day 1 through day 8.Increased superoxide and hydrogen peroxide generation (c/w increased risk for injury).STC1 Tg mice displayNo decrease of kidney function.No increase in superoxide and H2O2 production.No increase in vascular permeability.No increase in macrophage and T-cell infiltration after I/R. Conclusion: Tg overexpression of STC1 protects from I/R kidney injury; STC1 is a potential therapeutic target for the prevention/treatment of ischemia-related kidney injurySummary

    We summary *

  • ACKNOWLEDGEMENTS

    David Sheikh-Hamad, MD.Luping Huang, MD. PhD.Tatiana Belousova, MD.Jenny Pan, MD.Roohi Khan, MD.Yanlin Wang, MD. PhD. Luan Truong, MD. Funding support through the NIH

    I would like the thanks Dr. Hamad great support and instruction in this project.

    *

    *The mechanism of free radical generation in ischemia reperfusion injury shows in this figure.With the onset of ischemia, there is rapid proteolytic conversion ofxanthine dehydrogenase to xanthine oxidase which induce oxygen to generate superoxide free radical and hydrogen peroxide, all of these species can cause cellular and tissues injury*In our previous research we found that (read the slide)*This slide showed freshly isolated kidneys were sectioned andincubated in PBS containing 5 M MitoSOX Red reagent for 10 min. MitoSOXpermeates live cells and selectively targets the mitochondria. It is rapidly oxidized bysuperoxide and emits red fluorescence. kidney sliceswere rinsed in PBS, fixed in 4% paraformaldehyde, imbedded in OCT and 5 M sectionswere viewed under fluorescence microscope. The panel showed less superoxide generaation in STC1 Tg mice compare with Wild Type mice.**Base on our previous research and reports, we hypothesis (read the slide)First, we established mouse model of kidney I/R injury induced by clamping of bilateral renal pedicles for 30 min, Micewere sacrificed at 24h, 48h, 72h or 8d after surgical procedure, blood samples were obtainedfor creatinine measurement and kidneys were harvested for analysisof: histology; immunohistochemistry; Western blotting to assess the activities of MAPKsand STC1 protein expression; superoxide and H2O2. As this figure showed decreased creatinine clearence after kidney injury in wild type mice but not in STC1 transgenic mice.*Kidney tissues were harvested at 24h, 48h, 72h or 8d following I/R and kidneys, and PAS staini

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