The possible role of glutathione-S-transferase activity in diabetic nephropathy

Abstract

The most common cause of end stage renal disease is diabetic nephropathy. An early diagnosis may allow an intervention to slow down disease progression. Recently, it has been hypothesized that glutathione-S-transferase (GST) activity may be a marker of severity of chronic kidney disease. In particular, a lower GST activity is present in healthy subjects compared to patients with nephropathy. In the present review we illustrate the scientific evidence underlying the possible role of GST activity in the development of diabetic nephropathy and we analyze its usefulness as a possible early biomarker of this diabetic complication.

Diabetic nephropathy is the main cause of chronic kidney disease (CKD) worldwide, and represents one of the most significant long-term complications for morbidity and mortality in diabetic patients. The development of kidney injury in patients with type 2 diabetes mellitus (T2DM) is likely secondary to complex interactions of metabolic and hemodynamic factors. However, despite progress in the understanding of the pathophysiology of T2DM, the triggering molecular events leading to diabetic nephropathy are still unclear.^1,2 Furthermore, even in the presence of a more prevalent use of nephroprotective therapies, diabetic patients still have a high risk of developing end-stage renal disease (ESRD). ³ The understanding of the pathological processes leading to the decline of the glomerular filtration rate (GFR) and the diagnosis at an early stage of diabetic nephropathy may allow the identification of targets for treatment in a phase when it is most likely to be effective. Until recently, the presence and severity of microalbuminuria had been considered the main early prognostic marker of diabetic nephropathy given its association with the onset of macroalbuminuria ⁴ and, eventually, the development of ESRD.^5,6 However, recent studies have questioned the utility of microalbuminuria, as albumin excretion is more likely to return to normal levels than to progress to macroalbuminuria.^7,8 In addition, in approximately one-third of patients with newly onset microalbuminuria the decline in GFR antedates by several years the progression to macroalbuminuria. ⁹ Given the limitations of microalbuminuria, new early markers of diabetic nephropathy are strongly needed.

Potential new biomarkers of diabetic nephropathy

Diabetes mellitus is associated with enhanced oxidative stress and free radical-induced lipid peroxidation (LPO), which may be pivotal players in the development of diabetic nephropathy. Therefore, the extent of LPO and the antioxidant defense system have been proposed as potential markers of early kidney damage in diabetes. In a recent investigation, increased LPO and decreased levels of glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PDH), and glutathione-S-transferase (GST) were reported in the erythrocytes and reticulocytes of type 2 diabetic men compared with age-matched controls. In addition, in patients with diabetes mellitus LPO was greater in erythrocytes than in reticulocytes, suggesting that an enhanced LPO and an impaired antioxidant system promote early cellular senescence. ¹⁰

In agreement with these results, Annadurai et al. have recently reported that, compared to healthy subjects, diabetic patients have lower levels of antioxidants and higher levels of malondialdehyde (MDA), a marker of LPO status, and that these parameters are strongly correlated with haemoglobin A1c concentrations. ¹¹ Similarly, in 64 T2DM patients and 60 healthy subjects, GST and aldehyde dehydrogenase 1 (ALDH1) activity were strongly associated to the severity of diabetes. An increase of ALDH1 and GST activities may be a compensatory effect against the enhanced oxidative stress observed in this population. Importantly, a low activity of ALDH1 in patients with relatively high glucose and HbA1c levels may contribute to the pathogenesis of diabetic complications. ¹²

Also genetic studies support a link between the antioxidant system and diabetic nephropathy. Recently, Datta et al. have demonstrated an association between the deletion of the GST-coding genes GST-T1 and GST-M1 and diabetic nephropathy (DN), indicating a contribution of increased oxidative stress secondary to the deletion of these two genes. In particular, a double deletion of GST-T1 and GST-M1 and the consequent reduction of GST levels may lead to increased oxidative stress as confirmed by increased MDA levels reported in the study. ¹³ Interestingly, diabetic patients without nephropathy have an increased activity of antioxidant enzymes such as GPx, superoxide dismutase (SOD), and catalase (CAT) whereas this enhancement is not found in those with kidney disease. ¹⁴ Further light on the relationship between polymorphic variants of GST-M1, GST-T1, and GST-P1 and risk of developing T2DM has been shed by Bid et al. who found different associations according to genotype (GST-M1 null, GST-T1 present, and GST-P1 (I/I)) in a population from Northern India. ¹⁵

Experimental data are consistent with human studies. In a rat model of diabetes, Jiang et al. found that the expression of GST-M is significantly increased compared to control rats. As resveratrol treatment normalized GST-M, these authors concluded that GST-M expression in diabetic kidney is influenced by hyperglycemia or by other forms of glucotoxicity. ¹⁶ Interestingly, these findings suggest that resveratrol, inducing GST-M downregulation, could exert protective effects against diabetic nephropathy. ¹⁷ The role of hyperglycemia in the pathogenesis of diabetic nephropathy is also indicated by studies in patients with type 1 diabetes mellitus (T1DM). Recently, Holmquist and Liuba measured the ratio of α-GST urine excretion/creatinine in a sample of Swedish children (mean age, 14 years). Lower levels of this ratio appeared to be associated to decreasing elasticity and endothelial vasomotor function. Oddly, higher levels of α-GST urine excretion/creatinine were more frequent in patients with increased markers of systemic inflammation. The authors were not able to reconcile these discordant findings, and suggested that further studies should evaluate the diagnostic relevance of this ratio in T1DM. ¹⁸

Erythrocyte glutathione-S-transferases

Glutathione-S-transferases represent a superfamily of enzymes involved in cell protection and detoxification. A prominent function of these enzymes is the conjugation of GSH to toxic hydrophobic compounds provided by an electrophilic center. ¹⁹ This reaction facilitates toxin inactivation and renal elimination. ²⁰ Red blood cells express almost exclusively a single GST isoenzyme, GST-P1-1, ²¹ which represents more than 95% of the erythrocyte GST (e-GST) pool (Figure 1).

Figure 1.

Structure e-GST.

Over-expression of e-GST has been found in uremic patients under maintenance hemodialysis (MHD), ²² and e-GST has been proposed as novel biomarker for dialysis adequacy complementary to Kt/V_urea ²³ and in subjects affected by hyperbilirubinemia. ²⁴ Importantly, in a recent study we reported, for the first time, a significantly increased activity of e-GST also in patients with chronic kidney disease (CKD) in conservative therapy. In particular, we observed a positive correlation between e-GST activity and the severity of renal disease according to the ‘kidney disease outcome quality initiative’ (K-DOQI) classification. ²⁵ The role of e-GST in diabetic nephropathy has been investigated by Noce et al. who measured blood levels of e-GST in diabetic patients with and without CKD. ²⁶ Surprisingly, the levels of e-GST were significantly higher in nephropatic patients without diabetes compared to nephropatic diabetic patients, in almost every stage of CKD (Figure 2). It is reasonable to speculate that diabetes alone may not cause an over-expression of e-GST; however, the possibility that increased e-GST activity observed in non-nephropatic T2DM patients stems from a undiagnosed reduction in renal function cannot be ruled out. If this observation is confirmed, e-GST could be considered an early marker of renal damage for diabetic patients. ²⁶ Previous study ²⁷ demonstrated a higher prevalence of anemia in diabetic patients with CKD (stages IV–V) compared to non-diabetic patients with the same GFR. Further studies should investigate the possible correlation between e-GST activity and anemia or other disorders such as psoriasis.^28,29

Figure 2.

E-GST activity in CKD patients with and without T2DM. ²⁴

Of note, in this investigation, e-GST activity was significantly correlated with homocysteine (Hcy) levels. These findings are in agreement with previous studies conducted on haemodialysis patients without diabetes mellitus. Therefore, also Hcy could represent an early biomarker of renal dysfunction. ²⁶

Conclusion

Diabetic nephropathy represents one of the leading causes of ESRD. Even if albuminuria is the most commonly used biomarker of early kidney damage in diabetic patients, it has important limitations. The introduction of an accurate marker of early DN could lead to more effective treatment strategies to reverse kidney damage and prevent progression to advanced kidney disease. Recently e-GST has been proposed as a new possible marker for early diagnosis of DN. Further studies are necessary to validate this hypothesis.