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N. D. Vaziri M.D., MACP

Oxidative Stress and Inflammation in Chronic Kidney Disease: The Nature, Mechanisms, Consequences and Treatment. N. D. Vaziri M.D., MACP Division of Nephrology and Hypertension University of California Irvine, Irvine. Part 1- Oxidative Stress in CKD.

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N. D. Vaziri M.D., MACP

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  1. Oxidative Stress and Inflammation in Chronic Kidney Disease: The Nature, Mechanisms, Consequences and Treatment N. D. Vaziri M.D., MACP Division of Nephrology and Hypertension University of California Irvine, Irvine

  2. Part 1- Oxidative Stress in CKD - Oxidative stress is a constant feature of CKD - It is both a cause and a consequence of inflammation - Together oxidative stress & inflammation contribute to development & progression of CKD and the associated complications including atherosclerosis, CVD, EPO-resistant anemia, immune deficiency, cachexia, among others

  3. Production and Metabolism of Reactive Oxygen Species (ROS) .OH H2O + O2 ONOO CAT NO . + 2+ Fe SOD . H2O2 O2 O2 . OH e- O2 Cl, MPO • Mitochondria • Endoplasmic reticulum • Cyclooxygenase • Lipooxygenase • Uncoupled NOS • NAD(P)H Oxidase • Xanthine Oxidase • Cytochrome P-450 GPX H2O + GSSG HOCl O2+ 4e (H) 2H2O

  4. Oxidative Stress Oxidative Stress is a condition in which production of reactive oxidative species (ROS) exceeds the capacity of the antioxidant system

  5. Biochemical Consequences of Oxidative Stress In presence of oxidative stress, the uncontained ROS cause tissue damage/dysfunction by: • Directly attacking , denaturing &modifying structural and functional molecules (e.g. lipids, proteins, carbohydrates, DNA, RNA, NO, etc.) • Modulating activities of the redox-sensitive transcription factors (e.g. NFκB, AP-1) and signal transduction pathways (Activation of protein kinases e.g. ERK, P53 & ASK1, Ca ATPase release channels), thereby promoting inflammation, ER stress, fibrosis, apoptosis etc.

  6. Mechanisms of Oxidative Stress in CKD • A- Increased production of reactive oxygen species (ROS) • B- Impaired antioxidant defense system

  7. Factors Contributing to increased ROS Production & dissemination of oxidative stress • Activation of tissue angiotensin system • Hypertension • Inflammation • Uremic toxins (endogenous; exogenous) • Mitochondrial dysfunction • Accumulation of oxidation-prone lipoprotein remnants • Underlying conditions (e.g. diabetes, autoimmune diseases) • Increased tissue iron load (Fe shift, blood transfusion, excess IV Fe use) • Iatrogenic causes (blood/dialyzer interaction, dialysate impurities, excessive use of IV Fe, rejected transplant kidney, reaction to failed AV grafts)

  8. A- Sources/mechanisms of excess ROS production in CKD • Up-regulation/activation of ROS-producing enzymes (e.g.NAD(P)H oxidase, cyclooxygenase, lipoxygenase, etc) • Uncoupling of NO synthase (via monomerization of eNOS, depletion of tetrahydrobiopterin [BH4], accumulation of ADMA ) • Impairment of mitochondrial electron transport chain • Activation of leukocytes and resident cells • Dissemination of oxidative stress by circulating oxidized LDL & phospholipids via oxidation chain reaction

  9. NAD(P)H Oxidase The major source of ROS production in endothelial cells (NOX-II or gp91 phox ), VSMC (NOX-I and NOX-IV) and renal parenchymal cells (NOX-IV or Renox). - * NAD(P)H oxidase activation involves assembly of enzyme’s membrane-associated subunits (NOXs and p22) with cytosolic subunits (p47, p67 and rac-1).

  10. NAD(P)H oxidase is the major source of superoxide (O2-) in the kidney & vessel wall NOX-1: vascular smooth muscle cells NOX-3: colon NOX-4: renal cortex Subunits of NADPH oxidase NAD(P)H oxidase activation involves assembly of enzyme’s membrane-associated subunits (NOXs and p22) with cytosolic subunits (p47, p67 and rac-1).

  11. Up-regulation of NAD(P)H oxidase in the remnant kidney

  12. Up-regulation of Cyclooxygenase & lipoxygenase in remnant kidney * * Cox-2 * 12/15 Lipooxygenase Cox-1

  13. Increased ROS production by circulating granulocyte in ESRD patients

  14. Mechanisms of Oxidative Stress in CKD • A- Increased production of reactive oxygen species (ROS) • B- Impaired antioxidant defense system

  15. B- Factors contributing to Antioxidant Depletion Reduced Production of endogenous antioxidants (antioxidant enzymes, GSH, ApoA1, Albumin, LCAT, Melatonin, etc) Impaired activation of Nrf2 (the master-regulator of genes encoding antioxidant/detoxification molecules) Depletion of antioxidant molecules by ROS Diminished antioxidant activity of HDL Reduced intake of fresh fruits and vegetables (K restriction) Removal of water-soluble antioxidants by dialysis Anemia: (↓RBC antioxidants: GSH, GPX, PAF-AH, Phospholipids)

  16. Adaptive response to oxidative stress • Under normal condition, disruption of redox equilibrium by environmental or internal pro-oxidants triggers an adaptive response which results in up-regulation of antioxidant and cytoprotective enzymes and proteins. • In mammals, nuclear factor-erythroid 2 p45-related factors 1 & 2 (Nrf2) regulates constitutive expression & orchestrates transcriptional up-regulation of genes encoding these cytoprotective molecules.

  17. Nrf2/ARE pathway Reactive Oxygen Species (ROS) Actin Keap1 P P P Nrf2 Dissociation Nrf2 Cytoplasm Nucleus Activation Antioxidant proteins (e.g. GSTs, HO1) Nrf2 Nrf2 Small Maf Small Maf ARE

  18. (A) 6 weeks 2.0 1.2 3.0 1.4 1.6 1.0 2.5 1.2 1.2 0.8 2.0 1.0 Relative optical density 0.8 0.8 1.5 0.6 0.6 1.0 0.4 0.4 0.4 0.5 0.2 0.0 0.2 0.0 0.0 0.0 CTL CRF (B) 12 weeks Nrf2 Histone H1 Impaired Nrf2 Activity in CRF kidney Keap1 b-actin * Relative optical density CTL CRF Keap1 Nrf2 b-actin Histone H1 * Relative optical density Relative optical density *** CTL CRF CTL CRF Kim HJ, Vaziri ND. Am J Physiol Renal Physiol. 2010 Mar;298(3):F662-71.

  19. 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1.4 1.2 1.2 1.0 1.2 1.0 0.8 1.0 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0.0 0.0 0.0 Down-regulation of Nrf2 target gene products at 12 weeks NQO1 HO-1 b-actin b-actin * Relative optical density Relative optical density * CTL CRF CTL CRF GCLC GCLM b-actin b-actin * ** Relative optical density Relative optical density CTL CRF CTL CRF Kim HJ, Vaziri ND. Am J Physiol Renal Physiol. 2010

  20. 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1.2 1.2 1.0 1.0 0.8 0.8 0.6 0.6 1.2 0.4 0.4 1.0 0.2 0.2 0.8 0.0 0.0 0.6 0.4 0.2 0.0 Nrf2 target gene products at 12 weeks EC-SOD Cu,Zn-SOD Mn-SOD b-actin b-actin b-actin 1.2 1.0 0.8 ** Relative optical density 0.6 Relative optical density Relative optical density ** 0.4 0.2 0.0 CTL CRF CTL CRF CTL CRF Catalase Gpx b-actin b-actin * * Relative optical density Relative optical density CTL CRF CTL CRF Kim HJ, Vaziri ND. Am J Physiol Renal Physiol. 2010

  21. Role of HDL deficiency & dysfunction in CKD-associated oxidative stress

  22. Anti-oxidant/Anti-atherogenic Actions of HDL A- Reverse cholesterol - lipid transport B- EC migration & endothelial repair (via SRB-1) C- Antioxidant/anti-inflammatory actions a. ApoA-Imediated extraction of oxidized phospholipids from lipoproteins and cell membrane b. LCAT-mediated hydrolysis of proinflammatory oxidized phospholipids (AA at sn-2) c. Prevention of LDL oxidation and destruction of oxidized phospholipids by paraoxonase-1 & glutathione peroxidase (GPX) D- Inactivation of PAF and PAF-like phospholipids by PAF acetyl hydrolase(anti-inflammatory / anti-thrombotic)

  23. HDL- mediated Reverse Cholesterol Transport & Anti-oxidant/anti-inflammatory actions Mature HDL Nascent HDL LCAT ABCA1 HDL2 HDL3 FC CE FC CE Macrophage SRA1 LOX1 CD36 SR-B1 ApoB100 Ox-LDL FC CE PON GPX LCAT ApoA1 Liver HDL B chain ATP Synthase ROS LDL Bile Bile

  24. HDL Cholesterol ApoA-I

  25. Paraoxonase activity Glutathione peroxidase Activity Concentration

  26. HDL Antioxidant Activity

  27. Biomarkers of oxidative stress byproducts of ROS interaction with bio-molecules • Elevated plasma & tissue MDA • Elevated plasma, urine & tissue F2 isoprostane • Elevated plasma & tissue nitrotyrosine (NO oxidation) • Increased Protein carbonyls & oxidized thiols • Increased plasma & urine oxidized nucleic acids • Elevated plasma and tissue advanced glycoxidation end products (AGE)

  28. 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 1.6 1.2 0.8 0.4 0.0 Markers of oxidative stress in CKD 6 4.0 ** 5 3.0 4 3 2.0 Plasma MDA (nmol/mL) Reduced GSH/GSSG ratio * 2 1.0 1 0.0 0 CTL CRF CTL CRF ** * Mitochondrial TBARS (nmol/mg protein) Kidney tissue TBARS (nmol/mg protein) CTL CRF CTL CRF

  29. O2- +NOONOO- (peroxynitrite) ONOO- + Tyrosine  nitrotyrosine Protein Carbonyl

  30. Summary ROS production is markedly increased in the diseased kidney Increased ROS production is accompanied by impaired Nrf2 activation and consequent down-regulation of the antioxidant & cytoprotective molecules Studies are underway to explore the effect of a potent Nrf2 activator in CKD

  31. Part 2- inflammation in CKD Inflammation is invariably present in CKD

  32. Link Between Oxidative Stress and Inflammation Oxidative Stress NFκB Activation Antioxidant Depletion Ox LDL AGE Ox PL ↑ ROS Production Cytokines / Chemokines Leukocyte/Macrophage Activation (Inflammation)

  33. NFkB Activation

  34. PAI-1 NFkB activation MCP1 Phospho-IkB

  35. Causes of CKD-associated inflammation Oxidative stress Retained uremic metabolites & exogenous toxins - Co-morbid conditions (e.g. diabetes and autoimmune diseases) - Infections (blood access, PD catheters, hepatitis etc) - Iron overload Hypervolemia / Hypertension Increased pro-inflammatory properties of LDL Impaired anti-inflammatory properties of HDL Influx of impurities from dialysate compartment Complement/leukocyte activation by dialyzer/pump - Influx of pro-inflammatory products from the GI tract

  36. Role of the intestinal tract in the pathogenesis of inflammation

  37. Intestine and its barrier function Although anatomically situated in the most central region of the body, the GI tract is actually an extension of the external environment within the organism. The primary functions of the intestine include: absorption of nutrients; secretion of waste products; & serving as a barrier to prevent influx of microbes, harmful microbial byproducts and other noxious compounds into the host’s internal milieu.

  38. Trans-cellular and paracellularepithelial barriers Intestinal epithelial barrier structure

  39. Trans-cellular, cytosolic plaque, & acto-myosin ring in TJ assembly

  40. Evidence of the intestinal barrier dysfunction in uremia Presence of endotoxemia in uremic patients without detectable infection and its contribution to the prevailing systemic inflammation (Gonçalves et al, 2006; Szeto et al, 2008) Increased intestinal permeability to high MW PEGs in the uremic humans and animals (Magnusson et al, 1990,1991) Detection of luminal bacteria in mesenteric lymph nodes of the uremic animals (de Almeida Duarte et al 2004 ) Diffuse inflammation throughout the GI tract (esophagitis, gastritis, duodenitis, enteritis, colitis) in ESRD patients maintained on dialysis (Vaziri et al 1985)

  41. Hypothesis In view of the evidence for increased intestinal permeability in the uremic humans & animals and the critical role of the epithelial tight junction in the mucosal barrier function, I hypothesized that uremia may result in disruption of the intestinal tight junction complex

  42. Depletion of colonic tight junction proteins in uremia Ascending colon Descending colon Vaziri et al. Nephrol Dial Transplant. 2012 Jul;27(7):2686-93

  43. Comparison of TJ protein expression between control rats and rats with CRF induced by 5/6 nephrectomy

  44. Adenine induced-CKD model Comparison of TJ protein expression between control rats and rats with CRF induced by adenine

  45. Comparison of TJ protein mRNA expression between control and CRF rats

  46. Conclusions of the TJ studies - Uremia results in disintegration of the intestinal epithelial tight junction complex - This phenomenon can contribute to the systemic inflammation and account for the previously-demonstrated evidence of defective intestinal barrier function in humans and animals with advanced CKD

  47. Role of lipoprotein abnormalities Increased LDL pro-inflammatory activity and loss of HDL anti-inflammatory activity in ESRD

  48. 3.0 2.5 2.0 1.5 1.0 0.5 0 LDL p=0.003 ____ Inflammatory Index Normal LDL Uremic LDL ESRD patients’ LDL is highly pro-inflammatory

  49. -4.0 p=0.001 -3.5 -3.0 -2.5 HDL Anti-Inflammatory Index -2.0 -1.5 1.0 0.5 LDL + Normal HDL 0 LDL+ uremic HDL ESRD patients’ HDL is actually pro-inflammatory

  50. Treatment of CKD-associated oxidative stress • - All conventional therapies with proven efficacy in retarding CKD progression (i.e. RAS blockade , Glycemia & HTN control) reduce oxidative stress and inflammation • - Treatment with high doses of anti-oxidant vitamins are generally ineffective and may actually increase the risk of CVD and other complication • Experimental therapies currently in clinical trial : • I- AST-120, a specially formulated activated charcoal which limits absorption of the pro-oxidant gut–derived uremic toxins • II- The Nrf2 activator, Bardoxolone, which can lower oxidative stress and inflammation by raising expression of endogenous antioxidant enzymes and related molecules

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