Oxidative Stress and Inflammation in Chronic Kidney Disease: The Nature, Mechanisms, Consequences and Treatment

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

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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. NF?B, 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

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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)

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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

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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 enzymes membrane-associated subunits (NOXs
and p22) with cytosolic subunits (p47, p67 and
rac-1).
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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 enzymes membrane-associated subunits
(NOXs and p22) with cytosolic subunits (p47, p67
and rac-1).
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Up-regulation of NAD(P)H oxidase in the remnant
kidney
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Up-regulation of Cyclooxygenase lipoxygenase in
remnant kidney


Cox-2

12/15 Lipooxygenase

Cox-1
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Increased ROS production by circulating
granulocyte in ESRD patients
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Mechanisms of Oxidative Stress in CKD

• A- Increased production of reactive oxygen
  species (ROS)
• B- Impaired antioxidant defense system

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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)

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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.

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Nrf2/ARE pathway
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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 Mar298(3)F662-71.
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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
Nrf2 target gene products at 12 weeks
EC-SOD
Mn-SOD
Cu,Zn-SOD
b-actin
b-actin
b-actin
1.2
1.0
0.8

0.6
Relative optical density
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
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Role of HDL deficiency dysfunction in
CKD-associated oxidative stress
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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-I mediated 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)

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HDL- mediated Reverse Cholesterol Transport
Anti-oxidant/anti-inflammatory actions
Mature HDL
Nascent HDL
LCAT
ABCA1
HDL2
FC CE
HDL3
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
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HDL Cholesterol
ApoA-I
25
Paraoxonase activity
Glutathione peroxidase
Activity
Concentration
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HDL Antioxidant Activity
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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)

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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
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O2- NO?ONOO- (peroxynitrite) ONOO- Tyrosine
? nitrotyrosine
Protein Carbonyl
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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

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Part 2- inflammation in CKD

• Inflammation is invariably present in CKD

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Link Between Oxidative Stress and Inflammation
Oxidative Stress
NF?B Activation
Antioxidant Depletion
Ox LDL AGE Ox PL
? ROS Production
Cytokines / Chemokines
Leukocyte/Macrophage Activation (Inflammation)
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NFkB Activation
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PAI-1
NFkB activation
MCP1
Phospho-IkB
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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

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Role of the intestinal tract in the pathogenesis
of inflammation
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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 hosts
  internal milieu.

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Trans-cellular and paracellularepithelial
barriers
Intestinal epithelial barrier structure
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Trans-cellular, cytosolic plaque, acto-myosin
ring in TJ assembly
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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)

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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

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Depletion of colonic tight junction proteins in
uremia
Ascending colon
Descending colon
Vaziri et al. Nephrol Dial Transplant. 2012
Jul27(7)2686-93
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Comparison of TJ protein expression between
control rats and rats with CRF induced by 5/6
nephrectomy
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Adenine induced-CKD model
Comparison of TJ protein expression between
control rats and rats with CRF induced by adenine
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Comparison of TJ protein mRNA expression between
control and CRF rats
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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

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Role of lipoprotein abnormalities Increased
LDL pro-inflammatory activity and loss of HDL
anti-inflammatory activity in ESRD
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LDL
p0.003
____
Inflammatory Index
Normal LDL
Uremic LDL
ESRD patients LDL is highly pro-inflammatory
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-4.0
p0.001
-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
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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 gutderived 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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JPET 175828
J Pharmacol Exp Ther 2011 Jun337(3)583-90.
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JPET 175828
J Pharmacol Exp Ther 2011 Jun337(3)583-90.
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JPET 175828
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JPET 175828
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JPET 175828
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JPET 175828
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Conclusions

• CKD results in a vicious cycle of oxidative
  stress, inflammation and ER stress which work in
  concert to drive deterioration of kidney function
  and structure and contribute to the development
  and progression of CVD many other complications

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Acknowledgements
Venezuela
UCI
Dr B. Rodriguez-Iturbe Dr Y. Quiroz Dr M. Nava

• Dr Z. Ni, Dr Y. Bai
• Dr Y. Ding, Dr XQ Wang
• Dr DC Zhan Dr R. Sindhu
• Dr C. Barton Dr J. Zhou
• Dr M. Dicus Dr N. Ho
• Dr CY Lin Dr Z. Li
• F. Oveisi, F. Farbod
• Ehdai, L. Sepassi
• Dr K. Liang H.J. Kim
• Dt J Yuan Dr Subramanian
• Dr Aminzadeh N. Goshtasbi

UT Southwestern
Dr. J. Zhou
Korea
Dr. JR Koo Dr. CS Lim Dr JW Yoon
UCLA
Dr M. Navab
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• Thank you