Level of Cellular Damage of patients with renal Failure during Dialysis

 

Rasha Hasan Jasim, Kawther Mejbel Hussein

Department of Chemistry-Factually of Education for Girls-University of Kufa-Iraq.

 

ABSTRACT:

Background: Type II Diabetes Mellitus (T2DM) is characterized by insulin resistance at the level of skeletal muscle and fat tissue and muscle and liver, which leads to increase the level of glucose in the blood. This type of diabetes is not dependent on insulin, which affects adults or diabetes resistant ketosis, when the pancreas continues to produce insulin, but there is an imbalance in insulin receptor cells that does not respond to insulin, causing improper hepatic glucose metabolism. Dialysis is considered one of the most important strategies to deal with the decline of the kidneys from the performance of their vital functions, which leads to renal failure. Dialysis is an artificial mechanism by which nitrogen waste and liquid and toxic solvents are removed from the circulation. When the kidneys fail to do this properly, in all types of dialysis; blood is reacted with an artificial solution similar to human plasma and through a semipermeable membrane occurs the diffusion of liquids and dissolved substances. lt involves access to the blood circulation through either a venous or central venous artery catheter or a vaccination where the patient's blood pumping occurs through a blood removal device where two-way diffusion of atoms between dialysis and blood is allowed through a semipermeable membrane. Subjects: From the beginning of December 2018 to the end of May 2019, 158 individuals residents of Najaf and Karbala Governorates were included to participate in the current study. Method: Sandwich-ELISA technique was applied to estimate levels of Nitric Oxide, Superoxide Dismutase, GR in the sera samples of the study groups using kits that prepared by Elabscience Company, China. Results: The results of the present study recorded significant difference (p < 0.05) of NO, SOD, and GR concentrations measured in the samples of the two study groups. When comparing male and female subgroups implicitly, a significant difference (p = 0.000) of NO levels was recorded when patients of both genders compared together. Similarly, the study recorded a significant variation (p = 0.001) for NO levels when comparison was carried out between the males in the study subgroups, on the other side no such result was observed (p = 0.062) when the two women subgroups were compared together. The current study found a high significant difference (p = 0.000) when SOD in the samples of diabetic females was compared with their healthy partners in the control group, the highest mean of SOD (1.360 ng/ml) was observed in the group of diabetic females who underwent hemodialysis during the completion of the current work, while the highest concentration of a separate sample (4.493ng/ml) was found in a 66-year-old patient who had diabetes at the age of 35 and undergoing hemodialysis only for the second time. The study indicated a significant increase (p < 0.05) in the concentration of GR in males compared to females in both study groups. The highest mean of GR (1.561ng/ml) was observed in the group of healthy males, while the highest concentration of a separate sample (2.532ng/ml) was found in a 46-year-old patient who had diabetes at the age of 35 and undergoing hemodialysis since 6 months. In the patients group, positively significant correlation (r = 0.697 at p < 0.005) was observed for the concentrations of NO as well as GR to the age. More than four-fifths (92 of the total patient cases, at p < 0.005) of people with diabetes and dialysis as an adjunct to kidney function showed a positive correlation between NO and GR concentrations, while the relationship was shown to be negative between SOD and GR.

 

 

 

 

INTRODUCTION:

The adjustment of NO (NO) metabolic rate is mainly significant in type 2 diabetes, for the reason that activation of NOS has accomplished under insulin control by the Akt-pathway. Consequently, NO disturbances generation can be a result of insulin resistance that affects correspondingly the vascular response. Reported researches have shown that impaired NO metabolic rate has been found in T2DM, predominantly in the existence of nephropathy. A decreased urinary excretion of NO related products, like nitrites and nitrates, together termed as NOx were testified in type 2 diabetic patients with nephropathy. On the other hand, micro albuminuria has been linked with impaired endothelial function in type 2 diabetic subjects. Hyperglycemia may also be a factor in the lessened NO generation in T2DM, since high glucose inhibited endothelial NOS action in the glomeruli, through a protein kinase C–associated mechanism. Additionally, high glucose or the accompanying advanced glycosylation end products reduced NOS expression. Urinary NOx production has decreased in non diabetic renal disease. Correspondingly, increased plasma nitrate concentrations were testified in T2DMin addition to the metabolic syndrome. For the reason that the stimulation of NOS action by insulin is impaired in muscle of type 2 diabetic patients, examinations about response to the hormone of whole-body NO production in T2DM has key relevance [Cohen2005].

 

Augmented oxidative stress has the major role in the diabetes etiology besides its complications [Antonio 2000]. In diabetes, persistent hyperglycemia motivates the generation of ROS from numerous resources [Liang-Jun 2014], accordingly, diabetes typically causes augmented formation of ROS and debilitated antioxidant defenses. SOD catalyzes the transformation of O₂^- into H₂O₂.Under hyperglycemic circumstances, endothelial cells generate elevated levels of O₂^-. overproduction of O₂^- can inhibit glyceraldehyde-3-phosphate dehydrogenase that stands for a significant enzyme of the glycolytic pathway. This causes buildup of glucose and other intermediate metabolites of this alleyway and moves to other alternative alleyways of glucose metabolism accompanied by augmented creation of advanced glycation end products [Chia-We 2015].

 

Antioxidant enzymes stand for a foremost resource to protect in contradiction of reactive oxygen species produced by macrophages, neutrophils and eosinophils. These enzymes are mainly imperative in contradiction of chronic infections. GR (GR) (E.C. 1.8.1.7)[Kavita 2013] is one of a chain of enzymes which serves to maintain glutathione in the reduced form. In vitro, this enzyme can function with either NADH or NADPH as hydrogen donor [Zhu2018]. Glutathione (GSH) and GR stand for parts of the GSH redox cycle that keeps cells in contradiction of damage by oxidants. GR has a dominant role in glutathione metabolic rate and as such is a would-be target for different therapies.GR has FAD and a disulphide at its active site. The catalytic reaction necessitates drop of the site by NADPH, making a semiquinone of FAD, a sulphur radical and thiol. GR has involved some attention as a typical instance of flavin enzymes, and numerous efforts were prepared to regulate the order of attachment of substrates and releasing of its products. After reduction of the active site by NADPH, NADP is feasibly released after or before the catalytic steps including glutathione. The relation among reaction rates and substrate concentrations put forward primary release of NADP. Conversely, the competitive inhibition exposed by NADP headed for NADPH suggests release of NADP after condensed glutathione [Sergey 2012, Sáenz 2016, Yusen 2018]. Partial GR deficiency was first described in patients who had undergone drug-induced hemolysis, but whose red cells had normal G-6-PD activity. Subsequent studies showed, however, that subsequently partial GR deficiency was also observed in patients who had a variety of hematologic disturbances, both drug-induced and idiopathic, and a bewildering array of hematologic and nonhematologic diseases. Included in the disorders which were associated with deficiency of this red cell enzyme were diseases, such as hemophilia, leukemia, hemoglobin C disease, and Gaucher's disease-disorders which by no stretch of the imagination could be due to the enzyme deficiency [Sikanyika, 2019]. It was suggested, therefore, that GR deficiency might be secondary to some other disturbance common to these patients [Langbein  2017].

 

SAMPLES AND METHODS:

Samples: During the period from the beginning of December 2018 to the end of May 2019, 158 individuals residents of Najaf and Karbala Governorates were included in the current study. The participators were classified into two groups depending on their healthy to: patients with renal failure caused by diabetic complications undergoing hemodialysis and controls. The first group included 108 patients between the age of 20 and 80 (54.160±13.347), while the second group included 50 healthy persons between the age of 22 and 65 years (26.140±6.940). Total data about the study groups were summarized in Table 1.

 

METHOD:

Sandwich-ELISA technique was applied to estimate levels of Nitric Oxide, Superoxide Dismutase, GR in the sera samples of the study groups using kits that prepared by Elabscience Company, China.

 

Table 1: Levels (Mean±S.D.) of Age (Year) in The Study Individuals

Study Groups (n)	Gender (n)	Age (Year) Mean ± S.D.	Min.-Max. Range	p-value
Patients 108	Male 71	54.380 ± 12.827	24-80 56	0.786 For 1 vs 2 0.000 For 1 vs 3 0.808 For 3 vs 4 0.000 For 2 vs 4
	Female 37	53.730 ± 14.468	22-72 50
Controls 50	Male 38	26.370 ± 7.295	16-43 27
	Female 12	25.420 ± 5.900	20-38 18

1: Male Patients with Renal Failure, 2: Female Patients with Renal Failure, 3: Healthy Male Control, and 4: Healthy Female Control. The Mean Difference is Significant at 0.05 Level

 

RESULTS AND DISCUSION:

The results of the present study recorded a statistically significant difference (p = 0.000) of NO concentration measured in the samples of the two study groups, as shown in Tables 2.

 

Table 2: Levels (Mean ± S.D.) of NO Concentrations (µmol/L) in the Sera Samples of Renal Failure Patients and Controls Groups

Study Groups (n)	NO Concentrations (µmol/L) Mean ± S.D.	Min.-Max. Range	p-value
Patients 108	2.952 ± 0.932	0.110-4.463 4.353	0.000
Controls 50	1.618 ± 1.098	0.186-4.506 4.320

 

The Mean Difference is Significant at 0.05 Level:

Elevated glucose levels promote the production of reactive oxygen species as a result of auto-oxidation of glucose, metabolism, and the development of advanced glycosyl products [Brownlee 2001]. Uncontrolled blood sugar leads to a decrease in NO metabolism, which can be the cause of total and micro vascular complications [Dara 2017]. The dialysis procedure itself stimulates the cytokine induced by any synthase and also because of platelets that generate high concentrations of NO due to high urea. NO is an important metabolic product of the oxidation process. No excessive production can be cytotoxic, and is caused by the interaction of NO with reactive oxygen species and nitrogen, leading to the formation of peroxynitrite anion, tyrosine protein nitrogen, and the production of hydroxyl radical [NO in renal health NO is a cytotoxic molecule responsible for complications of dialysis and results in nitrogen stress in these patients, because it is a highly reactive free radical [Anila 2017]. The regulation of NO metabolism is particularly important in case of type 2 diabetes, because NO synthase (NOS) activation is controlled by insulin through the Akt pathway [Ranganath 2008]. High NO production may indicate insufficient blood purification, due to the common effect on the pathways of disposal through the renal duct. Therefore, changes in renal function, highlighted by changes in creatinine concentration, will be accompanied by changes in serotoxic NO [Reddy 2015].When comparing male and female subgroups implicitly, the study showed the absence of statistical differences (p > 0.05) when comparing male with female in the control group, while a significant difference (p = 0.000) of NO levels was recorded when patients of both genders compared together. Similarly, the study recorded a significant variation (p = 0.001) for NO levels when comparison was carried out between the males in the Study subgroups, on the other side no such result was observed (p = 0.062) when the two women subgroups were compared together, as demonstrated in Table 3. The renal blood vessels of men become more dependent on NO with age compared to gynecological diseases, suggesting that any renal disease that interferes with NO production may over time cause kidney damage to progress more quickly in men for women [Chien 2019].

 

Table 3: Levels (Mean ± S.D.) of NO Concentrations (μmol/L) in the Study Subgroups

Study Groups (n)	Gender (n)	NO Concentration (µmol/L) Mean ± S.D.	Min.-Max. Range	p-value
Patients 108	Male 71	3.062 ± 1.102	0.129-4.463 4.334	0.000 For 1 vs 2 0.001 For 1 vs 3 0.062 For 3 vs 4 0.086 For 2 vs 4
	Female 37	1.743 ± 0.573	0.110-2.732 2.622
Controls 50	Male 38	1.953 ± 1.168	0.186-4.506 4.320
	Female 12	1.506 ± 1.052	0.530-4.441 3.911

1: Male Patients with Renal Failure, 2: Female Patients with Renal Failure, 3: Healthy Male Control, and 4: Healthy Female Control. The Mean Difference is Significant at 0.05 Level

 

Significant difference (p < 0.05) was noticed at the two study's groups were compared together, when SOD concentration was examined, as shown in Table 4.

 

Table 4: Levels (Mean ± S.D.) of SOD Concentrations (ng/L) in the Sera Samples of Renal Failure Patients and Controls Groups

Study Groups (n)	SOD Concentrations (ng/L) Mean ± S.D.	Min.-Max. Range	p-value
Patients 108	1.278± 0.988	0.115-4.493 4.378	0.012
Controls 50	0.704±0.416	0.014 - 1.991 1.977

 

 

The Mean Difference is Significant at 0.05 Level:

Statistically, no significant differences (p > 0.05) were found when SOD comparing in both genders, implicitly; in the study groups. Similarly, the present study did not record significant differences in the statistical comparison between males with diabetes and their healthy peers in the control group.   In contrast to what was observed previous comparisons, the current study found a high significant difference (p = 0.000) when SOD in the samples of diabetic females was compared with their healthy partners in the control group, as illustrated in Table 5. The highest mean of SOD (1.360ng/ml) was observed in the group of diabetic females who underwent hemodialysis during the completion of the current work, while the highest concentration of a separate sample (4.493ng/ml) was found in a 66-year-old patient who had diabetes at the age of 35 and undergoing hemodialysis only for the second time. On the other hand, the lower mean concentration of this enzyme (0.741ng/ml) was recorded in the healthy male subgroup, as well as the lowest concentration of this enzyme (0.014ng/ml) was noticed in the sample of a healthy man at 35 years of age.

 

Table 5: Levels (Mean±S.D.) of SOD Concentrations (ng/L)in the Study Subgroups

Study Groups (n)	Gender (n)	SOD Concentrations (ng/L) Mean ± S.D.	Min.-Max. Range	p-value
Patients 108	Male 71	1.083 ± 0.879	0.115-4.493 4.378	0.066 For 1 vs 2 0.053 For 1 vs 3 0.093 For 3 vs 4 0.000 For 2 vs 4
	Female 37	1.360 ± 1.003	0.141-4.419 4.278
Controls 50	Male 38	0.741 ± 0.416	0.014-1.991 1.977
	Female 12	0.586 ± 0.211	0.264-1.602 1.338

1: Male Patients with Renal Failure, 2: Female Patients with Renal Failure, 3:Healthy Male Control, and 4: Healthy Female Control. The Mean Difference is Significant at 0.05 Level

 

       

Figure 1: Relationship Between Age and NO Concentration in Sera Samples of (A): Patients and (B): Healthy Individuals

 

In the patients group, positively significant correlation (r = 0.697 at p < 0.005) was observed for the concentrations of NO to the age as shown in Figure 1 A, while no such correlations were noted at this relation examined in the control group (Figure 1 B).

Despite the significant increase in levels of this enzyme in patients with type 2 diabetes mellitus with renal failure and in the different age groups, this increase was not consistent with the age of infected samples. Similarly, in the healthy individuals group where the study did not record any association between the level of enzyme and the age of healthy individual. Based on the results shown in Figure 2 A and B, the study was unable to find any statistically acceptable correlation for the age of study subjects with naturally produced SOD enzyme levels or as an immune response to renal failure.

 

 

Figure 2: Relationship Between Age and SOD Concentration in Sera Samples of (A): Patients and (B): Healthy Individuals

 

According to linear regression test that’s applied on the two study groups, respected positive correlation (r = 0.72 at p < 0.001) was observed when GR concentration was correlated to the age of cases in the group of diabetic patients with renal failure, as shown in Figure 3 A. On the other side, non significant negative correlation was recorded when the GR in the sera of healthy control samples was related to their age, Figure 3B illustrated the detailed results.

 

 

Figure 3: Relationship Between Age and GR Concentration in Sera Samples of (A): Patients and (B): Healthy Individuals

More than four-fifths (92 of the total patient cases, at p < 0.005) of people with diabetes and dialysis as an adjunct to kidney function showed a positive correlation between NO and GR concentrations, as illustrated in Figure 4 A. With the same manner, a significant positive correlation was shown in approximate 66% of the healthy individuals, when the same parameters were correlated (at p < 0.05) together in the controls group, as illustrated in the Figure 4 B.

 

 

Figure 4: Correlation of NO with GR Levels in The Sera Samples of (A): Patients and (B): Healthy Groups

 

Results of the present study showed the absence of statistical significance when the correlation between SOD and NO in the diabetic group was carried out as shown in Figure 5 A. On the other hand, the study illustrated a positive correlation (r = 0.58 at p < 0.05) between the two parameters measured in the controls group (Figure 5 B).

 

 

Figure 5: Relation of SOD with NO Concentrations in The Sera Samples of (A): Patients and (B): Healthy Groups

 

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Received on 10.03.2020           Modified on 19.05.2020

Accepted on 26.06.2020         © RJPT All right reserved