NOS Expression in Patients with Lupus Nephritis

Edgar León Esparza Ibarra, MSc

La Paz. February 18, 2005

Background
The Earth was originally anoxic
Metabolism was anaerobic

O2 started appearing ~2.5 x 109 years ago . O:O .
Free radicals:
o Any species capable of independent existence o A molecule with an unpaired electron

: : : :

ROS and RNS
Free radicals:

O2..OH

Superoxide Hydroxyl

RO2. Peroxyl RO. Alkoxyl

NO. Nitric Oxide NO2. Nitrogen dioxide

HO2. Hydroperoxyl

Exogenous sources of ROS and RNS 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 NO2 + Ultraviolet radiation --->NO + O .

Prostaglandin synthase NADPH oxidase . Flavoproteins Peroxisomes Mitochondria Electron transport Plasma Membrane Lipoxygenases. NOS isoforms Myeloperoxidase (phagocytes) Endoplasmic Reticulum Cytoplasm Transition metals Lysosomes Fe Cu Oxidases. Flavoproteins.Endogenous sources of ROS and RNS Microsomal Oxidation. CYP enzymes Xanthine Oxidase.

Can free radicals be second messengers?  Short lived (concentrations can change rapidly)  Enzymatically generated in response to stimulant  Specific in action (?) COO+ O2 COONADPH NADP+ COO+H3N C H +H3N C H +H3N C H (CH2)3 NH NOS C H2N NH2+ (CH2)3 NH C H2N + N OH H NOS (CH2)3 NH C O NH2 + NO L-Arginine Hidroxyarginine Citrulline .

colorless gas  Free radical  6-10 sec lifetime in vivo .What is Nitric Oxide? N O  Discovered in 1772 by Joseph Priestley  Clear.

Furchgott Dr. Louis J.Worthy of Nobel Prize Dr. Robert F. Ferid Murad Dr. Ignarro .

) . etc. inhibition of platelet aggregation. ion channels) Physiological response (relaxation of smooth muscles. phosphodiesterases.NO• signaling in physiology Agonists Nitric Oxide Synthase Ca+ O2-• NO• ONOO- Endothelial Cell PGI2 NO Binds to heme moiety of guanylate cyclase Conformational change of the enzyme EDHF K+ Increased activity (production of cGMP) Smooth Muscle Cell Modulation of activity of other proteins (protein kinases.

What is the role of Nitric Oxide in the human body? NO in the nervous system NO in the circulatory system NO in the muscular system NO in the immune system NO in the digestive system .

Normal tissues Cytotoxic effects due to a combination of • Elevated concentration (>10mM) • Formation of highly reactive peroxynitrite Up-regulation .BIOLOGICAL EFFECTS OF NO Regulatory Protective Deleterious Second messenger role requires • low concentrations of NO (<2mM) • occurs usually via guanylate cyclase.

which degrades cGMP and leads to enhanced vasodilation in erectile dysfunction. Sildenafil inhibits phosphodiesterase type 5.Today Aplications Inhale NO Drugs vs impotence Viagra® (Sildenafil) nitric oxide is released into the corpus cavernosum during sexual stimulation>cGMP mediated vasodilation and penis engorgement. .

31 Type II Type III Immunologically Vascular Endothelial Cells activated Macrophages iNOS Inducible No Effect 131KDa Chrom-17 17q11.Properties of NOS Isozymes Type I Tissue in which first described Tissue based terminology Expression Intracellular free Calcium Size Location of Gene Cerebellum Neuronal nNOS Constitutive Regulates 161KDa Chrom-12 12q24.2 eNOS Constitutive Regulates 133KDa Chrom-7 7q35-7q36 .112q24.

iNOS NF kB-Ik Bα .

.

upus Eritematoso Sistémico What is Lupus? Lupus (“wolf”) erythematosus (“redness”) Rim Granular Homogeneous Anti-DNAn Anti-DNAss Histonas Sm U1-RNP Antígeno DNA Histonas Mat E .

and Native Americans 80 % of all cases develop between the ages of 15 and 44 90% of all cases occur in females . Hispanics.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. Asians.

Systemic Lupus Erythematous (SLE) • Leading cause of death for patients is kidney related called lupus nephritis Minimal Mild Severe .

SLE • Signs of renal involvement Changes in urinary output Proteinuria Hematuria Fluid retention .

B. 3. E. 2.Anatomy of Kidney 1. 6. F. C. 4. Ascending Loop of Henle Descending Loop of Henle Peritubular capillaries Proximal tubule Glomerulus Distal tubule Renal vein Renal artery Ureter Medula Renal Pelvis Cortex . A. 5. D.

Glomerulus Efferent arteriole Macula densa cells Distal tubule Afferent arteriole yuxta glomerular cells Bowman´s capsule Mesangeal cells Urinary space Proximal tubule Podocytes Capillar .

Nephrol.Lupus Nephritis Classification (WHO)* I: No lesion II: Mesangial III: Focal proliferative IV: Diffuse proliferative V: Membranous VI: Sclerosis * J. Sec. 2004. Am. 15:241-250. .

NOS N O .Aim Know the expression of eNOS/iNOS in renal biopsies with lupus nephritis.

Materials and methods TUNEL (TdT) Biopsies LES (n=20) Control (n=10) Cuts Glomerular activity Nephritis class characterization Glomerular cronocity Hematoxiline -eosine .

1997. Thrombosis.Arteriosclerosis. GAPDH primers: 5'-GAA CAT CAT CCC TGC CTC TAC TG-3‘ sense 5'-GTT GCT GTA GCC AAA TTC GTT G-3‘ antisense . Human eNOS primers: 5'-CAGTGTCCAACATGCTGCTGGAAATTG-3' sense 5'-TAAAGGTCTTCTTCCTGGTGATGCC-3' antisense Human iNOS primers: 5'-GGCCTCGCTCTGGAAAGA-3‘ sense 5'-TCCATGCAGACAACCTT-3' antisense The Journal of Immunology. 2001. and Vascular Biology. 167: 75-81.17:3079-3082.

Materials and methods RT-PCR Electrophoresis (Photodocumentation) .

6 to class III and 3 to class II (World Health Organization). .Results 11 from 20 lupus renal biopsies were related to nephritis class IV.

Karyorrhesis Hematoxiline-eosine Yodure propidium TUNEL Assay .

a major correlation in biopsies with higher indices of activity/cronocity was observed. and therefore. .LES eNOS Control iNOS GAPDH • All biopsies expressed the constitutive isoform eNOS • Only biopsies with lupus nephritis expressed the inducible isoform NOS in 74 %. • The control GAPDH was present in all biopsies.

Conclusions and future work Our results suggest that the over-production of iNOS isoform is involved in the pathogenesis of lupus nephritis.  Immunohistochemistry of NOS. p53 and Hsp70  iRNA versus iNOS  Mouse model .

PhD and Rafael Herrera Esparza*. Cristina Rodríguez Padilla. Dr. MSc Esperanza Avalos Díaz. PhD and Pablo Zapata Benavides. Leonel Daza. MSc Ricardo Villalobos. Tania Zenteno Savín UAZ. PhD UANL. José Bollain y Goitia. Adrian López.Acknowledges CIBNOR. PhD .

Proposed Mechanism of Disease • iNOS generates excess NO • NO is reacted with Super oxide via superoxide dismutase. • The radical initiates a necrotic event enhancing the immune response. to generate the peroxy nitrile radical. .