OXIDATIVE STRESS IN PATIENTS WITH ACUTE STROKE AND IT’S EFFECT ON RENAL FUNCTION Dr Monem Makki Alshok Dr Moayed F. Al-Gazally Dr Ali Hasson Hussain

Stroke classified into two major types: 1- Ischemic stroke. is rapidly developing clinical signs of focal ("or global ) disturbance of cerebral function, with symptoms lasting 24 hours or longer, or leading to death, with no apparent cause other than of vascular origin". If the symptoms lasting less than 24 hours, the event is defined as a transient ischemic attack (TIA). A number of factors have consistently been shown to be associated with an increased risk of stroke. These well-documented risk factors for stroke are usually divided into nonmodifiable (age, gender, ethnicity) and modifiable factors (hypertension, IHD, diabetes…etc. Stroke classified into two major types: 1- Ischemic stroke. 2- Hemorrhagic stroke

Kidney Kidney in human contains about one million nephrons, which act as independent functional units. Each nephron contains a tuft of glomerular capillaries called the glomerulus, through which large amounts of fluid are filtered from the blood, and a long tubule in which the filtered fluid is converted into urine on its way to the pelvis of the kidney. The kidneys perform a variety of important functions that can be divided into : Excretory secretary Homeostatic

Renal failure Acute disease is usually reversible if managed appropriately whereas chronic renal failure is a progressive and irreversible process that leads to death in the absence of medical intervention. Recently, the Acute Kidney Injury Network (AKIN) recommended that the term acute kidney injury (AKI) replace the term ARF to include the entire spectrum of ARF

Free radicals: are atoms or molecules that contain one or more unpaired electrons, there are two types of free radicals: A. Reactive Oxygen Species (ROS) B. Reactive Nitrogen Species (RNS) Oxidative stress: is an imbalance between prooxidants and antioxidants in the cells which is manifested by elevated levels of free radicals. Lipid peroxidation: is a chain reaction between polyunsaturated fatty acids (PUFA) and ROS, it produces lipid peroxides and hydrocarbon polymers that are both highly toxic to the cell. Malondialdehyde: MDA is an end product of peroxidation of PUFA and related esters. MDA is a biological marker of lipid peroxidation caused by oxidative stress.

Antioxidants Enzymatic antioxidants include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and many supporting enzymes. The major nonenzymatic antioxidants are glutathione, some specialized proteins such as thioredoxins, and some vitamins such as ascorbic acid, α-tocopherol, and carotenoids including lycopene and β-carotene.

Superoxide dismutase (SOD): catalyzes the breakdown of the superoxide anion into oxygen and hydrogen peroxide. Catalase: Its function is assumed to be the destruction of hydrogen peroxide formed by the action of oxidases. Glutathione peroxidase (GPX): Using glutathione (GSH) as a reducing reagent, the GPX enzymes catalyze the reduction of H2O2 and organic peroxides (R-O-O-H) to water and the corresponding stable alcohol thus inhibiting the formation of free radicals. Glutathione: the most important action of GSH is chemical detoxification of hydrogen peroxide. This reaction, catalyzed by the selenium-requiring glutathione peroxidase, forming oxidized glutathione, which no longer has protective properties. The cell regenerates reduced glutathione in a reaction catalyzed by glutathione reductase, using NADPH as a source of reducing electrons.

Aim of the study

1- Study the role of oxidative stress in stroke patients through the measurement of lipid peroxidation marker (MDA) in sera of patients and compare the results with that of control group. 2- Study the changes in antioxidants in stroke patients through measurement of GPX, CAT, and GSH in sera of patients and compare the results with that of control group. 3- Study the effect of age on (MDA) and antioxidants in stroke patients and control group. 4- Assess kidney function of patients with acute stroke through measurement of BUN and SCr, and calculation of eGFR and BUN/SCr ratio, and compare the results with that of control group. 5- Study the correlation between kidney function tests and oxidants/antioxidants in stroke patients.

Materials and Methods

The patients group who subjected to this study were (74) patients. All of those patients were admitted to Merjan Teaching Hospital in Hilla city with clinical symptoms of acute stroke. The studied patients were subdivided into two groups according to type of stroke : 1. Ischemic stroke group: which comprises of 59 patients. 2. Hemorrhagic stroke group: which comprises of 15 patients. Forty one apparently healthy individuals were taken as a control. Both the sera from patients and controls are used for the measurements of the following parameters: total protein concentration (TP), serum creatinine concentration (SCr), blood urea nitrogen concentration (BUN), reduced glutathione (GSH) concentration, catalase (CAT) activity, glutathione peroxidase (GPX) activity, malondialdehyde (MDA) concentration .

Statistical analysis All statistical analyses were performed by using SPSS 15 software for Windows. Data were expressed as mean ± SD. The normality of the distribution of all variables was assessed by the Kolmogorov-Smirnov test. Student's t test and Pearson correlation analyses were used for normally distributed variables. Mann-Whitney U test and Spearman rank correlation test were used for nonparametric variables.

Results

Sampling characteristics Age distribution of patients with acute stroke was as shown in figure (1):

This figure shows that 74.94% of patients were in the age between 40 and 80 years of old. Also two patients lie in the range of 30-40 years, and two lie in the range of 90-100 years, and this is consistent with the fact that acute stroke may occurs at any age, but it occurs mainly in the age between 55- 85 years of old. Risk factors In our study, we found that all patients with acute stroke had at least one of the risk factors of stroke as shown in table(1).

Table (1): Order of risk factors among types of stroke. total Hemorrhagic stroke Ischemic stroke Risk factor % of patients No. of patients 70.3 52 93.3 14 64.4 38 Hypertension 45.9 34 26.7 4 50.8 30 Diabetes 41.9 31 40 6 42.4 25 Smoking 31.1 23 32.2 19 IHD 18.9 20 3 18.6 11 Previous stroke 10.8 8 6.7 1 11.9 7 TIA 4.1 3.4 2 VHD 1.4 1.7 Atrial fibrillation

Measurement of lipid peroxidation product (MDA) in patients and control groups. Table(2) shows that mean ± SD of MDA concentration in stroke patients and control groups was 7.40 ± 3.03 and 2.39 ± 0.72 µmol/l, respectively. Statistical analysis shows that there was a significant increase (p<0.001) in MDA concentration in stroke patients compared with the healthy control group. From this table we can also see that ischemic stroke patients have significantly increased MDA concentration compared to hemorrhagic stroke patients.

Table(2): The distribution of MDA among types of stroke. p-value mean ± SD No. Groups Parameter p<0.001** 7.40± 3.03 2.93± 0.72 74 41 Patients Control MDA (µmol/l) P<0.01** 7.87± 2.94 5.53± 2.69 59 15 Ischemic Hemorrhagic

For stroke patients 95% of the results lie between 6. 70 and 8 For stroke patients 95% of the results lie between 6.70 and 8.10 µmol/l (median= 7.36 µmol/l) , while in control group 95% of the results lie within 2.16-2.61 µmol/l (median= 2.33 µmol/l), as shown in figure (2).

Figure (2): The result of MDA in patients and control groups.

Effect of age on MDA concentration In patients group MDA concentration increases as age increases, but age group 50-60 years shows more increment in MDA concentration than age groups 60-70 and 70-80 years. Thus, there is a strong positive correlation between age and MDA concentration (r= 0.428, p< 0.01), as shown in figure (3) and (4). In control group also there was significant positive correlation between age and MDA concentration (r= 0.603, p<0.01), and MDA concentration increased as age increased but age group 40-50 shows more increment in MDA concentration than age groups 50-60, as shown in figures (5) and (6).

Figure(3): The correlation between age and MDA in patient group.

Figure(4): The effect of age on MDA concentration in patients group.

Figure (5): Effect of age on MDA concentration in control group.

Figure(6): The correlation between age and MDA in control group.

Measurement of GSH, GPX and CAT in patients and control groups The results in the table(3) show that acute stroke patients have highly significant decrease in GSH concentration, GPX and CAT activities compared to control group (P< 0.01), also patients with ischemic stroke have significantly decreased GSH concentrations (P< 0.05), and highly significant decrease in GPX and CAT activities (P<0.01) compared to patients with hemorrhagic stroke. Also the results show that there was nonsignificant difference in GSH concentration, GPX and CAT activities between males and females with acute stroke.

Table(3): GSH concentration, GPX and CAT activities in patients and control groups. Mean ± SD Groups P value CAT (U/mg protein) P value GPX (U/gm protein) GSH (µmol/l) P< 0.01 0.21 ± 0.09 0.35 ± 0.04 P<0.01 0.49 ± 0.19 0.91 ± 0.09 P<0.01** 4.48 ± 2.21 8.67 ± 1.60 Patients Control 0.19 ± 0.09 0.27 ± 0.06 0.46 ± 0.18 0.61 ± 0.21 P< 0.05* 4.16 ± 2.11 5.70 ± 2.21 Ischemic Hemorrhagic NS 0.22 ± 0.08 0.53 ± 0.20 4.87 ± 2.33 4.06 ± 2.01 Male Female

Correlations between age and GSH, GPX, and CAT The results in our study have shown that there were significant negative correlation between age and GSH, GPX, and CAT in patients group (r = -0.264, p> 0.05, r = -0.309, p < 0.01, r= -0.329, p<0.01, respectively), as shown in figures (7), (8), and (9). In control group the age was also negatively correlated with GSH and CAT (r= -0.679, p< 0.01, r= -0.506, r < 0.01, respectively), but the correlation between age and GPX was weak and statistically nonsignificant, but still negative( r= - 0.107, p> 0.05), as shown in figures (10), (11), and (12).

Figure(7): Correlation between age and Figure(8): Correlation between age GSH concentration in patients group. and GPX activity in patients group. .

Figure(9): Correlation between age and Figure(10): Correlation between age CAT activity in patient group. and GSH concentration in control group.

Figure(11): Correlation between age Figure(12): Correlation between and CAT activity in control group. age and GPX activity in control group.

Measurement of kidney function tests in patients and control groups According to the level of eGFR, the patients with acute stroke were divided into five groups as shown in figure(13). Figure (13): The cutoff points of eGFR in acute stroke patients.

The Results of our study have shown that there was highly significant increase in BUN and SCr concentrations in patients group compared to control group (p< 0.01), on the other hand patients group had significantly lowered eGFR levels compared to control group (P< 0.01), while BUN/SCr ratio didn’t differ in patients and control groups, as shown in table(4). Patients with ischemic or hemorrhagic stroke had nonsignificant differences (p>0.05) in BUN concentration and BUN/SCr ratio, while there was highly significant increases in SCr concentration and highly significant decreases in eGFR levels in patients with ischemic stroke compared to patients with hemorrhagic stroke (p< 0.01), as shown in table(5).

P-value Mean ± SD Groups Parameters P< 0.01** 37.45 ± 14.2 table(4): BUN, SCr, eGFR, and BUN/SCr levels in patients and control groups. P-value Mean ± SD Groups Parameters P< 0.01** 37.45 ± 14.2 17.95 ± 6.46 Patients Control BUN (mg/dl) 1.70 ± 0.71 0.85 ± 0.16 SCr (mg/dl) 42.19 ± 26.61 95.85 ± 21.25 eGFR(ml/min/1.73m2) NS 23.50 ± 9.16 21.31 ± 6.85 BUN/SCr

P-value Mean ± SD Groups Parameters BUN/SCr NS 38.74 ± 14.55 Table(5): BUN, SCr, eGFR, and BUN/SCr levels in ischemic and hemorrhagic stroke patients. P-value Mean ± SD Groups Parameters NS 38.74 ± 14.55 32.40 ± 12.17 Ischemic Hemorrhagic BUN (mg/dl) P< 0.05 1.78 ± 0.71 1.36 ± 0.63 SCr (mg/dl) P< 0.01** 37.56 ± 23.15 60.41 ± 32.03 eGFR(ml/min/1.73m2) 22.89 ± 8.91 25.91 ± 10.01 BUN/SCr

The Acute Kidney Injury Network (AKIN) defines AKI as an abrupt (within 48h) reduction in kidney function characterized by an absolute increase in serum creatinine of either ≥0.3 mg/dl (≥25 µmol/L) or a percentage increase of ≥50% or a reduction in urine output (documented oliguria < 0.5 ml/kg per h for >6 h). By using means of BUN and SCr of healthy control group (which are 17.95 and 0.85 mg/dl, respectively) as a baseline levels of these parameters in patient group, we found that 55 patients (74.32%) met AKIN criteria and defined as having AKI. Also We found that AKI was more prevalent in patients with ischemic stroke compared to patients with hemorrhagic stroke, where from 59 patients having ischemic stroke, 48 (81.40%) defined as having AKI , where as of 15 patients With hemorrhagic stroke, only 7 (46.70%) defined as having AKI.

The cutoffs of eGFR described in figure (13) were used to see the relation between eGFR level and AKI, we found that all patients with eGFR equal to 15-29 or ≤ 15 ml/min/1.73m2 having AKI, and no patient with eGFR≥ 90 ml/min/1.73m2 having AKI. Furthermore, only 5.90% of patients with eGFR equal to 60-89 ml/min/1.73m2 having AKI, where as 96% of patients with eGFR equal to 30-59 ml/min/1.73m2 having AKI. Also we found that 78.8% of patients with BUN > 17.95 mg/dl having AKI.

Correlation between oxidants/antioxidants and BUN, SCr, and eGFR. The results of our study show a highly significant positive correlation between MDA concentration and BUN and SCr levels, as shown in figures (14) and (15). Our results also show the presence of highly significant negative correlation between MDA concentration, and eGFR levels, and BUN/SCr ratio as shown in figures (16), (17).

Figure (14): Correlation between MDA Figure (15): Correlation between MDA and BUN. and SCr.

Figure (16): Correlation between MDA and eGFR. Figure (17): Correlation between MDA and BUN/SCr ratio.

Our results also show a highly significant negative correlation between antioxidants ( GSH, GPX, CAT), and BUN and SCr concentrations, and a highly significant positive correlation between antioxidants and eGFR levels, as shown in table (6): Table (6): Correlation between antioxidants and kidney function tests. eGFR SCr BUN parameter p-value r-value P< 0.01 0.515 − 0.508 − 0.468 GSH 0.595 − 0.604 − 0.547 GPX 0.572 − 0.568 − 0.348 CAT

Conclusions

1- Acute stroke is associated with elevated oxidative stress as indicated by elevated MDA concentration and decreased antioxidants GSH, GPX, and CAT levels. 2- Hypertension, smoking, diabetes and IHD are the major risk factors for stroke, with hypertension is a risk factor for hemorrhagic stroke more than ischemic stroke. 3- All patients with acute stroke have at least one of the risk factors of stroke. 4- MDA is significantly elevated and GSH, GPX, and CAT are significantly decreased in patients with ischemic stroke compared to those with hemorrhagic stroke.

5- MDA is significantly increased with increasing age and GSH, and CAT are significantly decreased with increasing age in patients and control. 6- kidney function is significantly compromised in patients with acute stroke and this is indicated by significantly increased BUN and SCr concentrations and significantly decreased eGFR. 7- Kidney function of patients with ischemic stroke is more compromised compared to those with hemorrhagic stroke. 8- Kidney function in patients with acute stroke may be directly or indirectly affected by the level of oxidative stress developed during the acute phase of disease , and this is indicated by significant positive correlation between MDA concentration and BUN and SCr levels, and strong negative correlation between MDA concentration and eGFR levels. 9- There is a cerebrorenal connection, that is the disease that affects the brain may also affect kidney and vice versa.

QUESTIONS AND DISCUSSION?