Presentation Edgar Esparza
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Transcript of Presentation Edgar Esparza
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8/13/2019 Presentation Edgar Esparza
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NOSExpression in Patients with Lupus
Nephritis
Edgar Len Esparza Ibarra, MSc
La Paz. February 18, 2005
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O:O:
:
:
:
..
Background
The Earth was originally anoxic
Metabolism was anaerobic
O2started appearing ~2.5 x 109
years ago
Free radicals:
oAny species capable of independent existence
o A molecule with an unpaired electron
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ROS and RNS
NO. Nitric Oxide
NO2. Nitrogen dioxide
Free radicals:
O2.- Superoxide
.
OH Hydroxyl
RO2. Peroxyl
RO. Alkoxyl
HO2.
Hydroperoxyl
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o Radiation
o Chemicals that react to form peroxides
o Chemicals that promote superoxide formation
o Chemicals that are metabolized to radicals
o Chemicals that release iron
Exogenous sources of ROS and RNS
NO2+ Ultraviolet radiation --->NO+ O
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Endogenous sources of ROS and RNS
Mitochondria
Lysosomes
Peroxisomes
Endoplasmic Reticulum
Cytoplasm
Microsomal Oxidation,
Flavoproteins, CYP enzymes Myeloperoxidase(phagocytes)
Electron transport
Oxidases,Flavoproteins
Plasma Membrane
Lipoxygenases,
Prostaglandin synthase
NADPH oxidase
Xanthine Oxidase,
NOSisoforms
Fe
Cu
Transition
metals
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Short lived (concentrations can change rapidly)
Enzymatically generated in response to stimulant
Specific in action (?)
Can free radicals be second messengers?
COO-
C
(CH2)3
NH
C
H2N
H
NH2+
+H3N
L-Arginine
NOS
NADPH
+ O2
NADP+
COO-
C
(CH2)3
NH
C
H+H3N
N+
H2NH
OH
Hidroxyarginine
COO-
C
(CH2)3
NH
H+H3N
+ NO
NOS
C
O NH2
Citrulline
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What is Nitric Oxide?
N O
Discovered in 1772 by Joseph Priestley
Clear, colorless gas
Free radical
6-10 sec lifetime in vivo
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Dr. Louis J. IgnarroDr. Ferid Murad Dr. Robert F. Furchgott
Worthy of NobelPrize
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Agonists
NO signaling in physiology
Endothelial
Cell
Smooth
Muscle Cell
NOPGI2 EDHF
K+
Ca+
Nitric Oxide Synthase
NO
Binds to heme moiety ofguanylate cyclase
Conformational change of
the enzyme
Increased activity(production of cGMP)
Modulation of activity ofother proteins (protein
kinases, phospho-diesterases, ion channels)
Physiological response(relaxation of smoothmuscles, inhibition of
platelet aggregation, etc.)
O2- ONOO-
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What is the role of Nitric Oxide in the
human body?
NOin the nervous system
NOin the circulatory system
NOin the muscular system
NOin the immune system
NOin the digestive system
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Protective Deleterious
Regulatory
BIOLOGICAL EFFECTS OF NO
Second messenger role requires
low concentrations ofNO(10mM)
Formation of highly reactive peroxynitrite
Up-regulation
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Today Aplications
Inhale NO
Drugs vs impotence Viagra
(Sildenafil)
nitric oxide is released into the corpuscavernosum during sexual stimulation-
>cGMP mediated vasodilation and penis
engorgement. Sildenafil inhibits
phosphodiesterase type 5, which degrades
cGMP and leads to enhanced vasodilation
in erectile dysfunction.
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Properties of NOSIsozymes
Type I Type II Type III
Tissue in which
first described
Cerebellum
Neuronal
Immunologically
activatedMacrophages
Vascular
Endothelial Cells
Tissue basedterminology
nNOS iNOS eNOS
Expression Constitutive Inducible Constitutive
Intracellular freeCalcium
Regulates No Effect Regulates
Size 161KDa 131KDa 133KDa
Location ofGene
Chrom-12
12q24.1-12q24.31
Chrom-17
17q11.2
Chrom-7
7q35-7q36
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iNOS
NF kB-Ik B
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What is Lupus?
Rim Granular Homogeneous
Lupus (wolf)
erythematosus
(redness)
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Incidence of Lupus
5 % of children born to people
with lupus develop lupus
10 % of people with lupus have a close
relative who has or may develop lupus
2-3 times more common in African-Americans, Hispanics,Asians, and Native Americans
80 % of all cases develop between the ages of 15 and 44
90% of all cases occur in females
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Systemic Lupus Erythematous (SLE)
Leading cause of death for patients iskidney related called lupus nephritis
Minimal
Mild
Severe
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SLE
Signs of renal involvement
Changes in urinary output
Proteinuria
Hematuria
Fluid retention
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Anatomy of Kidney
1. Ascending Loopof Henle2. Descending
Loop of Henle3. Peritubular
capillaries
4. Proximal tubule5. Glomerulus6. Distal tubule
A. Renal veinB. Renal artery
C. UreterD. MedulaE. Renal PelvisF. Cortex
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Glomerulus
Distal tubule
Afferent arteriole
Efferent arterioleBowmans capsule
yuxta
glomerular cells
Mesangeal cells
Urinary space
Proximal tubule
Podocytes
Capillar
Macula
densa cells
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I: No lesion
II: Mesangial
III: Focal proliferative
IV: Diffuse proliferative
V: Membranous
VI: Sclerosis
Lupus Nephritis
Classification (WHO)*
* J. Am. Sec. Nephrol. 15:241-250. 2004.
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Know the expression of eNOS/iNOS in renal biopsieswith lupus nephritis.
Aim
NOS
N O
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Materials and methods
Biopsies
LES (n=20)
Control (n=10)
Hematoxiline
-eosine
Nephritis class
characterization
Cuts
Glomerular activity
Glomerular cronocity
TUNEL
(TdT)
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GAPDH primers:
5'-GAA CAT CAT CCC TGC CTC TAC TG-3 sense
5'-GTT GCT GTA GCC AAA TTC GTT G-3 antisense
Arteriosclerosis, Thrombosis, and Vascular Biology.
1997;17:3079-3082.
The Journal of Immunology, 2001,
167: 75-81.
Human eNOS primers:5'-CAGTGTCCAACATGCTGCTGGAAATTG-3'sense
5'-TAAAGGTCTTCTTCCTGGTGATGCC-3'antisense
Human iNOSprimers:
5'-GGCCTCGCTCTGGAAAGA-3 sense5'-TCCATGCAGACAACCTT-3'antisense
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RT-PCR
Materials and methods
Electrophoresis
(Photodocumentation)
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Results
11 from 20 lupus renal biopsieswere related to nephritis class IV, 6
to class III and 3 to class II (World
Health Organization).
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Hematoxiline-eosine
Karyorrhesis
TUNEL Assay Yodure propidium
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LES Control
eNOS
iNOS
GAPDH
All biopsies expressed the constitutive isoform eNOS
Only biopsies with lupus nephritis expressed the inducible isoformNOSin 74 %, and therefore, a major correlation in biopsies with higherindices of activity/cronocity was observed.
The control GAPDH was present in all biopsies.
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Our results suggest that the over-production of
iNOS isoform is involved in the
pathogenesis of lupus nephritis.
Conclusions and future work
Immunohistochemistry of NOS, p53 and
Hsp70
iRNA versus iNOS
Mouse model
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CIBNOR.Dr. Tania Zenteno Savn
Acknowledges
UANL.Cristina Rodrguez Padilla, PhD and
Pablo Zapata Benavides, PhD
UAZ.Jos Bollain y Goitia, Adrian Lpez, MSc
Ricardo Villalobos,Leonel Daza, MScEsperanza Avalos Daz, PhD and
Rafael Herrera Esparza*, PhD
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Proposed Mechanism of Disease
iNOS generates excess NO
NO is reacted with Super oxide via superoxide dismutase,
to generate the peroxy nitrile radical.
The radical initiates a necrotic event enhancing the
immune response.