Presentation Edgar Esparza

8/13/2019 Presentation Edgar Esparza

1/32

NOSExpression in Patients with Lupus

Nephritis

Edgar Len Esparza Ibarra, MSc

La Paz. February 18, 2005


2/32

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


3/32

ROS and RNS

NO. Nitric Oxide

NO2. Nitrogen dioxide

Free radicals:

O2.- Superoxide

.

OH Hydroxyl

RO2. Peroxyl

RO. Alkoxyl

HO2.

Hydroperoxyl


4/32

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


5/32

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


6/32

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


7/32

What is Nitric Oxide?

N O

Discovered in 1772 by Joseph Priestley

Clear, colorless gas

Free radical

6-10 sec lifetime in vivo


8/32

Dr. Louis J. IgnarroDr. Ferid Murad Dr. Robert F. Furchgott

Worthy of NobelPrize


9/32

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-


10/32

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


11/32

Protective Deleterious

Regulatory

BIOLOGICAL EFFECTS OF NO

Second messenger role requires

low concentrations ofNO(10mM)

Formation of highly reactive peroxynitrite

Up-regulation


12/32

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.


13/32

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


14/32

iNOS

NF kB-Ik B


15/32


16/32

What is Lupus?

Rim Granular Homogeneous

Lupus (wolf)

erythematosus

(redness)


17/32

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


18/32

Systemic Lupus Erythematous (SLE)

Leading cause of death for patients iskidney related called lupus nephritis

Minimal

Mild

Severe


19/32

SLE

Signs of renal involvement

Changes in urinary output

Proteinuria

Hematuria

Fluid retention


20/32

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


21/32

Glomerulus

Distal tubule

Afferent arteriole

Efferent arterioleBowmans capsule

yuxta

glomerular cells

Mesangeal cells

Urinary space

Proximal tubule

Podocytes

Capillar

Macula

densa cells


22/32

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.


23/32

Know the expression of eNOS/iNOS in renal biopsieswith lupus nephritis.

Aim

NOS

N O


24/32

Materials and methods

Biopsies

LES (n=20)

Control (n=10)

Hematoxiline

-eosine

Nephritis class

characterization

Cuts

Glomerular activity

Glomerular cronocity

TUNEL

(TdT)


25/32

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


26/32

RT-PCR

Materials and methods

Electrophoresis

(Photodocumentation)


27/32

Results

11 from 20 lupus renal biopsieswere related to nephritis class IV, 6

to class III and 3 to class II (World

Health Organization).


28/32

Hematoxiline-eosine

Karyorrhesis

TUNEL Assay Yodure propidium


29/32

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.


30/32

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


31/32

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


32/32

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.

Presentation Edgar Esparza

Documents

Transcript of Presentation Edgar Esparza