Glycaemic control in type 2 diabetic patients with chronic kidney disease: the impacts on enzymatic antioxidants and soluble RAGE

Introduction

Chronic kidney disease (CKD) is an irreversible and progressive disease that is characterised by kidney damage and renal function decline. In a clinical setting, the renal function in CKD patients is measured in estimated glomerular filtration rate (eGFR) (Levey et al., 2003). Most CKD cases are caused by type 2 diabetes which is characterised by long-term hyperglycaemia attributed to insulin resistance and insulin insufficiency. Hyperglycaemia increases the risk of many diabetic complications, including diabetic kidney disease (Bash et al., 2008).

CKD is associated with enhanced oxidative stress that is triggered when the antioxidants are unable to counteract the harmful oxidative insults caused by excessive production of reactive oxygen species. The enzymatic antioxidants such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) form a part of the antioxidant defense against oxidative insults. Superoxide anion, a highly reactive ROS, is converted by superoxide dismutase SOD to hydrogen peroxide, which is then reduced to water by catalase and GPx (Fukai & Ushio-Fukai, 2011). The activities of enzymatic antioxidants are generally decreased in diabetic patients with CKD (Wong et al., 2016b) and antioxidant therapy has been shown to lower the risk of albuminuria in diabetic patients (Bolignano et al., 2017).

Receptor for advanced glycation end-products (RAGE) is a type of cell surface receptor of immunoglobulin superfamily that binds to an array of ligands, including advanced glycation end-products (AGEs) (Ramasamy, Yan & Schmidt, 2009). AGEs represent a heterogeneous group of molecules, including pentosidine and Nepsilon-(carboxymethyl)lysine, that are produced as a result of glycation of proteins, lipids or nucleic acids by reducing sugars (Gkogkolou & Böhm, 2012). Soluble form of RAGE (sRAGE) is the most commonly described variant of RAGE which circulates in the blood. Ligand-binding to sRAGE circumvents the deleterious events such as oxidative stress, inflammation and apoptosis arising from the RAGE-ligand binding (Kalea, Schmidt & Hudson, 2009). In fact, sRAGE also shows nephroprotective effects such as improvements in albuminuria and renal function in diabetic nephropathy (Wendt et al., 2003).

Haemoglobin A1c (HbA1c) is commonly used as a tool for monitoring diabetes and hyperglycaemia. HbA1c is a form of glycated haemoglobin which is an indicator of average plasma glucose concentration over three months (The International Expert Committee, 2009). Controlling diabetes and hyperglycaemia has beneficial effects against kidney disease. Long-term intensive glycaemic control has been shown to reduce the risk of kidney failure (Wong et al., 2016a). Besides, tight glycaemic control could also lessen the risks of myocardial infarction, onset and progression of albuminuria in diabetic kidney disease (Ruospo et al., 2017).

Despite the favourable outcomes of well-controlled diabetes, the effect of glycaemic control on enzymatic antioxidants and sRAGE in diabetic CKD patients is hitherto unknown. Therefore, the aim of this study was to investigate the impact of glycaemic control status on the activities or levels of GPx, SOD and sRAGE in CKD patients. The present study sheds light on whether poor glycaemic control is associated with diminished activities of enzymatic antioxidants and increased sRAGE levels in diabetic CKD.

Materials & Methods

Results

Discussion

Enzymatic antioxidants and sRAGE have central roles in counteracting oxidative stress and ameliorating pathological features in diabetic nephropathy. The SOD and sRAGE have been shown to alleviate fibrosis and albuminuria as well as improve renal function in rats with diabetic nephropathy (Wendt et al., 2003; Kuo et al., 2015). Maintaining the blood glucose level within the normal range can decrease the risk of albuminuria, which is a risk factor for renal progression (Ruospo et al., 2017). Recent study also showed that higher HbA1c levels were associated with increased cardiovascular mortality in diabetic kidney disease patients (Chang et al., 2016).

In general, the present study showed that activities of enzymatic antioxidants and sRAGE levels were comparable among diabetic CKD patients with poor and good glycaemic control. This suggests that the levels of glycaemic control do not account for the quantitative alterations in GPx, SOD and sRAGE in diabetic CKD patients. Regardless of glycaemic control status, diabetic CKD patients manifested diminished SOD activities and increased sRAGE levels. Thus, the presence of CKD is an important factor that explains such changes of SOD and sRAGE in diabetic patients whereas the different levels of glycaemic control do not affect these parameters in diabetic CKD patients. According to these findings, the presence of CKD may override the effect of glycaemic control in altering the enzymatic antioxidant activities and sRAGE levels.

In this study, CKD patients manifested lower plasma SOD activity than non-CKD patients. This indicates that the patients with renal dysfunction are associated with enhanced oxidative stress. Although this is a speculation, downregulation of SOD expression and enhanced oxidative stress have been reported in murine models with chronic kidney failure that were induced by 5/6 nephrectomy (Vaziri et al., 2003; Ding et al., 2015). The present study showed that SOD activities were higher in patients with well-controlled diabetes. The interaction analysis corroborates the association between increased SOD activities and non-CKD patients with well-controlled diabetes. Therefore, an improvement in glycaemic control in diabetic patients could lead to an increase in SOD activities that helps counteract the increasing oxidative stress in diabetes mellitus or hyperglycaemia. Therefore, an upregulation of SOD could potentially slow down the progression of diabetic CKD in patients with well-controlled diabetes. On the other hand, the presence of CKD negates the effect of glycaemic control on the changes of SOD activities in diabetic CKD patients. This shows that renal dysfunction is associated with lower SOD activities that could not be modulated through controlling hyperglycaemia.

The present study did not show overall differences in GPx activities among the six groups. Based on the results of two-way ANOVA, the diabetic CKD patients manifested lower GPx activities than non-CKD patients, irrespective of their glycaemic control status. This supports the view that GPx activities are decreased in patients with renal dysfunction (Zachara, 2015). Of note, the progression of kidney disease is associated with GPx activity decline (El-Far et al., 2005), indicating the changes in GPx activities depend on the levels of renal function. Although poorly-controlled diabetes is associated with lower GPx activities (Komosińska-Vassev et al., 2005), the comparable GPx activities among CKD patients with poor and good glycaemic control in this study points out that presence of CKD would predispose the diabetic patients to lower activities of enzymatic antioxidants.

Consistent with the current findings, renal failure has predominant contribution over type 2 diabetes towards the augmentation of oxidative stress characterised by the reduction of enzymatic antioxidant activities and the elevation of oxidative damage markers (Kuppusamy et al., 2005). Therefore, the presence of kidney dysfunction is the mainstay of heightened oxidative stress in type 2 diabetic patients. This is evident in type 2 diabetic patients with nephropathy whose enzymatic antioxidants such as GPx, SOD and glutathione reductase were depleted while the levels of malondialdehyde, an oxidative damage marker, were elevated (Kumawat et al., 2013). Our findings also suggest that good glycaemic control does not restore the activities of enzymatic antioxidants in diabetic CKD patients whose antioxidant capacities are commonly diminished. Well-controlled diabetes also does not contribute to an improvement in parameters such as urea, serum creatinine and eGFR in CKD patients. Nevertheless, the risk of albuminuria can be alleviated through tight glycaemic control and antioxidant treatment (Bolignano et al., 2017; Ruospo et al., 2017).

The comparable sRAGE levels in diabetic CKD patients with poor and good glycaemic control in the present study highlights that the presence of CKD negates the impact of glycaemic control on sRAGE levels. These findings concur with a review by Koyama, Yamamoto & Nishizawa (2007) which stated that the sRAGE levels are greatly affected by the presence of CKD in type 2 diabetic patients. Although the current analyses did not show significant interaction effect between CKD and diabetes on sRAGE, it has been postulated that the increased sRAGE levels in diabetes and renal dysfunction may be caused by the increased levels of AGEs (Prasad, 2014). In diabetic milieu, hyperglycaemia results in a concerted increase in RAGE expression and the levels of AGEs (Yao & Brownlee, 2010). The diabetes-induced reactive oxygen species and AGEs induce matrix metalloproteinase-9 which cleaves the cell–surface receptor to form sRAGE, elevating the levels of sRAGE in diabetic patients (Zhang et al., 2008; Prasad, 2014). In addition, the unsatisfactory renal function in the CKD patients may also explain elevated sRAGE levels due to reduced excretion (Kalousová et al., 2006).

The AGEs and sRAGE are mainly excreted by the healthy kidneys (Vlassara, 1994; Kalousová et al., 2007). Under the circumstances of diabetes and kidney disease, the formation of AGEs accelerates and more sRAGE molecules are produced to neutralise the AGEs. However, excess AGEs and sRAGE are unable to be removed due to the declining renal function. In the present study, diabetic CKD patients with poor glycaemic control manifested higher sRAGE levels. This could be attributed to limited excretion and upregulation of sRAGE to thwart the deleterious effects of AGEs. Consistent with this premise, treatment of the diabetic mice with sRAGE reduced the levels of AGEs in vasculature, but did not improve glycaemia in the mice (Park et al., 1998). Chronic hyperglycaemia is associated with increased generation of AGEs and oxidative damage products and exhaustion of antioxidants (Whiting et al., 2008; Yao & Brownlee, 2010). Apparently, well-controlled diabetes can lowers the production of oxidative damage products such as malondialdehyde (De Souza Bastos et al., 2016). These findings supported that sRAGE treatment and good glycaemic control hold promise in ameliorating oxidative stress.

Antidiabetic drugs have been shown to modulate the levels of antioxidants and sRAGE. The treatment with glyburide (sulfonylurea) restored the activities of enzymatic antioxidants such as glutathione-S-transferase and glucose-6-phosphate dehydrogenase in the liver tissues of diabetic rats (Bugdayci et al., 2006). The type 2 diabetic patients who were treated with metformin (biguanide) had lower malondialdehyde levels and higher total antioxidant status (Abdulkadir & Thanoon, 2012). A recent in vitro study also demonstrated that metformin could reverse the aldehyde modification-induced inhibition of SOD and upregulate its antioxidant activities in diabetic patients (Lankin et al., 2016). Besides, metformin also prevents oxidative burst through a reduction of reactive oxygen species and the augmentation of enzymatic antioxidant activities in lipopolysaccharide-stimulated human macrophages (Bułdak et al., 2014). The sRAGE levels were raised in type 2 diabetic patients who were treated with a combination of antidiabetic medications such as gliclazide (sulfonylurea), metformin and insulin (Devangelio et al., 2007). Of note, diabetic patients may also use other medications to treat the complications or comorbidities. For instance, diabetic patients who have hypertension and kidney disease may be given perindopril (angiotensin-converting-enzyme inhibitor) that could increase sRAGE levels (Forbes et al., 2005), while the patients with hypercholesterolaemia may be prescribed with statins that could increase the activities of enzymatic antioxidants (Davignon, Jacob & Mason, 2004). Therefore, it is challenging to investigate the modulatory effects of medications on antioxidants and sRAGE due to the vast range of possible combinations and confounding factors.

There are several limitations in the present study. There is a lack of age-matched healthy controls for the comparison of GPx, SOD and sRAGE. In this study, the individuals without CKD and diabetes who were age-matched with the patients were affected by other diseases such as hypertension and cardiovascular diseases. The present study did not show significant differences in these parameters between diabetic patients and non-diabetic patients. In addition, there is a lack of data on AGEs and proteinuria. The levels of AGEs increase as a result of elevated oxidative stress during the course of CKD progression (Stinghen et al., 2016). Besides, proteinuria also deteriorates with CKD progression (Ruggenenti et al., 1998). It is possible that AGEs and proteinuria could be modulated by glycaemic control in diabetic CKD patients. Thus, the impact of glycaemic control on AGEs and proteinuria in diabetic CKD should not be overlooked.

In this study, the glycaemic control levels were set according to the HbA1c targets indicated for general population (Hartz et al., 2006). However, it is recommended to use HbA1c target between 7.5% and 9.0% as the desired glycaemic outcome for the older study participants (Lipska et al., 2016). The reason for imposing less-tight glycaemic control in the older population with diabetes is to minimise the risks and hazards of hypoglycaemic episodes, depression and low body mass index (Panduru et al., 2017).

Conclusion

Glycaemic control did not account for the changes of the activities or levels of GPx, SOD and sRAGE in diabetic CKD patients. Reduced enzymatic antioxidant capacity and increased sRAGE level are the distinctive characteristics of CKD which are hard to be modulated through good glycaemic control. The presence of CKD would override the effect of glycaemic control on GPx, SOD and sRAGE, whereas the effect of glycaemic control status in determining SOD activities was only pronounced in non-CKD patients. Therefore, reduction of oxidative stress in diabetic CKD patients may be achieved through other means such as antioxidant supplementation and sRAGE therapy.

Supplemental Information