Multi-Nutrient Supplement Ameliorates Diabetic Nephropathy in Rats

INTRODUCTION

Diabetic nephropathy (DN) is a common end stage complication of diabetes mellitus (DM).¹ The average incidence of DN is high (3% per year) during the first 10 to 20 years after onset of diabetes.² Previous studies have indicated that DN is the vital player in increasing mortality and morbidity in developed countries based on explosive increase in the number of people diagnosed with DN. Till date there are no appropriate treatment options available for DN and most of the treatment options available to follow an approach involving the reduction of glucose, regulation of blood fat and use of angiotensin converting enzyme inhibitor (ACEI) and angiotensin-receptor blocker (ARB) agents. 

Many studies have been proven the protective role of the individual constituents of multi-nutritional supplement against diabetic complications. The multi-nutritional supplement selected in our study includes alpha lipoic acid, gamma linolenicacid,benfotiamine, l-methyl folate, methylcobalamin and pyridoxine hydrochloride. alpha-lipoic acid (LA) is an endogenously produced coenzyme that plays an essential role in mitochondrial dehydrogenase reactions and act as an antioxidant.³ Treatment with LA reduces the oxidative stress markers in plasma of patients with diabetes mellitus and with poor glycemic control⁴ . There isevidence that administration of LA ameliorates diabetic nephropathy.⁵ γ-linolenic acid (GLA) has shown to abrogate experimental diabetic nephropathy by exerting anti-inflammatory and anti-fibrotic effects6 .Benfotiaminehas shown to prevent the progression of diabetic complications probably by increasing tissue levels of thiamine diphosphate and by inhibiting advance glycation end products.⁷ The active metabolite of folate, l-methylfolate, stabilizes and enhances production of tetrahydrobiopterin, a cofactor of endothelial nitric oxide synthase (eNOS).⁸ It counteracts eNOS uncoupling and associated superoxide generation, upregulation of inducible nitric oxide synthase (iNOS), and formation of the highly reactive oxidant peroxynitrite in vascular endothelium.⁹ Methylcobalamin (a coenzyme form of vitamin B12, cobalamin),which prevents reduced glutathione (GSH) depletion and oxidative-nitrosativestressin several diabetes complications.¹⁰ Pyridoxal phosphate act as a metal chelator, known to inhibit formation of advanced glycation end products (AGEs) and display antioxidant properties in several tissue sitesfor DN.¹¹ On the basis of the aforementioned findings, multi-nutritional supplement more effectively counteract oxidative-nitrosative stress, inflammation and AGEs formation, which play a pivotal role in pathogenesis of DN. Therefore in this study we evaluated the effect of multinutritional supplement therapy onoxidative stress and manifestations of DN using streptozotocin induced diabetes in rats, a generally accepted model of type 1diabetes.

2. MATERIALS AND METHODS

2.1 Chemicals:

Streptozotocin (MP Biomedicals Pvt. Ltd.) and Multinutritional supplement (Lupin Ltd.) were procured. All other chemicals and reagents used were of analytical grade.

2.2 Experimental animals:

In- house bred male Wistar rats of appropriate age weighing 250-300g were used in the study. The animals were housed in propylene cages in an air-conditioned room at 24±1o C with a 12 hrs light/dark cycle and free access to water and standard diet ad libitum. The use of animals for the experiments was approved by the Institutional Animal Ethics Committee (IAEC approval number: AACP/IAEC/Feb 2018/01) and Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA) guidelines were followed.

2.3 Experimental design:

Overnight fasted rats were rendered diabetic by administration of STZ 50mg/kg (dissolved in cold citrate buffer pH 4.5) intraperitoneal. Glucose (10%w/v) solution was provided a day after STZ injection to avoid hypoglycemic shock.12 After 72 hours of STZ injection, animals with fasting blood sugar >250mg/dl were considered as diabetic after which they were divided into two groups with 6 rats in each group composed of one un-treated diabetic group and other diabetic rats treated with nutritional supplement orally for 6 week. Normal non-diabetic rats were given normal saline orally as control. Insulin (3 IU/kg, s.c.) was administered twice in a week to diabetic rats to prevent mortality throughout the study.

Multinutritional supplement contains ([benfotiamine (100mg/kg)+gamma linolenic acid (23.24mg/kg) +Alpha lipoic acid (17.14mg/kg) +L-methyl folate (0.165mg/kg)+ methyl cobalamin (0.258mg/kg) + Pyridoxal-5-phosphate (0.516mg/ kg)]). This dosage of multi-nutritional supplement was calculated by converting human dose to animal dose as per the animal body weight.¹³

2.4 Measurement of body weight:

Body weight was measured at the beginning and every week throughout the study using digital balance (Essae® DS-252).

2.5 Kidney function test determination:

After 8 week of treatment, individual rats of each group were placed in metabolic cages with free access to food and water to obtain 24-h urine collection. Urinary albumin levels were detected using assay kits by autoanalyzer according to the manual provided by the manufacturer. Urinary albumin excretion rate (UAER), an indication of albuminuria, was calculated by the formula, UAER (mg/24 h) = 24 h urine volume × urinary albumin (mg/

dl). Overnight-fasted animals were anesthetized; blood was withdrawn through retro-orbital puncture and collected in clot activator Vacutainer then centrifuged at 3000 RPM for 10 min to separatethe serum. FBG, glycosylated hemoglobin, blood urine nitrogen, and serum creatinine (Scr) were determined by auto analyzer. All kits were used in accordance with manufacturer instructions. Then, animals were sacrificed, and their kidneys were isolated, weighed and homogenized for biochemical estimation. The kidney index was calculated using the formula 100 × kidney weight/ BW.

2.6 Measurement of oxidative stress:

Kidneys were washed with saline, chopped on ice, and a homogenate (10%, w/v) was prepared with 0.1 M phosphate buffer and centrifuged at 3000 rpm for 10 min at 4°C using Sorvall refrigerated centrifuge and the supernatant obtained was used for biochemical estimations. Catalase (CAT) activity was detected by the method of Aebi14, glutathione (GSH) was determined by themethod of Moron et al.15 and superoxide dismutase (SOD) wasdetermined by the method of Misra and Fridovich.16 Lipid peroxidation or malondialdehyde (MDA) formation was determined by the method of Slater and Sawyer.¹⁷

2.7 Statistical analysis:

Values are expressed as mean ± standard error of mean (SEM).Statistical significance with respect to DN control was evaluated using one-way ANOVA followed by Tukey’st-test using Graph Pad Prism software (Version 5.0, Graph Pad Prism Software Inc., San Diego, CA).

RESULTS

3.1 Effect of nutritional supplement on % change in the body weight of diabetic rats:

The percentage reduction in the body weight of the diabetic rats was significantly higher as compared to the normal rats. Treatment with nutritional supplement significantly improved the loss of body weight in diabetic rats. (Table 1)

3.2 Effect of nutritional supplement on renal function tests of diabetic rats:

Urinary albumin, urinary albumin excretion rate, BUN level and serum creatinine of diabetic rats were significantly higher than that of the normal rats. Treatment with nutritional supplement significantly reduced the albumin excretion levels of diabetic rats. (Table 1)

Kidney index of diabetic rats was significantly higher as compared to the normal rats. Treatment with nutritional supplement significantly reduced the kidney index of diabetic rats.(Table 1)

3.3 Effect of nutritional supplement on fasting serum glucose level and glycosylated haemoglobin of diabetic rats:

The fasting serum glucose level and percentage of glycosylated haemoglobin of diabetic rats was significantly higher when compared to that of the normal rats. Treatment with nutritional supplement significantly reduced the levels of serum glucose and glycosylated hemoglobin of diabetic rats.(Fig.1 and Fig. 2)

3.4 Effect of nutritional supplement on antioxidant enzyme and lipid peroxidation in kidney homogenate of diabetic rats:

SOD activity, catalase activity and reduce glutathion in kidney homogenate of diabetic rats was significantly lower when compared to the normal rats. Treatments with multi-nutritional supplement significantly increased the SOD, catalase and reduced glutathione levels in diabetic rats. (Fig.3)

A lipid peroxidation (MDA) level of diabetic rats was significantly higher as compared to the normal rats. Treatment with nutritional supplement did not shown any significant effect on MDA levels of diabetic rats.(Fig.4)

DISCUSSION

In this study, diabetes was induced by the administration of STZ which resulted in hyperglycemia. After 6 weeks, there was a significant reduction in the body weight of the diabetic rats as compared to the normal rats. Studies have proven that the reduction in the body weight is, because the insufficient insulin prevents the body from getting the glucose into the body’s cells from the blood to use as energy. When this occurs, the body starts burning fat and muscle for energy, thereby; causing a reduction in the overall body weight.18 Treatment with nutritional supplement significantly improved the body weight in diabetic rats.

Further, nutritional supplement also significantly reduced the serum glucose levels showing anti-hyperglycemic effect. L-methyl folate, alphalipoic acid, gamma linolenicacid,andbenfotiamine, components of multi-nutritional supplement have been already reported for their anti-hyperglycemic effect. Thiamine acts as a coenzyme for transketolase (TK) pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase complexes, enzymes which play a fundamental role in intracellular glucose metabolism.¹⁹ Gammalinolenicacidexertsantidiabetic effect by improving insulin resistance.²⁰ During diabetes, the excess glucose present in the blood reacts with hemoglobin to form HbA1C,²¹ this serves as an indicator of metabolic control in diabetes.²² In the present study, the diabetic rats showed higher level of HbA1C compared to normal rats, indicating their poor glycemic control. Diabetic rats treated with multi-nutritional supplementsignificantly decreased the level of HbA1C, which might be the result of an improvement in the glucose metabolism by treatment. Chronic hyperglycemia results in renal damage.

In this study,there was an increased urinary albumin level and UAER in diabetic rats which mean that the kidneys abnormally spill a tiny amount of protein into the urine from the blood that indicates the abnormal functioning of the kidneys. Treatment with multi-nutritional supplement significantly reduced albumin urea in diabetic rats.

Normally, kidney filters creatinine and other waste products out of blood. These waste products are then removed from the body through urine. In diabetic rat, serum creatinine and BUN level was increased which indicates damaged kidneys. Maintenance of these renal function parameters, closer to those in control rats by multi-nutritional supplement treatment suggests that multinutrients play a role, either directly or indirectly, in providing protection against diabetic nephropathy or delay its development.

There is substantial evidence from previous reports that oxidative stress is an important mediator in the progression of DN.²³ In the present study, we observed that in the diabetic control group, there was an increased ROS generation as evidenced by increased level of MDA, a marker of lipid peroxidation, and depletion of free radical scavengers like SOD, CAT, and GSH. Multinutritional supplement administration reduced ROS generation and restored antioxidant status. Supplementation with 35 mg/kg α-lipoic acid for 12 weeks prevented the increase of proteinuria in the diabetes group, and lessened renal pathological changes which were associated with diabetic nephropathy via decreasing oxidative stress.²⁴ Folic acid has an important role in reducing oxidative stress, improving endothelial function, and preventing apoptosis.²⁵ GLAexerted neuroprotective effect by free radical scavenging activity²⁶

CONCLUSION

Our results clearly demon¬strated that multinutrient treatment reduces the elevated serum glucose, metabolic abnormalities, urinary pro¬tein excretion and ameliorates renal damage in STZ-induced DN in rats. Moreover, multinutrients significantly exert protective effect against ROS induced injury through regulation of oxidative stress. We conclude that the multinutrient supplement therapy can be a promising approach to delay the progression and development of diabetic nephropathy

CONFLICT OF INTEREST

All authors declare none conflict of interest.