Role of Fenugreek (Trigonella foenum graecum) on Ischemia Reperfusion Induced Renal Damage in Uni-Nephrectomized Diabetic Rats

Introduction

Diabetic nephropathy (DN) is a major long-term complication of diabetes mellitus. Clinically there is a development of microalbuminuria with progression to overt proteinuria, increased in blood pressure, and reduced renal function. Excessive deposition of extracellular matrix protein in the glomeruli and subsequent mesangial expansion are the main structural alterations in diabetic nephropathy. Diabetic kidney disease is a major cause of end-stage renal disease in many countries.¹ Ischemia-reperfusion (I/R) combined with hyperglycaemia could also be important in the development of diabetic nephropathy. Ischemia-reperfusion causes oxidative stress which is involved in the development and progression of diabetic complications such as retinopathy, neuropathy, and nephropathy. Oxidative stress is significantly increased in diabetes because prolonged hyperglycemia increases the generation of free radicals.²

Abnormally high levels of free radicals and the simultaneous decline of antioxidant defense mechanisms lead to the damage of cellular organelles and enzymes. Antioxidant compounds, which are able to scavenge free radicals or modulate oxidative stress, are beneficial and have a higher therapeutic value with antidiabetic and antioxidant potential. Majority of patients with type-II diabetes are affected by the end-stage renal disease (ESRD). It has been considered that about 25-40% of patients with Type-I or Type-II diabetes develop diabetic nephropathy within 20-25 years of the onset of disease. The major clinical treatment for diabetic nephropathy targets hyperglycemia and hypertension. The novel target which is linked to glucose-dependent pathways are the major focus of new therapies directed against diabetes-induced renal damage.³

Fenugreek (Trigonella foenum graecum L. Family: Leguminosae), a spice rich in dietary fibres has traditional history of medicinal use in the management of diabetes is a potent source of antioxidants.⁴ Previous studies have shown that T foenum graecum seeds have hypoglycemic, hypocholesterolemic, and hyperinsulinemic effects in patients with types 1 and 2 diabetes mellitus and experimental diabetic animals. The water extract of T foenum graecum seeds lowers blood glucose, reduces levels of glycated haemoglobin, and reduces lipidemia in streptozotocin (STZ) induced diabetic rats in a dose-dependent manner.⁵ In addition to its hypoglycemic effects, the T graecum seeds have also been reported to restore the altered antioxidant status in various tissues due to diabetes-induced oxidative stress.^6,7 T foenum graecum seeds extract has been reported to prevent both lipid peroxidation and red blood cell oxidative hemolysis.⁸ An aqueous acetate extract of the T foenum graecum seeds significantly decreased the content of catalase (CAT) and superoxide dismutase (SOD) activities in the liver, heart, and kidneys of rats that were fed a cholesterol-rich diet.⁹ An aqueous extract of fenugreek seeds significantly decreased kidney/body weight ratio, serum creatinine ratio, blood urea nitrogen, 24 h content of urinary protein and creatinine clearance in STZ induced diabetic rats as compared to non-treated group.² Fenugreek protects heart tissue enzymes such as lactate dehydrogenase (LDH), creatinine kinase (CK), aspartate transaminase (AST) and alanine transaminase (ALT), decreased levels of serum lipoproteins like VLDL, LDL and increase in HDL, decreased levels of triglycerides (TG), free fatty acids (FFA) and cholesterol (CHO) in isoproterenol-induced myocardial infarction in rats.¹⁰ Orally administered fenugreek seeds can protect gastric mucosa by its antisecretory action dose-dependently in ethanol-induced gastric ulcer. Moreover, alkaloid and flavanoids rich fractions of Fenugreek seeds have antinociceptive and anti-inflammatory actions.¹¹

Fenugreek seeds mainly contain 4-hydroxy isoleucine (4 HI), trigonelline, galactomannan with flavonoids, carotenoids, coumarin, protein, saponins and lipids.¹² The reconstructed composition of fenugreek seeds, IND01 which contains mainly 4-HI (40%), trigonelline (30), and galactomannan (30%) were reported to have synergistic antihyperglycaemic interaction with synthetic antihyperglycaemic molecules. The exact composition responsible for the beneficial effect on DM induced renal damage is yet challenging.¹³

Material and Methods

Drugs and chemicals

Fresh Fenugreek seeds were procured from herbal markets, Bengaluru and authenticated by FRLHT. Streptozotocin (STZ) was procured from SIGMA ALDRICH, Bangalore and all other chemicals and reagents used were of analytical grade and procured from approved vendors.

Animals

After approval of IAEC (IAEC/ABMR CP/2013-2014/02), Sprague-Dawley rats weighing 180-250 gm were obtained from the animal house of Acharya & B.M. Reddy College of Pharmacy, Bangalore, Karnataka. They were kept in colony cages at an ambient temperature of 25 ± 2 ºC and 45-55 % relative humidity with 12:12 h light: dark cycles. They had free access to a standard rodent pellet diet and drinking water. The food was withdrawn 18-24 h before the surgical procedure; however, water ad libitium.

Preparation of hydro-alcoholic extract of Fenugreek seeds

Seeds were shade dried and ground, powdered, and passed through sieve no 20. The coarse powder was collected. The powder was packed and extracted with ethanol: water (70:30) at 65-70 ºC for 72 h using Soxhlet column. The solvent was removed from the extract by using vacuum evaporator and hot air oven. The dried extract was kept in china dish and % yield was calculated. The extract of Fenugreek was dissolved in distilled water. Two concentrations 200 mg/kg and 400 mg/kg were chosen for the study administered by orally to the rats.¹⁴

Experimental method

Right uninephrectomy under anaesthesia with thiopentone sodium (45 mg/kg, i.p) was conducted. The rat was placed on its ventral surface, left to right of the investigator. A dorsoventral incision was made into the abdominal cavity, down the sides of the rat near to the coastal border of the thorax. The kidney was freed of connective tissue and was pulled out gently, preferably by grasping the periental fat. The adrenal gland which was attached loosely to the anterior pole of the kidney by connective tissue and fat was gently freed by tearing the attachment. A single ligature was placed around the renal blood vessel and the ureter as far from the kidney as possible, toward the midline, but without damaging or occluding any collateral blood vessel that may be encountered. The ligature was tied securely with a double reef knot and the blood vessels are transacted next to the kidney which was removed. The kidney was cleaned of excess tissue, blotted, and weighed. The incision was closed in the usual way.^15,16

Experimentally induced diabetes mellitus

After 18 h fasting male Sprague-Dawley rats (180-250 gm) were induced by i.p. injection of a single dose of STZ (45 mg/kg) followed by the i.p. administration of nicotinamide (110 mg/kg) after 15 minutes. STZ was dissolved in cold citrate buffer (pH 4.5) immediately before use and solution was made fresh daily. STZ has been shown to induce a diabetic state in 72 h. The rats were allowed access to sucrose (5% v/v) in drinking water for the next 24 h to prevent hypoglycaemia. Three days after STZ injected, rats with blood glucose levels 250 mg/dl were used for the studies.¹⁷

Experimental design

Sixty four male Sprague-Dawley rats were divided into eight groups of six animals in each group as uni-nephrectomized non-diabetic group and 10 animals in the diabetic group. Group I animals underwent all surgical procedures without I/R (sham), group II rats received normal saline (I/R control), group III rats received Fenugreek seeds extract (200 mg/kg, p.o.), group IV rats received Fenugreek (400 mg/kg, p.o.) and are same as for diabetic group also.

Twenty four hours/day urine was collected on the 14^th day and centrifuged. On the 15^th day, the left kidney was isolated after ischemia for 45 minutes and reperfusion for 5 hours. At the end of the experiment, blood was collected from the carotid artery, centrifuged at 5000 rpm for 20 minutes and serum samples were collected.

After sacrificing, the kidney was retracted and washed with 0.9% of normal saline. The weight of the kidney was noted and accordingly 10% w/v homogenate was prepared using homogenizer with 1 M potassium phosphate buffer at ice-cold temperature. The homogenate was poured into centrifugation tubes and centrifuged at 10,000 rpm at 4 ºC for 15 min. The supernatant was taken to perform the biochemical estimations.

Measurement of physiological, biochemical, and renal functional parameters

Urine output, body weight, and left kidney weight were measured. Serum creatinine, BUN, albumin, albuminuria, LDH, and total protein were estimated by using commercial kits (Swimmed diagnostic) according to the method described by Jaffe’s kinetic method, bromo cresol green (BCG), modified biuret methods in creatinine, albumin and total protein, respectively.^{18,19,20,21,22}

Determining antioxidant enzymes activities

The activities of SOD and catalase in tissue homogenate were determined by Kakkar et al, 1984 and Aebi et al; 1974, respectively.^23,24

Measurement of lipid peroxidants

The lipid peroxidation end product malondialdehyde (MDA) in tissue and serum was measured by the method of Ohkawa et al. ^25,26

Estimation of the inflammatory biomarker

Inflammatory biomarker myeloperoxidase (MPO) was measured by the method of Mullane et al. ²⁷

Histopathology

The kidneys from control and experimental groups were fixed with 10% formalin and embedded in paraffin wax and cut into a longitudinal section of 5 μM thickness. The sections were stained with haematoxylin and eosin dye for histopathological observation. The change in inflammation structure was observed and compared.

Statistical analysis

The results were expressed as (Mean ± SD). Biochemical parameters were determined by factorial One-way ANOVA.

The individual group was compared against I/R control using Dunnet’s test. P <0.05 was considered statistically significant. Statistical analysis was performed using Prism software (Version 10).

Results

In the present study, there was significant (P<0.05) increase in BUN, albuminuria, non-significant increase in creatinine, a significant increase in LDH (non-diabetic group only), and a significant decrease in albumin were observed in Sham groups compared to I/R control groups.

Treatment with Fenugreek at the dose of 200 mg/kg and 400 mg/kg reversed significant decrease (P<0.05) in BUN, albuminuria, LDH. There were no significant changes in creatinine levels but a significant increase in albumin level in diabetic and non- diabetic groups were seen when compared with I/R control group.

The values are expressed as Mean ± SD (n=5). ***=p<0.05 as compared to I/R control, ns= non-significant as compared to I/R control. I/R=Ischemia and reperfusion.

Effect of TG treatment on lipid peroxidation

There was significant (P<0.05) increase in MDA level in tissue and serum were observed in Sham groups compared to I/R control groups.

Treatment with Fenugreek (200 mg/kg, 400 mg/kg) decreased the concentration of MDA significantly (P<0.05) in tissue (diabetic and non- diabetic groups) and serum (Diabetic group only) compared to I/R control groups.

Effect of TG on antioxidants enzyme activities

It was observed that there was significant (P<0.05) decrease in antioxidants enzymes SOD and catalase levels in tissue were observed in I/R control groups compared to Sham groups.

Treatment with Fenugreek (200 mg/kg, 400 mg/kg) Significantly (p<0.05) increased in tissue SOD and catalase levels in comparison to I/R control groups, these observations were same in both diabetic and non- diabetic groups.

The values are expressed as Mean ± SD (n=5). ***=p<0.05 as compared to I/R control, ns= non-significant as compared to I/R control. I/R=Ischemia and reperfusion.

The values are expressed as Mean ± SD (n=5). ***=p<0.05 as compared to I/R control I/R=Ischemia and reperfusion.

Effect of TG on inflammation

In this study demonstrated that there was a non-significant increase in inflammatory biomarkers myeloperoxidase (MPO) level in Sham groups compared to I/R groups in both diabetic and non- diabetic groups. Treatment with Fenugreek (200 mg/kg, 400 mg/kg) non-significantly changed the MPO level in tissue compared to I/R control groups in diabetic and non- diabetic groups.

right of the investigator. A dorsoventral incision was made into the abdominal cavity, down the sides of the rat near to the coastal border of the thorax. The kidney was freed of connective tissue and was pulled out gently, preferably by grasping the periental fat. The adrenal gland which was attached loosely to the anterior pole of the kidney by connective tissue and fat was gently freed by tearing the attachment. A single ligature was placed around the renal blood vessel and the ureter as far from the kidney as possible, toward the midline, but without damaging or occluding any collateral blood vessel that may be encountered. The ligature was tied securely with a double reef knot and the blood vessels are transacted next to the kidney which was removed. The kidney was cleaned of excess tissue, blotted, and weighed. The incision was closed in the usual way.^15,16

Histopathological changes in the kidney

Histopathological observations made in the present study revealed that Ischemia reperfusion leads to mesangial proliferation, inflammation, and tubular haemorrhage in the tissue and on treatment with Fenugreek (400 mg/kg) alleviated these histopathological changes.

In this study demonstrated that there was a non-significant increase in inflammatory biomarkers myeloperoxidase (MPO) level in Sham groups compared to I/R groups in both diabetic and non- diabetic groups. Treatment with Fenugreek (200 mg/kg, 400 mg/kg) non-significantly changed the MPO level in tissue compared to I/R control groups in diabetic and non- diabetic groups.

Discussion

Diabetic nephropathy is one of the major microvascular complications of diabetes mellitus. Enhanced oxidative stress contributes a major role in pathological progress of diabetic nephropathy through overproduction of ROS, reduced level of antioxidants enzymes, and the formation of lipid peroxides.

A high degree of oxidative stress in the renal tissue is indicated in the mesangial expansion of the extracellular matrix or increased glomerular filtration rate, proteinuria, and glomerulosclerosis. Tsukhara et al reported that a high degree of oxidative stress increases the level of urine albumin excretion, urinary protein, and other functional changes like levels of serum creatinine and BUN. Structural abnormalities include an increase in the thickness of GBM between the glomerulus capillary and the pedicle of the podocyte in the diabetic kidney.

Hellberg and Kallskog reported that ischemia-reperfusion causes aggregation of polymorphonuclear cells in the glomeruli and stimulates for the release of chemical mediators viz. free radicals, which are then filtered and injured tubular cells from the luminal side of the tubule. Hence deciduation of tubular cells into lumen takes place which results in tubular occlusion and decreased renal function.

The present study was designed to investigate the renoprotective effect of Fenugreek on ischemia reperfusion-induced renal damage in uni-nephrectomized Diabetic rats.

This study revealed that diabetes produced a significant reduction in rat weight as compared to the non-diabetic group. Uninephrectomy when combined with diabetes mellitus produced a significant reduction in rat weight than in diabetes mellitus alone. Weight loss despite normal or increased appetite is a common feature of DN in the intact and uni-nephrectomized diabetic group.

Treatment with Fenugreek has shown restoration of 24 h urine volume slightly towards normal value. This effect maybe because of the control of glomerular pressure, systemic pressure, and renal blood flow.²

Antioxidant enzymes were induced to protect against cellular and tissue injury. An imbalance between the production of ROS and antioxidants is believed to be involved in diabetes-induced renal failure.

Fenugreek contains flavonoids and phenolic compounds is a potent source of antioxidants have exerted an influence on the activity of radical scavenger enzymes that had been diminished under ischemia-reperfusion, resulting in increased activity of SOD, a catalyst of the generation of the H2O2 from superoxide anion increased activity of catalase which eliminates H2O2, inhibits the production of hydroxyl ions.

In the present study, SOD activity decreased significantly in normal and diabetic control groups subjected to ischemia-reperfusion injury. This may be due to the excessive formation of superoxide anions. This reduction in SOD activity was significantly (P<0.05) reversed by different doses (200, 400 mg/kg) of Fenugreek seeds extract in the diabetic group, (400 mg/kg) only in non-diabetic group.

The activities of H₂O₂ scavenging enzyme CAT was also decreased significantly (P<0.05) in normal, diabetic control groups subjected to I/R injury. The decline in the enzyme level may be explained by the fact that excessive superoxide anions may inactivate SOD, thus, resulting in an inactivation of the H₂O₂ scavenging enzyme.

Treatment with Fenugreek (200 and 400 mg/kg) increased the levels of CAT enzyme and considerably improves cellular antioxidative defense against oxidative stress.

Malondialdehyde (MDA) is a major aldehyde product of lipid peroxidation and increased malondialdehyde content may contribute to increased generation of free radicals. Gowda TSS and Anuradha CV reported that induction of diabetes causes significant elevation of MDA level as compared with the control group.^28,3 In the present study, treatment with Fenugreek decreased in the tissue MDA level in the treatment groups of both diabetic and non-diabetic groups. Decreased serum MDA was observed in diabetic treatment groups only when compared with respective sham and I/R control groups (no significant changes in the non-diabetic group).

Myeloperoxidase (MPO) functions as a major enzymatic catalyst for initiation of lipid peroxidation at sites of inflammation. As per earlier research, Fraser DJ reported during ischemia, the circulatory cells secrete inflammatory cytokines in the damaged areas and cause inflammation.²⁹

Treatment with fenugreek seed extract possesses the non-significant decrease in the MPO level in treatment groups compared to sham and I/R control groups of diabetic and non-diabetic groups.

A raised urinary albumin excretion rate is considered to be the strongest predictor of the development of DN. The greater extent of blood pressure control, decrease glomerular capillary permeability to proteins and reduced filtration surface area might be the possible explanation.¹

In this study, in treatment with Fenugreek possess a significant increase in serum albumin level with drug therapy to compare to I/R control group of both diabetic and non-diabetic group.

In diabetes, basement membranes, especially in the microvasculature, are subjected to alteration of structure and function such as diffuse and sometimes nodular thickening. The greatly expanded mesangial matrix associated with these pathological lesions obliterating many capillary lumina results in the reduction of the surface area available for filtration. Thus, in turn, would lead to the accumulation of creatinine in blood and a subsequent decrease in creatinine clearance.

In this present study treatment groups showed a non-significant reduction of creatinine level in plasma compared to I/R control group of diabetic group. The non-diabetic group did not show any difference compared to I/R control group.

BUN is an important marker of renal function this is influenced by protein metabolism.²⁷ The treated groups had shown a significant reduction in blood protein which indicates the improvement in renal function. Increased blood urea levels were observed in I/R of both diabetic and non-diabetic group. Treatment with fenugreek seeds extracts significantly reduced the elevated levels of blood urea in UNDD and UNDND rats.

Histopathological observations made in the present study revealed that Ischemia-reperfusion leads to Mesangial proliferation, inflammation, and Tubular haemorrhage in the tissue and on treatment with Fenugreek (400 mg/kg) alleviated these histopathological changes.

Arora S reported that oral administration of fenugreek seeds powder for three weeks in alloxan-induced diabetic rats against oxidative stress stabilize glucose homeostasis and free radical metabolism in the kidney. Fenugreek seeds have been shown to have potent antioxidant effects and protect many vital organs in the body against oxidative stress induced by streptopozptocin.³⁰

The exact composition responsible for the beneficial effect of DM induced renal complications is yet challenging. Individual constituents of Fenugreek seeds like 4- Hydroxyisoleucine, Glactomannan, Trigonellin, demonstrated the potent anti-diabetic effect in animals. The earlier literature supported the hypothesis for anti-inflammatory and/or antioxidant activity as a potential mechanism of renoprotective action of Fenugreek.³¹

Taken together, this study suggested that Fenugreek protects kidney function against functional and morphological injuries via its antioxidants and anti-inflammatory activities.

Conflict of Interest

The authors declare no conflict of interest.