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
- 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
Production and Metabolism of Reactive
Oxygen Species (ROS)
.OH
ONOO
CAT
+
NO
O2
O2
•Mitochondria
• Endoplasmic reticulum
•Cyclooxygenase
•Lipooxygenase
•Uncoupled NOS
•NAD(P)H Oxidase
•Xanthine Oxidase
•Cytochrome P-450
2+
SOD
H 2O 2
Cl, MPO
e-
.
H2O + O2
.
OH
.
O2
GPX
HOCl
O2+ 4e (H)
Fe
2H2O
H2O + GSSG
Oxidative Stress
Oxidative Stress is a condition in which
production of reactive oxidative species (ROS)
exceeds the capacity of the antioxidant system
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.
Mechanisms of Oxidative Stress in CKD
• A- Increased production of reactive oxygen
species (ROS)
• B- Impaired antioxidant defense system
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)
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
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).
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).
Up-regulation of NAD(P)H oxidase in the remnant kidney
Relative Protein Abundance
Up-regulation of Cyclooxygenase & lipoxygenase in remnant kidney
*
160
140
120
100
80
60
40
20
0
CTL
1
CRF
Relative Protein Abundance
Cox-2
*
1400
1200
1000
800
600
400
200
0
Relative Protein Abundance
CTL
1
CRF
*
3000
2500
12/15 Lipooxygenase
2000
1500
1000
500
0
CTL
1
Cox-1
CRF
Increased ROS production by circulating
granulocyte in ESRD patients
Mechanisms of Oxidative Stress in CKD
• A- Increased production of reactive oxygen
species (ROS)
• B- Impaired antioxidant defense system
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)
Adaptive response to oxidative stress
• Under normal condition, disruption of redox
equilibrium by environmental or internal prooxidants 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.
Nrf2/ARE pathway
Reactive Oxygen Species (ROS)
Actin
Keap1
Nrf2
Dissociation
Nrf2
P
Nrf2
P
Cytoplasm
Small
Maf
Nucleus
Activation
Small
Maf
Nrf2
ARE
P
Antioxidant proteins
(e.g. GSTs, HO1)
Impaired Nrf2 Activity in CRF kidney
(A) 6 weeks
Nrf2
Keap1
Histone H1
b-actin
2.0
1.2
Relative optical density
Relative optical density
1.4
1.0
0.8
0.6
0.4
0.2
0.0
1.2
0.8
0.4
0.0
CTL
CTL
CRF
Keap1
Histone H1
b-actin
1.2
3.0
1.0
0.8
0.6
***
0.4
0.2
Relative optical density
Nrf2
Relative optical density
(B) 12 weeks
*
1.6
CRF
*
2.5
2.0
1.5
1.0
0.5
0.0
0.0
CTL
CRF
CTL
CRF
Kim HJ, Vaziri ND. Am J Physiol Renal Physiol. 2010 Mar;298(3):F662-71.
Down-regulation of Nrf2 target gene products at 12 weeks
HO-1
NQO1
b-actin
b-actin
1.4
1.2
1.0
0.8
0.6
*
0.4
0.2
Relative optical density
Relative optical density
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.0
CTL
CTL
CRF
GCLC
GCLM
b-actin
b-actin
1.2
1.0
0.8
**
0.6
0.4
0.2
0.0
CTL
CRF
Relative optical density
Relative optical density
*
CRF
1.2
*
1.0
0.8
0.6
0.4
0.2
0.0
CTL
CRF
Kim HJ, Vaziri ND. Am J Physiol Renal Physiol. 2010
Nrf2 target gene products at 12 weeks
Cu,Zn-SOD
Mn-SOD
EC-SOD
b-actin
b-actin
b-actin
1.2
**
0.6
0.4
0.2
1.4
Relative optical density
0.8
Relative optical density
1.0
1.0
0.8
0.6
**
0.4
0.2
CTL
CTL
CRF
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.0
0.0
CTL
CRF
Catalase
Gpx
b-actin
b-actin
1.0
*
0.8
0.6
0.4
0.2
Relative optical density
1.2
1.2
Relative optical density
Relative optical density
1.2
1.0
*
0.8
0.6
0.4
0.2
0.0
0.0
CTL
CRF
CTL
Kim HJ, Vaziri ND. Am J Physiol Renal Physiol. 2010
CRF
CRF
Role of HDL deficiency & dysfunction in
CKD-associated oxidative stress
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)
HDL- mediated Reverse Cholesterol Transport
& Anti-oxidant/anti-inflammatory actions
Mature HDL
Nascent HDL
LCAT
HDL2
ABCA1
HDL3
FC
FC
CE
CE
Macrophage
SRA1
LOX1
CD36
SR-B1
Ox-LDL
CE
Liver
PON
B chain ATP
Synthase
GPX
ROS
LCAT
ApoA1
Bile
LDL
HDL
FC
ApoB100
HDL Cholesterol
ApoA-I
Paraoxonase activity
Glutathione peroxidase
Activity
Concentration
HDL Antioxidant Activity
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)
Markers of oxidative stress in CKD
**
3.0
2.0
1.0
6
Reduced GSH/GSSG ratio
Plasma MDA (nmol/mL)
4.0
0.0
4
3
*
2
1
0
CTL
CRF
CTL
1.6
0.7
0.6
0.5
0.4
0.3
0.2
0.1
CRF
**
*
Mitochondrial TBARS
(nmol/mg protein)
Kidney tissue TBARS
(nmol/mg protein)
5
1.2
0.8
0.4
0.0
0.0
CTL
CRF
CTL
CRF
O2- +NOONOO- (peroxynitrite)
ONOO- + Tyrosine  nitrotyrosine
Urine 8-hydroxy-2'-deoxyguanosine excretion
8-oxo-dG (ng/24 hr)
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
CTL
CRF
Protein Carbonyl
Summary
• ROS production is markedly increased in the
diseased kidney
• Increased ROS production is accompanied by
impaired Nrf2 activation and consequent downregulation of the antioxidant & cytoprotective
molecules
• Studies are underway to explore the effect of a
potent Nrf2 activator in CKD
Part 2- inflammation in CKD
Inflammation is invariably present in CKD
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)
NFkB Activation
NFkB activation
PAI-1
MCP1
1200
1000
800
600
400
200
3000
2500
2000
1500
1000
500
0
0
CTL
1
CRF
CTL
1
CRF
Relative Protein Abundance
Relative Protein Abundance
Relative Protein Abundance
Phospho-IkB
1800
1600
1400
1200
1000
800
600
400
200
0
CTL
1
CRF
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
Role of the intestinal tract in the
pathogenesis of inflammation
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.
Trans-cellular and paracellular
epithelial barriers
Intestinal epithelial barrier structure
Trans-cellular, cytosolic plaque, & actomyosin ring in TJ assembly
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)
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
Descending colon
Ascending colon
Depletion of colonic tight junction proteins in uremia
Vaziri et al. Nephrol Dial Transplant. 2012 Jul;27(7):2686-93
Comparison of TJ protein expression between control rats and
rats with CRF induced by 5/6 nephrectomy
Adenine induced-CKD model
Comparison of TJ protein expression between control rats and
rats with CRF induced by adenine
10
Relative Occludin
mRNA expression
Descending colon
5
Relative Claud-1
mRNA expression
5
CTL
0
CTL
CRF
Relative Claud-1
mRNA expression
*
0
CRF
4
Relative Z01
mRNA expression
Ascending colon
Relative Occludin
mRNA expression
10
5
0
Relative Z01
mRNA expression
10
6
CTL
0
CTL
CRF
**
4
2
CRF
0
CTL
CTL
Comparison of TJ protein mRNA expression between control
and CRF rats
Figure 5
CRF
7
4
2
0
CRF
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
Role of lipoprotein abnormalities
Increased LDL pro-inflammatory activity and
loss of HDL anti-inflammatory activity in
ESRD
3.0
LDL
Inflammatory Index
2.5
p=0.003
____
2.0
1.5
1.0
0.5
0
Normal
LDL
Uremic
LDL
ESRD patients’ LDL is highly pro-inflammatory
HDL Anti-Inflammatory Index
-4.0
p=0.001
-3.5
-3.0
-2.5
-2.0
-1.5
1.0
0.5
0
LDL +
Normal HDL
LDL+
uremic HDL
ESRD patients’ HDL is actually pro-inflammatory
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
JPET # 175828
J Pharmacol Exp Ther 2011
Jun;337(3):583-90.
JPET # 175828
J Pharmacol Exp Ther 2011 Jun;337(3):583-90.
JPET # 175828
JPET # 175828
JPET # 175828
JPET # 175828
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
Acknowledgements
UCI
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
A. Ehdai,
L. Sepassi
Dr K. Liang H.J. Kim
Dt J Yuan
Dr Subramanian
Dr Aminzadeh N. Goshtasbi
Venezuela
Dr B. Rodriguez-Iturbe
Dr Y. Quiroz
Dr M. Nava
UT Southwestern
Dr. J. Zhou
Korea
Dr. JR Koo
Dr. CS Lim
Dr JW Yoon
UCLA
Dr M. Navab
Thank you