Impact of exercise on renal function, oxidative stress, and systemic inflammation among patients with type 2 diabetic nephropathy

Background Diabetic nephropathy (DN) is a prevalent microvascular diabetic complication all over the world. Objective This study was designed to measure oxidative stress, systemic inflammation and kidney function response to exercise training in patients with type 2 diabetic (T2DM) nephropathy. Material and Methods Eighty obese T2DM patients (50 males and 30 females), their body mass index (BMI) mean was 33.85±3.43 Kg/m2 and the mean of diabetes chronicity was 12.53±2.64 year participated in the present study and enrolled two groups; group I: received aerobic exercise training and group II: received no training intervention. Results The mean values of creatinine, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and malondialdehyde (MDA) were significantly decreased, while the mean values of interleukin-10 (IL-10), glutathione peroxidase (GPx) and glutathione (GSH) were significantly increased in group (A) after the aerobic exercise training, however the results of the control group were not significant. In addition, there were significant differences between both groups at the end of the study (P<0.05). Conclusion There is evidence that aerobic exercise training modulated oxidative stress and inflammatory cytokines and improved renal function among patients with diabetic nephropathy.


Introduction
Diabetic nephropathy (DN) considered as the most serious diabetic complication; while renal replacement is required for the majority of subjects with chronic renal disease among patients with T2DM 1,2 , where poor glycemic control 3 is related to abnormal oxidative stress and systemic inflammation that induce progressive diabetic renal lesion 4,5 . Hyperglycemia induces oxidative stress and inflammation 6 . In addition, poor glycemic control induces abnormal level of oxidative stress markers 7 . In the other hand, oxidative stress induce dysfunction of β-cell that lead to insulin resistance development, diabetes and its associated microvascular complications 8,9 , so that pa-tients with T2DM are under oxidative stress because of prolonged exposure to hyperglycemia 10 . Researches proved that hyperglycemia that induced systemic inflammation and oxidative stress which induce DN 11,12 . Hyperglycemia in diabetic patients leads to mitochondrial dysfunction, advanced glycation end processes and other factors, and generate the reactive free radicals, then triggers the DNA fragmentation that lead to cell death 13 . However, Navarro et al. found an increase in the gene expression for pro-inflammatory cytokine in patients with DN 14 . Several studies reported that there was a significant elevation in inflammatory cytokines in T2DM with DN and there is an association between their levels and the incidence & the course of renal lesion among diabetics [15][16][17] . Hyperglycemia also causes oxidative stress, decreases the regeneration of glutathione (GSH) from oxidized GSH and reduces the availability of nicotinamide adenine dinucleotide phosphate 18,19 . However, several reports stated that there was reduced level of GSH in diabetes as-sociated with systemic inflammation [20][21][22] . In addition, in β-cell dysfunction may be related to abnormal GSH level induce long-term complications of diabetes 23 . Moreover, low GSH is related to DNA oxidative damage in T2DM 24 . Many studies reported decline in the level of SOD in diabetic tissue and blood 25,26 . While, study performed by Lucchesi and colleagues to observe the oxidative balance of diabetic rats reported diminished activity of SOD and other antioxidative enzymes in the liver tissue 27 . In the other hand, several studies reported an increased MDA level in patients with T2DM 28,29 . In addition, Baynes and Ramesh et al. reported that lipid peroxidation in diabetes induced many secondary chronic complications including atherosclerosis and neural disorders 30,31 . Physical activity has several health benefits and plays an important role in treatment of chronic disorders. However, regular physical activity improves glucose control, blood lipid profile, insulin sensitivity and endothelial function that help to prevent diabetic complications 32 . Moreover, physical activity may reduce the risk and progression for diabetic nephropathy 33 . This study was designed to measure oxidative stress, sys-temic inflammation and kidney function response to exercise training in patients with type 2 diabetic nephropathy.

Materials and Methods Subjects
Eighty obese T2DM patients ( 50 males and 30 females), their body mass index (BMI) mean was 33.85±3.43 Kg/ m2and the mean of diabetes chronicity was 12.53±2. 64 year participated in the present study and enrolled two groups; group I: received aerobic exercise training and group II: received no training intervention. Exclusion criteria included smokers, kidney insufficiency, congestive heart failure, pregnant female patients, hepatitis and respiratory failure. Clinical evaluations and laboratory analysis were performed by independent assessors who were blinded to group assignment and not involved in the routine treatment of the patients. The CONSORT diagram outlining the details of the screening, run-in and randomization phases of the study and reasons for participant exclusion can be found in figure (1). Informed consent was obtained from all participants. This study was approved by the Scientific Research Ethical Committee, Faculty of Applied Medical Sciences at King University. Measurements and procedures A. Measurement of oxidative stress markers and anti-oxidant status For all participants serum (from 10 ml blood in plain vial) and plasma (from 5 ml blood in EDTA vial) were separated from the sample within 30 min of collection and was stored in pyrogen free polypropylene cryo-tubes at (-80ºC) until analysis. Assessment of lipid markers for peroxidation as malondialdehyde (MDA) was determined according to Buege and Aust 34 . However, Anti-oxidant status, glutathione (GSH) that was determined by the method of Beutler and colleagues 35 , in the other hand, glutathione peroxidase (GPx) was measured by the method of Nishikimi and colleagues 36 .

C. Aerobic exercise training program
Patients in group (A) were submitted to a 40 min aerobic session on a treadmill (the initial, 5-minute warmup phase performed on the treadmill at a low load, each training session lasted 30 minutes and ended with 5-minute recovery and relaxation phase) either walking or running, based on heart rate, until the target heart rate was reached, according to American College of Sport Medicine guidelines. The program began with 10 min of stretching and was conducted using the maximal heart rate index (HRmax) estimated by: 220-age, with exercise intensity was 70-80% of HRmax 34 .

Statistical analysis
The mean values of the investigated parameters obtained before and after three months in both groups were compared using paired "t" test. Independent "t" test was used for the comparison between the two groups (P<0.05).

Results
Eighty obese patients with type 2 diabetes mellitus completed the screening evaluation. The baseline characteristics of the participants are shown in table (1). Most participants (60%) were men. Forty participants were assigned group (A) (n = 40; 24 males and 16 females) and group (B) (n =40, 26 males and 14 females). None of the baseline characteristics differed significantly between the two groups is listed in table (1). The mean values of creatinine, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and malondialdehyde (MDA) were significantly decreased, while the mean values of interleukin-10 (IL-10), glutathione peroxidase (GPx) and glutathione (GSH) were significantly increased in group (A) after the aerobic exercise training(table 2), however the results of the control group were not significant (table 3). In addition, there were significant differences between both groups at the end of the study (table  4).    MDA: Malondialdehyde; GSH: Glutathione; GPx: Glutathione peroxidase; TNF-α: tumor necrosis factor -alpha; IL-6: Interleukin-6; IL-10: Interleukin-10; (*) indicates a significant difference between the two groups, P < 0.05.

Discussion
Diabetic nephropathy (DN) is a worldwide prevalent medical problem affecting 20-40% of T2DM and characterized with high rate of morbidity and mortality as the DN is a principal etiology of renal failure [35][36][37] 50 . The three possible mecha¬nisms of exercise anti-inflammatory effects include reduction in visceral fat mass 51 ; reduction in the circu¬lating numbers of pro-inflammatory monocytes 52 and an increase in the circulating numbers of regulatory T cells 53 . Moreover, Hong and colleagues show that cardiorespiratory fitness is associated with reduced low grade inflammation which may in part be mediated by enhancing the ability of immune cells to suppress inflammatory responses via adrenergic receptors 54 .
Concerning results of oxidative stress markers, results of our study agreed with other authors who reported that a six-months aerobic exercise was able to decrease lipid peroxidation, as well as to increase GSH and catalase activity in T2DM patients 55,56  There are 2 mechanisms that underlie the anti-oxidative of aerobic exercise training. The first mechanism is that improvement in glycemic control associated with aerobic exercise training may result in a decrease in oxidative stress. Aerobic exercise training improves insulin sensitivity 63 and glycemic control 64 . Hyperglycemia can induce oxidative stress via several mechanisms including glucose autoxidation, formation of advanced glycation end products, and activation of the polyol pathway 65 . Chugh et al. reported previously that 6 weeks of glycemic control with sulfonylurea resulted in an improvement of glycemic control and a reduction in serum malondialdehyde, a reliable measure of lipid peroxidation 66 . The other mechanism is that a decrease in oxidative stress caused by aerobic exercise training may lead to an improvement in gly-cemic control. Aerobic exercise may increase antioxidant activity and reduce oxidative stress. Elosua et al. reported that aerobic exercise training increased the activity ofthe endogenous antioxidants, glutathione peroxidase, and glutathione reductase and decreased oxidized low-density lipoprotein concentration 67 . There is evidence that oxidative stress is associated with insulin resistance, as Urakawa et al. demonstrated that plasma isoprostane levels were negatively correlated with glucose infusion rates in men 68 .
These results therefore indicate that improved insulin sensitivity and glycemic control induced reduction in oxidative stress caused by aerobic exercise training.
Concerning renal function, results of the present study proved that aerobic exercise training improved creatinine in patients with DN, the possible cause for improving renal function following aerobic training may be due mod- The current study has important strengths and limitations. The major strength is the supervised nature of the study. However, all exercise sessions were supervised. Moreover, the study was randomized; hence, we can extrapolate adherence to the general population. In the other hand, the major limitations is only obese type 2 diabetic patients enrolled in the study, so the value of this study only related to obese patients with type 2 diabetic nephropathy, also small sample size in both groups may limit the possibility of generalization of the findings in the present study. Finally, within the limit of this study, aerobic exercise training is recommended for modulation of oxidative stress and inflammatory cytokines and improved renal function among patients with diabetic ne-phropathy. Further researches are needed to explore the impact of weight reduction on quality of life and other biochemical parameters among obese patients with type 2 diabetic nephropathy.

Conclusion
The current study provides evidence that aerobic exercise training modulated oxidative stress and inflammatory cytokines and improved renal function among patients with diabetic nephropathy.