Renoprotective Effects of Reconstructed Composition of Trigonella foenum-graecum L. Seeds in Animal Model of Diabetic Nephropathy with and without Renal Ischemia Reperfusion in Rats
Subhash L. Bodhankar,
Prasad A. Thakurdesai
Diabetic nephropathy is the kidney complication of diabetes mellitus and leading to end stage renal disease. In the past, reconstructed antidiabetic combination from Trigonella foenum-graecum (fenugreek) seeds (IND01) containing 4-hydroxyisoleucine (40%), trigonelline (30%) and galactomannan (30%) showed excellent antihyperglycemic activity. The objective of the present study was to evaluate the renoprotective efficacy of IND01 in animal model of diabetes with and without ischemia reperfusion injury. The diabetes was induced by alloxan in dose of 160 mg kg-1, intraperitoneally (early nephropathy model, without ischemia reperfusion) or 70 mg kg-1, i.v. (model with ischemia reperfusion). In both models, effects of oral treatment of IND01 (50, 100 and 200 mg kg-1, daily once for 30 days) were observed on biochemical parameters (creatinine clearance) and urine (blood urea nitrogen). On day 30, rats were sacrificed and histology was performed on isolated kidneys. Alloxan administration with or without ischemia reperfusion showed symptoms of severe nephropathy (decreased creatinine clearance, increased BUN, presence of glomerular matrix formation, tubular necrosis, interstitial inflammation and fibrosis). The daily oral administration of IND01 (50-200 mg kg-1) showed potent and mild renoprotective effects on biochemical parameters against diabetic rats without ischemia (early nephropathy) and with ischemia model respectively. IND01 showed moderate protection from histological abnormalities in kidney of alloxan-induced rats without ischemia reperfusion injury (early nephropathy model). However, such protection was not offered by IND01 in alloxan induced rats with ischemia reperfusion injury. In conclusion, IND01 showed renoprotection in animal model of early nephropathy probably by effective glycemic control.
to cite this article:
Sachin Arora, Subhash L. Bodhankar, V. Mohan and Prasad A. Thakurdesai, 2012. Renoprotective Effects of Reconstructed Composition of Trigonella foenum-graecum L. Seeds in Animal Model of Diabetic Nephropathy with and without Renal Ischemia Reperfusion in Rats. International Journal of Pharmacology, 8: 321-332.
Received: January 23, 2012;
Accepted: March 31, 2012;
Published: June 13, 2012
Diabetic nephropathy (DN) is the kidney disease that occurs as a result of
diabetes mellitus (DM). DN is the single most common disorder leading to renal
failure (Sampanis, 2008). Between 20 and 40% of patients
with diabetes, eventually develop DN, which is the leading cause of end-stage
renal disease (ESRD) and needs dialysis or kidney transplant (Sampanis,
2008). Both clinical and experimental data suggest that hyperglycemia increases
the risk of acute renal failure (Wald et al., 1990).
Conversely, ischaemiareperfusion (I/R) combined with hyperglycemia could
also be important in the development of diabetic nephropathy (Melin
et al., 2003). After many years of DM, the delicate filtering system
in the kidney gets destroyed, becoming leaky to large blood proteins such as
albumin that are lost in urine. Its incidence and prevalence has increased to
alarming proportion in the past decade despite recent therapeutic advances.
In patients with longer lasting DM, particularly in the so-called decompensated
DM, the occurrence of specific and non-specific chronic complications are common
(Brilla, 1997). DN is a result of interaction of metabolic
and haemodynamic factors. Glucose-dependant pathways are activated within the
diabetic kidney. The causes for this activation include increased oxidative
stress, renal polyol formation, advanced glycated end-products (AGEs) accumulation
and presclerotic cytokines, such as transforming growth factor-β1 (TGF-β1).
These pathways eventually lead to increase in renal albumin permeability and
extra cellular matrix build-up, which results in increasing proteinuria, glomerulosclerosis
and tubulointerstitial fibrosis.
DN patients need to maintain the vital functions and improve the quality of
life. This objective is only possible by reducing the need of dialysis and improving
their renal functions to normal levels. Currently, Angiotensin Converting Enzyme
(ACE) inhibitors and angiotensin-II (AT-II) receptor blockers are used to treat
DN. Although, these drugs are useful, they need to be monitored as they may
have detrimental effects in some people. Novel targets, which are linked to
glucose dependent pathways, are the major focus of new therapies directed against
diabetes induced renal damage. It is likely that resolution of DN will require
synergistic therapies to target multiple mediators of this disease (Bachar
and Lichtstein, 1993). Hence, the nephropathic population of patients is
in dire need for alternate medication.
Fenugreek (Trigonella foenum-graecum L. Family: Leguminosae), a spice
rich in dietary fibres has traditional history of medicinal use in the management
of diabetes. Anecdotal report suggest a 3000 year old history of medicinal use
for fenugreek in Egypt, Southern Europe, India, Asia and northern Africa (WHO,
2007). In recent decades, several health beneficial physiological attributes
of fenugreek seeds have been reported in animal studies as well as human trials
(Ulbricht et al., 2007; Kang
et al., 2008). Trigonella foenum-graecum seeds have previously
been shown to have hypoglycemic and hypocholesterolemic effects on type 1 and
type 2 DM patients (Sharma et al., 1990; Gupta
et al., 2001; Yadav et al., 2008)
and experimental diabetic animals (Jelodar et al.,
2005; Abdelatif et al., 2012). Trigonella
foenum-graecum seed extract showed improvement in diabetic rats in a dose-dependent
manner (Xue et al., 2007). The parameters like
kidney to body weight ratio, blood glucose, glycated haemoglobin (HBA1c), triglycerides,
total cholesterol and higher-density-lipoprotein-cholesterol lipid peroxidation
and antioxidant status and hemorheological properties were improved in alloxan-induced
diabetic rats (Xue et al., 2007).
Recently, efficacy of fenugreek oil in to ameliorate DM and improvement in
renal toxicity is reported (Hamden et al., 2010).
Oral administration of fenugreek seed powder for 3 weeks in alloxan-induced
diabetic rats stabilized glucose homeostasis and free radical metabolism in
kidney (Thakran et al., 2003). Further, protective
effects of fenugreek seed powder on kidney histology of toxicant-induced kidneys
are reported (Thakran et al., 2004; Sushma
and Devasena, 2010). However, the exact composition responsible for beneficial
effect on DM induced renal complications is yet challenge.
Fenugreek seed mainly contain 4-hydroxyisoleucine (4-HI), trigonelline, galactomannan
with flavonoids, carotinoids, coumarins, proteins, saponins and lipids (Al-Habori
et al., 2001; Basch et al., 2003).
Individual constituents of fenugreek seeds like 4-HI (Sauvaire
et al., 1998; Broca et al., 2000;
Broca et al., 2004; Narender
et al., 2006; Singh et al., 2010),
trigonelline (Mishkinsky et al., 1967; Shah
et al., 2006a), galactomannans (Hannan et
al., 2003; Hannan et al., 2007; Kamaljit
et al., 2011) demonstrated potent antidiabetic effect in animals.
4-HI has been found to be a major free amino acid in the seeds (Sauvaire
et al., 1976). Trigonelline is a major alkaloid constituent of the
fenugreek seeds (Mishkinsky et al., 1967). Galactomannans
are polysaccharides consisting of mannose backbone with galactose side groups.
The defatted fenugreek seed contains about 40% of 4-HI, 30% of trigonelline
and 30% of galactomannan. In the past, we have reported antihyperglycemic activity
of reconstructed composition of fenugreek seeds, IND01, which contains 4-HI
(40%), trigonelline (30%) and galactomannan (30%) (Shah
et al., 2006b). IND01 also reported to have synergistic antihyperglycemic
interaction with synthetic antihyperglycemic molecules like pioglitazone and
glyburide (Shitole et al., 2009). However, effects
of IND01 on renal complications of diabetes are yet unknown. The aim of present
work was to evaluate effects of long-term treatment of IND01 in kidneys of diabetic
rats with or without renal I/R.
MATERIALS AND METHODS
Drugs and chemicals: The preparation of IND01 involve the extraction,
isolation and characterization of 4-HI, trigonelline and low molecular weight
galactomannan and was carried out as per reported methods (Shah
et al., 2006a,b; Shah
et al., 2009). These isolated phytochemicals were mixed in proportion
of 40:30:30, respectively to get reconstructed composition product, IND01. The
test solution of IND01 was freshly prepared in by dissolving in distilled water
in concentration of 100 mg mL-1 and was administered to animal based
on body weight. Alloxan monohydrate (Sigma-Aldrich, USA.), pioglitazone (Ajantha
Pharma Pvt. Ltd., India) and insulin: (Mixtard 30 HM, 40 IU mL-1
Novo Nordisk) and anaesthetic ether (TKM Pharma, Hyderabad, India) were purchased
from respective vendors. Biochemical kits for estimation of creatinine (Jaffe
method), urea (Enzymatic method) and glucose (Accurex Biomedical Pvt. Ltd.,
Mumbai, India) were purchased respective vendors.
Animals: Male Wistar rats (150-200 g) were purchased from National Toxicology
Centre, Pune. During the experiment, rats were housed at standard housing condition
like temperature of 25±1°C, relative humidity of 45-55% and 12 h.
light: 12 h dark cycle. Research protocol was approved by Institutional Animal
Ethics Committee (IAEC) of Poona College of Pharmacy, Bharati Vidyapeeth Deemed
University, Pune. The rats had free access to food pellets (Amrut Laboratory,
Navmaharashtra Chakan Oil Mills Ltd., Sangli, India) and tap water ad libitum
during the experiments.
Early nephropathy in diabetic rats (Mishra et al.,
2010): All rats except those of Group I, i.e., normal or Non-diabetic,
were treated with alloxan (160 mg kg-1, i.p.) to induce hyperglycemia
and diabetes. Serum Glucose (SG) levels of rats were determined after 48 h of
injection (day 0 of study) using Accurex glucose estimation kits. Rats with
serum glucose above 300 mg dL-1 were selected. The selected diabetic
rats were divided into group II to VI of 6 rats each. Rats of group II and III
were orally gavaged with distilled water (1 mL kg-1) and standard
antidiabetic agent, pioglitazone (10 mg kg-1) once a day for 30 days,
respectively. Rats from group IV, V and VI were orally gavaged with IND01 at
dose of 50, 100 and 200 mg kg-1, respectively once a day for 30 days.
The biochemical parameters of all rats were measured on 0, 15 and 30th day and
histological studies were done on kidney samples after sacrifice on 30th day.
Renal I/R injury in diabetic rats (Melin et al.,
2002): All rats except those of Group A, i.e., normal or Non-diabetic,
were treated with alloxan (70 mg kg-1, i.v.) to make them diabetic.
Serum Glucose (SG) levels of rats were determined after 48 h of injection (day
0 of study) using Accurex glucose estimation kits. Rats with SG levels above
300 mg dL-1 were selected for this study. Selected rats were randomly
divided into groups of 6 rats (Group B to G). Seven days before induction of
renal I/R, Group A, B and C had received once daily treatment of vehicle (distilled
water) for 7 days before I/R surgery. Rats in Group D were pretreated with insulin
(8 IU, s.c.) daily for 7 days. Rats from group E, F, G were pretreated with
IND01 (50, 100 and 200 mg kg-1, p.o. daily once), respectively for
After completion of 7 days of pretreatment (Day 0 of study), renal I/R was
induced in the rats of group C to G. Group B rats, called as sham control, had
small cut in skin but no I/R induction. Group A rats (Normal) neither had I/R
or cut to the skin. The rats form group C, D. E. F and G were anaesthetized
by intraperitoneal (i.p.) injection of thiopental sodium in a dose of 50 mg
kg-1. During the operation the animals were placed on a servo-controlled
heating pad keeping the body temperature at 37.5°C. A left flank incision
was made and the left renal artery was located and dissected free from its surrounding
structures. After a recovery period of 10 min, a small midline incision was
made to isolate left renal artery and clamped for 30 min. Subsequently the wound
in the abdomen was sutured, povidone iodine ointment was applied and animals
were housed individually in cages. The rats were then allowed to recover and
dosing was continued as mention above till day 30th day after surgery. Blood
and urine samples were collected on 0, 10, 20 and 30th day after surgery for
biochemical and renal function estimations.
Measurement of serum biochemical and renal function parameters (Sharma
et al., 2006): On selected days, rats were placed in metabolic
cages (Techniplast, Milan, Italy) and 24 h urine was collected. On each of these
days, rats were anaesthetized by anaesthetic ether and blood was withdrawn by
retro-orbital puncture using micro capillary tubes. Serum was obtained by centrifuging
the blood at 7000 rpm. at 4°C. The creatinine levels and Blood Urea
Nitrogen (BUN) were measured in serum and 24 h urine samples, respectively.
Creatinine clearance (CCr) was calculated by reported method (Cockcroft
and Gault, 1976):
Histopathology of kidneys in diabetic rats (Melin et
al., 1997; Melin et al., 2002): On
30th day, after collection of urine and serum, the animals were sacrificed.
Their kidneys were removed, stored in 10% formalin until dissected. At the time
of dissection, kidneys were divided into two halves (parallel to major axis)
and washed. These tissues were processed for 12 h using isopropyl alcohol and
xylene and embedded in paraffin. Paraffin embedded tissues were sectioned (5
μm thickness) and stained using haematoxylin and eosin (H and E). Histological
changes in the glomeruli, tubules, interstitium and blood-vessels were recorded
for each specimen by photomicrographic examination with light microscope (Olympus,
USA) fitted with camera (Nikon E200). Renal changes were scored using a scale
of none (-), mild (+), moderate (++) and severe (+++) damage.
Statistical analysis: Data was expressed as Mean ± SEM for each
biochemical parameter. Data for CCr and BUN was separately analyzed by two way
ANOVA followed by Bonferroni Posttest for each model. p<0.05 was considered
Effect of IND01 on creatinine clearance (CCr) in alloxan-induced DN in rats
without I/R: Alloxan (160 mg kg-1, I.p.) injection significantly
reduced CCr as compared with non-diabetic (normal) rats within 48 h.
|| Effect of IND01 on creatinine clearance (CCr) and blood urea
nitrogen (BUN) in alloxan-induced diabetic rats without ischemia reperfusion
|aValues in the bracket are dose in mg kg-1,
per oral, All rats from all groups except normal group were made diabetic
with alloxan (160 mg kg-1, i.p.) treatment, Values are expressed
as mean±SEM. (n = 6), Data was analyzed by two-Way ANOVA followed
by Bonferroni Posttest for each parameter separately. # p<0.5, ## p<0.01
and ### p<0.001 as compared with non-diabetic rats; *p<0.05, **p<0.01,
***p<0.001 as compared to diabetic control rats on respective days
|| Effect of IND01 on creatinine clearance (CCr) in alloxan-induced
diabetic rats with ischemia reperfusion (I/R)
|All rats from all groups except normal group were made diabetic
with alloxan (70 mg kg-1, i.v.) treatment, Values are expressed
as mean±SEM, (n = 6), Data was analyzed by two-Way ANOVA followed
by Bonferroni Posttest for each parameter separately. # p<0.5, ## p<0.01
and ### p<0.001 as compared with non-diabetic rats; $ p<0.05, $$ p<0.001,
$$$ p<0.001 as compared with diabetic sham rats; *p<0.05, **p<0.01,
***p<0.001 as compared to diabetes with I/R rats on respective days
This fall suggests induction of kidney dysfunction by alloxan. The fall in
CCr was sustained during the study period of 30 days (Table 1).
Daily once treatment of pioglitazone (10 mg kg-1, p.o.) and IND01
(200 mg kg-1, p.o.) in alloxan-induced rats (group 2) improved CCr
from 15th days of treatment. Lower dose of IND01 (50 and 100 mg kg-1,
p.o.) treatment improved CCr on day-30.
Effect of IND01 on Blood Urea Nitrogen (BUN) in alloxan-induced DN in rats without I/R: Sustained increase (p<0.001) in BUN levels were observed in alloxan-treated (diabetic) rats as compared normal (non-diabetic rats). Moreover, BUN levels were significantly higher on day 30 from baseline (day 0) (Table 1). Pioglitazone treatment significantly (p<0.001) lowered BUN levels from day-15 onwards. IND01 treatment showed significant decrease in BUN levels only after 30 days of treatment (Table 1).
Effect of IND01 on creatinine clearance (CCR) in alloxan-induced DN in rats
with I/R: Alloxan (160 mg kg-1, i.p.) pretreatment brought significantly
sharp fall in CCr compared with non-diabetic (normal) as well as diabetic sham
control rats within 48 h. This fall suggests induction of reduced kidney function
in diabetic rats. The fall in CCr was sustained in the study period of 30 days.
Daily treatment of Insulin (8 IU, s.c.) and IND01 (100 and 200 mg kg-1,
p.o.) to renal I/R in diabetic rats showed improved CCr form at baseline (48
h. after diabetes induction). Insulin produced significant CCr improvement compared
with diabetes with I/R rats till day 20 of study. Improvement in IND01 treated
rats did not sustain beyond 10 days (dose 200 mg kg-1). On day-30,
neither Insulin nor IND01 showed significant improvement in CCr compared with
diabetes rats with I/R (Table 2).
Effect of IND01 on Blood Urea Nitrogen (BUN) in alloxan-induced DN in rats
with I/R: Sustained increase (p<0.001) in BUN levels in alloxan treated
rats were found as compared normal (non-diabetic rats) and BUN levels were significantly
higher form baseline (day 0) to 30-days (Table 3). Pioglitazone
treatment significantly (p<0.001) lowered BUN levels from day-15 onwards.
IND01 treatment showed significant decrease in BUN levels only after 30 days
of treatment (Table 3). At baseline (48 h. after diabetes
induction), BUN levels in ischemic-injured diabetic rats were found as significantly
higher (p<0.001) compared to non-diabetic and diabetic sham control rats.
Significantly (p<0.001) higher BUN levels were observed in ischemic-injured
diabetic rats during the study period of 30 days.
||Photomicrograph of section of Kidneys showing glomerulus of
alloxan-induced diabetic rats after 30 days of treatment with (a) Normal
(b) Alloxan (160 mg kg-1, i.p.) (c) Pioglitazone (10 mg kg-1,
p.o.) (d) IND01 (50 mg kg-1, p.o.) (e) IND01 (100 mg kg-1,
p.o.) and (f) IND01 (200 mg kg-1, p.o.), Sections were stained
with hematoxylin and eosin (HE) at magnification of 100 X
|| Effect of IND01 on blood urea nitrogen (BUN) in alloxan-induced
diabetic rats with ischemia reperfusion (I/R)
|All rats from all groups except normal group were made diabetic
with alloxan (70 mg kg-1, i.v.) treatment. Values are expressed
as mean±SEM. (n = 6), Data was analyzed by two-Way ANOVA followed
by Bonferroni Posttest for each parameter separately, # p<0.5, ## p<0.01
and ### p<0.001 as compared with non-diabetic rats, $ p<0.05, $$ p<0.001,
$$$ p<0.001 as compared with diabetic sham rats, *p<0.05, **p<0.01,
***p<0.001 as compared to diabetes with I/R rats on respective days
Insulin (8 IU, s.c., once a day) treatment did not show significant rise in
BUN as shown by ischemic-injured diabetic rats (Table 2).
On 30-days of pre-treatment, IND01 (50 or 100 mg kg-1, p.o., once
daily), did not show any reduction in BUN in Ischemic-injured rats. However,
at dose of 200 mg kg-1, p.o., IND01 showed mild (p<0.05) reduction
from raised BUN levels as compared with ischemic-injured diabetic rats. Effect
of IND01 on histology of kidneys in alloxan-induced DN in rats without I/R:
The histological observations of sections of kidneys of alloxan treated rats
showed hyperplasia of mesangium, glomerular basement membrane thickening, tubular
atrophy and interstitial inflammation of mild to moderate intensity confirming
induction of nephropathy (Table 4 and Fig. 1).
These changes were absent in rats treated with pioglitazone and IND01 (200 mg
kg-1) which confirms renoprotective action as well as antihyperglycemic
effects. However, lower doses of IND01 (50 and 100 mg kg-1), caused
certain degree (although mild in intensity) of hyperplasia of mesangium and
glomerular basement membrane enlargement. Except normal (non-diabetic) rats,
moderate tubular atrophy, tubular dilation and interstitial inflammation was
noted in all the kidney samples (including kidneys of IND01 and pioglitazone
treated diabetic rats), which suggest irreversible nature of tubular atrophy
in diabetic nephropathy.
Effect of IND01 on kidney histopathology in alloxan-induced DN in rats with
I/R: The histological observations of sections of kidneys of ischemic-injured
diabetic rats showed moderate to severe (++ to+++) glomerular cellularity, glomerular
matrix formation, tubular necrosis with dilatation, interstitial inflammation
|| Photomicrograph of section of Kidneys showing glomerulus
on day-30 in rats alloxan-induced diabetic rats without renal I/R in diabetes
sham (a) and with I/R (b), respectively are shown. The kidneys of rats with
7-day pre - and 30 day post I/R treatment with (c) Insulin (8 IU, s.c.),
(d) IND01 (50 mg kg-1, p.o.), (e) IND01 (100 mg kg-1,
p.o.), (f) IND01 (200 mg kg-1, p.o.) in diabetes with renal I/R
induction are also shown. Sections were stained with hematoxylin and eosin
(HE) at magnification of 100 X
|| Effect of IND01 on histopathology of sections of kidney in
early DN in alloxan-induced diabetic rats
|All rats from all groups except normal group were made diabetic
with alloxan (160 mg kg-1, i.p.) treatment, +:- Mild ++:- Moderate,
+++:- Severe -- :- Absent
|| Effect of IND01 on histopathology of sections of kidney in
I/R induced in alloxan-induced diabetic rats
|All rats from all groups except normal group were made diabetic
with alloxan (70 mg kg-1, i.v.) treatment, +: Mild + +: Moderate
+ + +: Severe, -- : Absent figure legends
Diabetic sham control rats also showed mild changes in kidney morphology (Table
5 and Fig. 2). In our study, insulin treatment caused
complete prevention in kidney sections with respect to glomerular cellularity
and matrix formation during 30 days of period whereas IND01 treatment prevented
these changes partially. Mild to moderate tubular necrosis with dilatation,
interstitial inflammation and fibrosis were observed in all the kidney sections,
which suggest irreversible nature of tubular atrophy. These changes were severe
in Ischemic-injured diabetic rats. The rats treated with Insulin or IND01 for
30 days showed only mild changes compared to rats having diabetes and I/R (Table
One of the specific chronic complications is represented by the finding of diabetic nephropathy that is characterized by proteinuria, frequent hypertension and slow gradual alteration of renal functions. In a narrow sense of the word, diabetic nephropathy is referred to as microangiopathic impairment of kidneys.
Fenugreek as a diet supplement causes a marked decrease in symptoms of diabetes
in terms of polydypsia, polyuria, urine sugar, renal hypertrophy and glomerular
filtration rate (Shetty and Salimath, 2009). In the
present study, we investigated effects of reconstructed composition of Trigonella
foenum-graecum L. seeds. Each ingredient in fenugreek extract has different
chemical constituents with complex molecular formulae, which must be beneficial
for different aspect of diabetes management (Hassanzadeh
et al., 2011). Another challenge is consistency in quality and efficacy
of composition (Ansari and Inamdar, 2010; Sarwar
et al., 2011). The present study was an attempt to study optimized
composition for the therapeutic effects in the animal model of renal complication
of DM. IND01 is reconstructed composition which contains active ingredients
of fenugreek seeds in same proportion as they found in nature.
In the present study, alloxan with or without renal ischemic-injury produced
severe biochemical or histological in rats kidneys. CCr levels were decreased
and BUN levels were increased in diabetic as well as renal I/R with diabetic
rats. These results are consistent with earlier reports on alloxan model (Spadella
et al., 1998) and effect of pioglitazone (Stendig-Lindberg,
In the present study, consistent decrease in CCr in the diabetic rats with and without renal I/R was observed. IND01 showed dose-dependent improvement in CCr in alloxan induced diabetes rats after 30 days of treatment. In the diabetic rats with renal I/R , pretreatment of IND01 for 7 days could offer only moderate reversal of CCr. The protective effects of IND01 on CCr were found be decline over the period of 30 days (Table 3). On the other hand, administration of insulin (8 IU, s.c.) prior to I/R in diabetic rats offered consistent improvement in CCr during the study period in diabetic rat with I/R (Table 3).
CCr is an effective means of assessing renal function and useful indicator
of glomerular filtration rate (GFR). CCr is the removal of creatinine from the
body and defined as the volume of blood plasma that is cleared of creatinine
per unit time. The pathophysiology of DN involves glucose that binds irreversibly
to proteins of kidneys and AGEs, that can form complex cross-links over years
of hyperglycemia. DN can cause renal damage by stimulation of growth and fibrotic
factors via receptors for AGEs. Increased glomerular capillary pressure occurs
early in diabetes and is associated with hyperfiltration at the glomerulus.
The glomerular mesangium expands, initially by cell proliferation and then by
cell hypertrophy. Increased mesangial stretch and pressure, as well as high
glucose levels, can stimulate this expansion. Besides, the role of glycosylation
of proteins is involved in alloxan induced DN model in animals (El-Mekawi
et al., 1993). Higher level of glycosylation of urinary proteins
after alloxan induced diabetes rats after 7 days of treatment had been reported
earlier (El-Mekawi et al., 1993). Therefore,
ability of any compound in reversing renal dysfunction point to good control
of glycosylation of protein. In present study, the protection from renal dysfunction
by IND01 in early-stage diabetic nephropathy can be attributed to probable fall
in AGEs and improved glycemic control. Fenugreek has been reported to improve
glycemic control and glycated haemoglobin (HBA1c) status in diabetes patients
(Gupta et al., 2001; Kassaian
et al., 2009) and animals (Devi et al.,
2003; Xue et al., 2007). Galactomannan from
fenugreek is especially reported for this purpose (Srichamroen
et al., 2008).
Similar effects were noted in terms of BUN measurements in the present study.
IND01, pioglitazone and insulin showed significant improvement of BUN levels
in the doses tested against diabetes with or without renal I/R. However, onset
of action to achieve the protection was quickest for Insulin (48 h after diabetes
with renal I/R) followed by pioglitazone (15 days in diabetes without I/R) and
IND01 (30 days in both models). IND01 at a dose as low as 50 mg kg-1,
p.o., showed potent protective effects in diabetic rats (without I/R). In presence
of renal I/R in diabetic rats, IND01 could only produce mild effects at highest
dose (200 mg kg-1) after 30 days of treatment. This observation is
consistent with earlier report that insulin offers renal protection from I/R
when improved metabolic control is achieved before renal I/R (Melin
et al., 2002). The control of hyperglycemia before I/R is important
for renoprotective action of insulin and IND01 in diabetes in presence of renal
BUN measurement is another kidney function assessment test. Urea is a by-product of protein metabolism and is formed in the liver. Urea is excreted in urine and undergoes tubular reabsorption. Because of tubular atrophy in DM, tubular reabsorption of BUN is affected. Therefore, high amount of BUN in the diabetic rats with or without renal I/R indicate impairment of tubular reabsorption which was also observed in our study.
The changes in kidneys that can be evaluated histopathologically depend on
the duration of diabetes and how diabetes was treated. In short-lasting untreated
diabetes, character of diabetic nephrosis is found to be manifested microscopically
in diabetic patients (Farquhar et al., 1959;
Meoro et al., 1999; Rosai and
Ackerman, 2004) as well as alloxan-induced rats (Bartosikova
et al., 2003). In the present study, sections of kidneys of diabetic
rats with and without I/R showed cellularity, glomerular matrix formation, tubular
necrosis with dilatation, interstitial inflammation and fibrosis. The intensity
of kidney damage was severe (+++) with I/R whereas moderate damage (++) was
observed without I/R. Daily oral administration of IND01 (50-200 mg kg-1)
for 30 days seems to provide moderate (++) protection from histological abnormalities
in kidneys of early experimental DM (without I/R). However, such protection
was not offered by IND01 in diabetes with I/R model.
Alloxan is known to produce tubular atrophy, interstitial inflammation, hyperplasia
of mesangium and glomerular basement membrane enlargement. These renal changes
are because of high glucose levels present for longer period in the diabetic
rats. There is growing evidence that hyperglycemia results in altered renal
oxygen metabolism and decreased renal oxygen tension and that these changes
are linked to altered kidney function (Shafrir and Gutman,
1993). Current evidence suggests the selective cytotoxicity of alloxan is
because of efficient uptake, oxidant production by redox coupling of alloxan
with intracellular reductant (ascorbate and thiols) coupled with low levels
of glutathione peroxidase in the islets (Malaisse, 1982).
Much of the evidence concerning the role of oxidative stress in the induction
of DM comes from the studies of alloxan which produces diabetes in experimental
animals (Wolff, 1993; Balkis et
al., 2008). Alloxan selectively destroys the islets of Langerhans by
oxidant production, enhances lipid peroxidation and susceptibility to oxidative
stress associated with depletion of antioxidants in liver, kidney and pancreas
(Anuradha and Ravikumar, 2001). Alloxan toxicity in
vitro and in vivo can be inhibited by many free radical scavengers
and lipid-soluble antioxidants (Malaisse, 1982; Wolff,
1993). Therefore, alloxan-induced DM served as pathological bio-model for
testing a substance with supposed antioxidative activity in vivo.
Fenugreek seeds have been shown to have potent antioxidant effects and protect
many vital organs in the body against oxidative stress induced by alloxan (Ravikumar
and Anuradha, 1999; Anuradha and Ravikumar, 2001;
Al-Wabel et al., 2008; Al-Matubsi
et al., 2011; Premanath et al., 2011).
Therefore, antioxidant potential can also envisaged as possible mechanism for
renal protection shown by IND01 in the present study. However, antioxidant activity
in fenugreek seeds was primarily attributed to the presence of flavonoids and
polyphenols (Dixit et al., 2005). The absence
of flavonoids and/or polyphenols in test composition (IND01) in present study
pointed towards mechanisms other than anti-oxidant potential.
On the other hand, sustained hyperglycemia caused by alloxan administration
results into many biochemical and histological abnormalities and lead to nephropathy
in rabbits (Winiarska et al., 2008), mice (Nordquist
et al., 2008) and rats (Macedo et al.,
2007). Further, chronically hyperglycemic alloxan diabetic rats showed reduced
glomerular filtration rates renal plasma flow (p-aminohippurate clearance) and
extracellular fluid volume associated with urinary Na+ losses which
eventually reduces GFR (Di Loreto et al., 2004).
It has been postulated that glomerular hyperfiltration or elevated GFR in early
diabetes may eventually cause glomerular damage, leading to a progressive loss
of renal function (diabetic nephropathy). Diabetic rats with long-term moderate
hyperglycemia, however, do not develop characteristic glomerular lesions of
human diabetic nephropathy and, in fact, develop only minimal glomerular injury
even after 1-year of diabetes (ODonnell et al.,
1988). Long-term diabetes in animals could accelerate the formation of the
intercellular matrix of glomerular loops in proliferative glomerulitis in rabbits,
resulting in accelerated glomerulosclerosis (Wanibuchi et
al., 1991) and further worsens the nephropathy. Thus, although the diabetic
rat with moderate hyperglycemia (induced by alloxan or streptozotocin) may be
useful to study the mechanisms in early diabetes, it may not be an appropriate
model of renal failure in IDDM (ODonnell et al.,
On the other hand, I/R combined with hyperglycaemia can mimic important mechanisms
to develop ESRD and/or diabetic nephropathy in diabetic animals (Melin
et al., 2003). Hyperglycemia is known to aggravate the renal injury
and causes acute renal ischemia in the rat (Podrazik et
al., 1989). An exceptional susceptibility to unilateral renal I/R injury
resulting in inflammation, fibrosis, atrophy of the kidney and ESRD had been
demonstrated in the diabetic rat (Melin et al., 2002).
Studies also showed that a brief I/R results in a progressive injury leading
to end-stage renal failure (similar to ESRD) in diabetic animals (Melin
et al., 1997; Melin et al., 2002).
In DM animals renal I/R caused massive induction of apoptosis in the renal medulla
after six hours as well as inflammation, fibrosis, renal atrophy and anuria
within four weeks (Melin et al., 2002). Several
mechanisms have been suggested to explain why hyperglycaemia increases I/R injury.
Intracellular oxidative has been proposed as a unifying explanation for most
metabolic alterations in diabetes (Brownlee, 2001) I/R
is also a state where oxidative stress has been implied in case of renal ischemic
injury (Chien et al., 2001). It could be speculated
that the combined oxidative stress from these two sources (diabetes and I/R)
could be especially harmful. The increased inflammatory response after I/R in
diabetes may play a role in increased sensitivity to I/R in diabetes (Panes
et al., 1996). The beneficial effects of IND01 on CCr in both the
models and mild reduction shown in basement membrane enlargement (in diabetic
I/R model) were observed in present study. The results support hypothesis for
antiinflammatory and/or antioxidant activity as potential mechanism of renoprotective
action of IND01 Besides the glomerular changes, interstitial fibrosis and infiltration
of inflammatory cells are important features of diabetic nephropathy (Bohle
et al., 1991). In fact, the degree of tubulointerstitial injury is
as closely related to the decline in function as are the glomerular alterations
(Bader et al., 1980). The tubulointerstitial
fibrosis and infiltration of inflammatory cells observed in diabetic rats after
renal I/R share similarities with diabetic nephropathy (Melin
et al., 1997). In the past, aqueous extract of fenugreek has been
reported to have antiinflammatory activity (Vyas et
al., 2008; Subhashini et al., 2011).
Fenugreek leaves extract has been shown potent anti-inflammatory effects in
animals of inflammation (Ahmadiani et al., 2001).
Because of presence of alkaloids and absence flavonoids, saponins, or steroids
in fenugreek leaves extract, authors suggested alkaloid as a responsible constituent
for antiinflammatory potential of fenugreek. Therefore, the protective effect
shown by IND01 can be partly attributed by presence of an alkaloid constituent,
trigonelline. In the past, many natural and synthetic analogs of leucine (an
amino acid) also showed antiinflammatory potential (Somasundaram
et al., 1983; Turek et al., 1991;
Bator et al., 1992; Serebruany
et al., 1995). Therefore, possible contribution of 4-hydroxyisoleucine
in the renoprotective action (through antiinflammatory mechanism) against diabetes
cannot be ruled out.
In conclusion, IND01 showed potential for renal protection against diabetic rats with or without renal I/R. The effects seem to be medicated through effective glycemic control.
The authors would like acknowledge Dr. S. S. Kadam, Vice-Chancellor and Dr. K.R. Mahadik, Principal, Poona College of Pharmacy, Bharati Vidyapeeth University, Pune, India and Sunil Bhaskaran, MD, Indus Biotech Private Limited, Pune for providing necessary facilities to carry out the study.
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