الرحمن الله بسمالرحيم

Some Studies on Immune Response of ESRD Patients

Presented by :

Vet. Samar Kamel Mohamed

Under the supervision of

Prof. Dr. Hassan Abd EL-Halim Amer Professor of Biochemistry and Chemistry of Nutrition.

Faculty of Veterinary Medicine, Cairo University

Adel M. El-Behairy Mohamad Ali Warda Professor of Biochemistry and Professor of Biochemistry and Chemistry of Nutrition. Chemistry of Nutrition.

Faculty of Vet. Medicine, Cairo Univ. Faculty of Vet. Medicine, Cairo Univ.

Dr. Mohamed Hassan ShaheenHead of Laboratory Medicine Dept.

Maadi Armed Forces Hospital

Introduction

Chronic Renal Failure (CRF): Is defined as the renal injury of more prolonged nature which

often leads to progressive and irreversible destruction of nephron

mass, causing permanent reduction in glomerular filtration rate

sufficient to produce detectable alterations in well-being and

organ function.

Several comorbidities occur in CRF including: 1-Biochemical alterations in blood parameters:

(uremia, hyperuricemia, hypoalbuminemia, electrolytes

imbalance, metabolic acidosis, disorders in calcium and

phosphorus metabolism, dyslipidemia) 2-Anemia. 3- Hypertension. 4- Development of cardiovascular diseases. 5- Oxidative stress.

Anemia is defined as a decrease in the number of circulating

red blood cells (RBCs), reduction in the amount of

hemoglobin (Hb) in the RBCs, or combination of both.

Despite new therapeutic options and treatment strategies,

anemia remains one of the major complications of CKD,

especially in patients undergoing chronic hemodialysis (HD).

Anemia of Chronic diseases

The average normal ranges of Hb varies by age and gender, in

adults are 14 to 18 g/dl in males, and 12 to 16 g/dl in females .

The presence of anemia in these patients reduces quality of life

and contributes to symptoms of advanced renal failure, such as

fatigue, reduced exercise tolerance, depression and dyspnea.

Anemia is associated with worsening of cardiovascular

morbidity and accelerated rate of kidney damage, and it is an

independent predictor of mortality in CKD patients

Renal anemia

Anemia is universal in end stage renal disease (ESRD)

patients.

Renal anemia is defined as an Hb concentration of < 11.5

g/dl in women, and < 13.5 g/dl in men.

Primary cause of renal anemia is relative lack of

erythropoietin hormone (EPO).

Various secondary causes can contribute to renal anemia,

including : Deficiency of iron Gastrointestinal bleeding Active blood loss Haemolysis Aluminium overload HypothyroidismSevere hyperparathyroidismInflammatory conditions (acute and chronic inflammations

which suppress erythropoiesis in the bone marrow) Shortened red blood cell survival due to uremia Deficiencies of folate and vitamin B12

Inflammation and oxidative stress

Inflammation and oxidative stress causes anemia in patients

with CKD by several mechanisms include:

The depletion of redox capacity and oxidation of membrane

phospholipids in erythrocytes, both of which lead to a

shortened life span of these cells.

Increased production of hepcidin hormone, that inhibits both

intestinal absorption and mobilization of iron stores and

induction of erythropoietin resistance.

Treatment of Anemia

Anemia is corrected with the administration of erythropoiesis stimulating agents (ESAs), the therapeutic goal is to reach a Hb concentration between 11.0 and 12.0 g/dl.

Correction of anemia in CKD patients, especially undergoing HD yields numerous benefits:

1) Higher tolerance for physical activity. 2) Improvement of cardiovascular functions. 3) Reduced the need for blood transfusions. 4) Better quality of life. 5) Reduced hospitalization. 6) Lower mortality.

ESA hyporesponsiveness

ESA resistance has been defined by the European Renal

Association-European Dialysis and Transplant Association

(ERA-EDTA) as being present when patients do not achieve

the recommended Hb target level (11–12 g/dl), despite a

treatment with ESAs over several months with maintained

doses of rhEPO higher than 300 IU/Kg/ week of epoetin.

Main factors causing EPO - resistant anemia:

1- Malnutrition

It is a relative common complication in chronic HD patients

and may lead to EPO-resistant anemia.

2- Hyperparathyroidism

Secondary hyperparathyroidism is associated with bone

marrow fibrosis, increased hemolysis, decreased

erythropoiesis, reduce RBCs production and survival.

As well as, reduce release and impaired response to EPO.

3- Iron deficiency

In HD patients iron deficiency can be :

Absolute (eg, chronic blood retention in the dialysis

circuit, malnutrition, gastrointestinal bleeding, and frequent

blood collections).

Functional (i.e., limitation of bone marrow erythropoietic

activity by inability to mobilize sufficient iron from body

storage sites); in this situation the body’s total iron stores

may be normal.

There is a clear interconnection between inflammation and

oxidative stress in ESRD patients on maintenance HD.

Oxidative stress forms part of the inflammation mechanism.

HD is associated with an oxidative imbalance, in which

oxidation of different lipids and proteins are predominant.

As well as, loss of some antioxidants during HD may

contribute to this disorder

4- Inflammation and oxidative stress

Aim of the work  The study was designed to :

1) Correlate the hemodialysis process in ESRD with the inflammatory

markers on cellular level.

2) To address the potential coherent relation between hemodialysis

and global inflammatory response in the form of C-reactive

protein.

3) To screen the immune status by measuring serum immunoglobulin

fractions and plasma protein foot-printing together with fluctuation

in interlukin6 level that shades patient’s immune status.

4) All the previous changes will be judged together with

plasma osmolality and other blood chemistry parameters

(including: Blood urea , serum creatinine, uric acid, total iron,

calcium, phosphorus, total proteins, albumin, total bilirubin,

alanine aminotransferase, aspartate aminotransferase, alkaline

phosphatase, total cholesterol, triacylglycerol, and blood

glucose level).

The correlation of the hemodialysis process in ESRD with the

inflammatory markers on cellular level will be performed in

the form of:

1. Modulation of antioxidant enzyme (superoxide dismutase)

expression.

2. Expected cell membrane deterioration (malondialdehyde level).

3. Accelerated apoptosis in the form of DNA-fragmentation.

Materials &

Methods

Experimental Design: From 200 ESRD outpatients , the study was focused on

80 ESRD categorized into 2 groups:

Group IEPO poor

responder patients

Group IIEPO good

responder patients

10 ml blood were taken just before and after the HD session

Blood collected in three types of vacutainer tubes

EDTA-blood

containing sodium citrate without anticoagulant

Serum

centrifugation

Blood Samples

containing EDTA

Citrated Plasma

Blood Samples (cont.)

Citrated Plasma Estimation of plasma osmolality

EDTA-blood samples

Detection of leucocytic oxygen consumption

Parathrmone level

Complete blood count

Blood Samples (cont.)

EDTA-blood samples

Malondialdehyde level

DNA- fragmentation

Superoxide dismutase

Blood Samples (cont.)

Serum samples

Protein pattern analysis

Ferritin levelInterlukin6 (IL6)

C-reactive protein Total iron

Blood Samples (cont . )

Serum samples

Protein profile

Electrolytes

Liver function tests

Kidney function tests

SaltsLipids