Nephroprotective Effect of Coenzyme Q10 alone and in Combination with N-acetylcysteine in Diabetic Nephropathy

Diabetes mellitus (DM) is a group of heterogeneous disorders characterized by hyperglycaemia linked to insulin deficiency or decreased tissue sensitivity (Maitra, 2015; Masharani, 2008). The two types of DM are type 1 DM (T1DM) and type 2 DM (T2DM).

T2DM or non-insulin-dependent diabetes mellitus (NIDDM), a multifactorial disorder develops as a result of insulin resistance with relative insulin deficiency and is most common among adults. T2DM features a strong genetic component in addition to the acquired pathogenic causes associated with secretion and resistance to insulin (Kohei, 2010).

Premature deaths due to DM are associated with macro and microvascular complications. The primary microvascular complications of DM are diabetic nephropathy (DN), diabetic neuropathy and diabetic retinopathy (Pal et al., 2014; Geraldes et al., 2009).

DN is the foremost cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD). It is a progressive and irreversible loss of renal function involving glomerular hyperfiltration, incipient nephropathy, microalbuminuria, overt proteinuria and ESRD (Mogensen et al., 1983). DN induces renal structural changes like an accumulation of extracellular matrix and glomerular mesangial expansion, interstitial fibrosis and basement membrane thickening (Vora & Ibrahim, 2003).

Hyperglycaemia plays a crucial role in the development of DN. It has been shown that persistent hyperglycaemia in DM can induce OS by several mechanisms that involve, glucose auto-oxidation, activation and acceleration of polyol pathway, protein glycation through non-enzymatic means and reduced antioxidant defense. Hyperglycaemia is believed to act by activation of Protein kinase C (PKC) pathway, generation of reactive oxygen species (ROS) and over-expression of transforming growth factor-β (TGF-β) (Schena & Gesualdo, 2005; Brownlee, 2001).

The normal kidney owing to its high metabolic activity generates substantial OS that is stabilized by an antioxidant defense system. Uncontrolled blood glucose level shifts this equilibrium to a pro-oxidant state resulting in tissue injury and vascular anomalies It has been shown that the mechanisms involved in the development of the DN induce considerable OS by one or other means (Vasavada & Agarwal, 2005).

At present, none of the therapies used for DN have proved efficacious due to the involvement of diverse etiologic factors in the progression of DN. Therefore, it is difficult to select the best possible treatment approach and therapeutic agent. Consequently, effective and novel therapeutic strategies are required in the treatment of DN. Studies have demonstrated the beneficial effects of antioxidant therapies in DN (Mahajan et al., 2019).

CoQ10 (ubiquinone) is an endogenous, vitamin-like lipophilic substance serving as a natural antioxidant. CoQ10 plays a key role in the mitochondrial electron transport chain and scavenges free radicals to protect the β cells from the harmful effects of OS by its antioxidant properties (Hodgson et al., 2002; Rosenfeldt et al., 2007). Studies have also shown that CoQ10 is effective for glycaemic control among individuals with T2DM and reduces HbA_1c levels (Maheshwari et al., 2014). NAC is a glutathione precursor and a powerful antioxidant. NAC has been shown to impart protection to the β-cells against cellular damage. NAC attenuates hyperglycaemia and improves glucose intolerance. This action of NAC is linked to glucose-induced insulin release (Haber et al., 2003; Ahmad et al., 2012; Shimizu et al., 2005).

So far, no earlier studies have reported the synergistic effect of CoQ10 and NAC in the treatment of DN. Hence, we have selected these antioxidants and an effort was taken to study their combined effect in nephroprotection against experimentally induced DN. Therefore, the current research was aimed to explore the nephroprotective effect of CoQ10 and NAC alone and in combination in STZ-NAD-induced DN.

The aim of the present study was to investigate the effect of CoQ10 and NAC alone and in combination on STZ-NAD induced rat model of DN. In the current investigation, STZ-NAD administration to rats resulted in classical features of DM, like hyperglycaemia, retarded growth, polyuria, proteinuria, structural and functional abnormalities in the kidney along with the increased OS. These observations concerned with DN were consistent with previous findings (Garjani et al., 2011; Maheshwari et al., 2014). STZ-NAD induced renal damage was revealed from increased MDA level, reduced SOD and CAT activities and decreased GSH content in renal tissue. Also, renal MPO activity and nitrite content were increased significantly in the rats with DN. On the other hand, the treatment of rats with DN by the antioxidants CoQ10 or NAC improved the renal parameters with restriction of hyperglycaemia and restoration of anti-oxidant enzymes. Especially when the combined treatment was given, more beneficial outcomes were observed.

DN is rapidly becoming the major cause of the ESRD globally. Chronic hyperglycaemia is known to play a key role in all the diabetic complications including DN. Also, persistent hyperglycaemia is linked to the early and sustained OS generation during DM. OS and uncontrolled hyperglycaemia together play a decisive role in the pathogenesis of tubuloglomerular abnormalities (Mahajan et al., 2019).

Thus, one of the different strategies to alleviate DM or its complications is by using antioxidants. Various endogenous or exogenous antioxidants were found useful in diabetic complication including DN due to their ability to scavenge the free radicals and modulate various signalling pathways to restore the renal function. Antioxidants like vitamin E and C, CoQ10, NAC, α-lipoic acid, taurine and others are found effective against oxidative cell damage (Ahmadi et al., 2013; Jemai et al., 2009). Therefore, antioxidants could be used effectively as a complementary therapy in OS induced complications like DN. Recent animal and human studies have shown the beneficial effects of CoQ10 on elevated HbA_1C, urea, and creatinine in DM (Maheshwari et al., 2017; Zhang et al., 2019). Studies with NAC revealed its effectiveness against renal impairment associated with diabetes as indicated by a reduced urinary protein and urinary thiobarbituric acid reactive substances (Shimizu et al., 2005; Lee et al., 2016).

In the current research, a significant decline in body weight % in diabetic rats was noted. This reduction in body weight could be due to the excessive degradation of tissue proteins and increased muscle wasting. It was also evident that insulin deficiency contributes to the decreased protein synthesis and low serum total protein levels in diabetic rats (Rajkumar & Govindarajulu, 1991).

The results of the study showed that in diabetic rats treated with CoQ10, NAC or their combination, body weight % improved significantly probably due to the protective effect of these antioxidants in controlling muscle wasting, enhanced glucose uptake, reduced insulin resistance and inhibition of gluconeogenesis (Rajkumar et al., 1999; Amin et al., 2014; Midaoui et al., 2008).

The kidney to body weight ratio of the diabetic rats increased significantly due to renal hypertrophy, which is the key feature in initial alteration by DM. It was previously shown that over-utilization of glucose, glycogen accumulation, and increased lipogenesis in the renal tissue during DM lead to kidney hypertrophy (Teoh et al., 2010; Mogensen, 1999). Treatment of diabetic rats with CoQ10, NAC or their combination effectively attenuated kidney hypertrophy.

Persistent hyperglycaemia has direct linkages the development of DN. Elevated blood glucose affects the endogenous antioxidant defense system and aggravates the development of DN through intracellular ROS, lipid peroxidation and leakage of urinary protein (Algenstaedt et al., 2003). In the current investigation, persistent hyperglycaemia was developed in diabetic rats until week 12 of the study period. Also, all the diabetic animals showed increased HbA_1c. Diabetic animals treated with CoQ10, NAC or their combination for 8 weeks restricted hyperglycaemia and HbA_1c.

Studies showed that patients with T2DM often have a deficiency of CoQ10 as marked by considerably lower levels of CoQ10 in plasma in comparison to healthy individuals, which in turn impair body's defense against hyperglycaemia induced OS (Ates et al., 2013; Hasegawa et al., 2005; Sourris et al., 2012; Mezawa et al., 2012). Mezawa et al showed the beneficial effects of CoQ10 in increasing insulin production and/or insulin secretion. Additionally, CoQ10 might attenuate OS in mitochondria and improve the functioning of β-cell along with enhanced insulin sensitivity (Anwar et al., 2014). Consequently, exogenous administration of CoQ10 could potentially attenuate mitochondrial dysfunction induced by OS, thus improving glycaemic control in T2DM (Alam et al., 2014).

Several clinical and experimental studies revealed the beneficial effects of NAC against insulin resistance and associated complications. In most of these studies, NAC reduced hyperglycaemia maybe by improving insulin sensitivity and enhancing peripheral glucose uptake by its antioxidant properties (Ammon et al., 1992; Ho et al., 1999). In this study, oral administration of NAC (300 mg/kg, p.o.) to the diabetic rats restricted hyperglycaemia than the diabetic control group but didn’t reduce blood glucose levels in comparison to the observations from similar group at week 0. Thus, the combined treatment employed herein may have a synergistic effect of CoQ10 and NAC in reducing hyperglycaemia and HbA_1c.

It was shown that in DM, excessive excretion of total protein and albumin in urine leading to their reduced serum levels contributed to the pathogenesis of DN (Roy et al., 2010). CoQ10, NAC or their combination treatment restored serum total protein and albumin by preventing their urinary excretion. The combination treatment with CoQ10 and NAC in diabetic rats was found more effective than CoQ10 or NAC alone in restoration of serum total proteins and albumin.

The most common characteristics in the development of DN are the elevated serum creatinine (SCr) and declined creatinine clearance (Ccr) (Dabla, 2010). The present study showed the significant elevation in the SCr, and decline in CCr in diabetic groups than normal rats pointing to the declined renal function and development of DN. However, diabetic rats that received CoQ10 (10 mg/kg), NAC (300 mg/kg) or their combination decreased SCr and improved CCr, particularly in the animals treated with the combination of the antioxidants. In addition to the chronic hyperglycaemia, polyuria is another feature of DM arising from osmotic diuresis. The significant increment in the urine output (mL/day) noted in this study may be due to glucosuria associated osmotic diuresis. In this study, severe renal impairment in the diabetic rats was identified by substantial rise in the urinary total proteins and UAER. Decreased serum albumin and increased UAER in diabetic complications were linked to the rapid progression of renal disease.

Earlier reports demonstrated that DM induced renal dysfunction is associated with a gradual reduction in serum albumin level. Albuminuria, resulting from damage to the glycosaminoglycans in the basement membrane and increased pore size may be linked to the decreased serum albumin (Mora-Fernández et al., 2014; Haraldsson & Sörensson, 2004). Consequently, the reduction of proteinuria could be beneficial for improving renal function and to prevent the progression of DN towards ESRD (Oktem et al., 2006). Combined oral administration of CoQ10 and NAC to the diabetic rats was found effective in reversing the urinary protein loss declining UAER.

OS is the key element in the development and progression of DN and associated renal injury. Diabetic state is believed to induce OS by an imbalance between the normal antioxidant defense and elevated ROS production. It has been shown that antioxidant treatment in DN improves renal function by directly acting against oxidative tissue damage (Mahajan et al., 2019). Moreover, exogenous administration of antioxidants restricts the progression of DN by effective inhibition of ROS and scavenging the preformed intracellular ROS (Agrawal & Sadhukhan, 2015).

We noted impaired oxidative stability in diabetic rats as indicated by the elevated MDA level and reduced activity of antioxidant enzymes SOD and CAT. It is believed that during DM hyperglycaemia-induced OS increases lipid peroxidation (Idris et al., 2001). MDA, the product of lipid peroxidation is responsible for the cellular injury. As an aldehyde, MDA links sugar and protein to form glycated proteins. Such structural and functional abnormalities in proteins might be responsible for diabetic complications. Thus, increased levels of HbA_1c may have some linkages with increased lipid peroxidation (Krhač & Lovrenčić, 2019). There was a considerable decrease in lipid peroxidation and an elevation in SOD, CAT activity, along with increased GSH content in the renal tissue of rats treated orally with a combination of CoQ10 and NAC.

Studies correlated MPO and DM with a positive relationship between the raised MPO activity and the pathogenesis of DN (Rovira-Llopis et al., 2013). The MPO-hydrogen, a peroxide-chloride system, leads to a variety of chlorinated protein and lipid adducts that may cause dysfunction in the different compartments of the kidney (Prabhakar, 2004). In the present study, oral combination therapy of diabetic rats with CoQ10 and NAC significantly reduced renal MPO activity in the renal tissue than the CoQ10 and NAC alone treated rats.

NO is implicated in pathogenesis of DN since it modulates renal structure and function. In diabetes, abnormal NO production is linked to the progression of the kidney damage. Studies showed that most of the cytotoxicity attributed to NO is due to peroxynitrite, produced from the reaction between NO and the superoxide anion (Stamler et al., 1992; Kisic et al., 2016). In the present study, there was a significant elevation in nitrite content in the renal tissue of diabetic rats as compared to the normal rats. However, the diabetic rats treated with a combination of CoQ10 and NAC effectively reduced renal nitrite levels.

In conclusion, the results presented in this study provide valuable information supporting that treatment with antioxidants might prevent or delay the renal damage associated with DN. Oral administration of antioxidants, that is, CoQ10 and NAC possesses a significant nephroprotective effect against STZ-NAD induced DN. A combination therapy with CoQ10 and NAC is more promising as it improves renal function and reduces OS in the rats subjected to DN. Nephroprotective effect shown by the combination treatment in this study may be attributed to hypoglycaemic and antioxidant properties of these compounds that attenuated OS and enhanced renal function in diabetic rats. Finally, it was concluded that the combined administration of CoQ10 with NAC might attenuate or delay the progression of DN.