Chemico-Biological Interactions 188 (2010) 237245

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Impact of d-pinitol on the attenuation of proinammatory cytokines,

hyperglycemia-mediated oxidative stress and protection of kidney
tissue ultrastructure in streptozotocin-induced diabetic rats
Selvaraj Sivakumar, Periyasamy Palsamy, Sorimuthu Pillai Subramanian
Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamil Nadu, India

a r t i c l e i n f o a b s t r a c t

Article history: Oxidative stress plays a crucial role in the progression and development of diabetes and its complications
Received 15 May 2010 due to chronic hyperglycemia. The present study was aimed to investigate the kidney tissue protective
Received in revised form 2 July 2010 nature of d-pinitol, a cyclitol present in soybean, by assessing the key markers of hyperglycemia-mediated
Accepted 12 July 2010
oxidative stress, proinammatory cytokines and ultrastructural alterations in streptozotocin-induced
Available online 17 July 2010
diabetic rats. Oral administration of d-pinitol (50 mg/kg body weight/day) for 30 days to diabetic group of
rats showed a signicant elevation in the level of total protein and signicant decline in the levels of blood
Keywords:
urea, serum uric acid, creatinine and advanced glycation endproducts (AGEs) and kidney proinamma-
d-Pinitol
Diabetic kidney
tory cytokines such as TNF-, IL-1, IL-6, NF-B p65 subunit and nitrite. Further, d-pinitol administration
Lipid peroxidation elicited a signicant attenuation in the activities of kidney enzymatic antioxidants such as superoxide dis-
Oxidative stress mutase (SOD), catalase, glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione
Streptozotocin reductase (GR) and the levels of kidney non-enzymatic antioxidants such as vitamin E, vitamin C and
reduced glutathione (GSH) in the diabetic group of rats, with a concomitant decline in the levels of
kidney lipid peroxides, hydroperoxides and protein carbonyls. The histological and ultrastructural obser-
vations on the kidney tissues also conrmed the renoprotective nature of d-pinitol. Thus the present study
demonstrated the renoprotective nature of d-pinitol by attenuating the hyperglycemia-mediated proin-
ammatory cytokines and antioxidant competence in kidney tissues of streptozotocin-induced diabetic
rats.
2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction AGEs formation leading to the development of secondary diabetic

Diabetes mellitus is one of the most common chronic metabolic
disorder and a major contributor to the development of cardiovas-
cular disease. The total number of people with diabetes is projected
to rise from 171 million in 2000 to 366 million in 2030 [1]. It is
characterized by supraphysiological glucose due to the deciency
in insulin secretion or insulin receptor or postreceptor events
leading to the disturbance in the metabolism of carbohydrates, pro-
teins and fats [2]. Moreover, the impaired metabolism leads to the
progression and aggravation of oxidative stress through several
mechanisms, such as glucose autoxidation, protein glycation and
Abbreviations: AGEs, advanced glycation end-products; ANOVA, analysis of vari-
ance; ELISA, enzyme linked immunosorbent assay; GPx, glutathione peroxidase; GR,
glutathione reductase; GST, glutathione-S-transferase; GSH, reduced glutathione;
LPO, lipid peroxidation; LSD, least signicant difference; SEM, standard error of
mean; SOD, superoxide dismutase.
Corresponding author. Tel.: +91 44 22300488; fax: +91 44 22300488.
E-mail addresses: subbus2020@yahoo.co.in, ppalsamy@gmail.com
(S.P. Subramanian).
complications such as nephropathy, retinopathy, neuropathy and
macro and microvascular damages [3].
Diabetic nephropathy is a single leading cause of end stage renal
disease and is a medical catastrophe worldwide in which oxida-
tive stress plays a decisive role in the kidney impairments such as
acute and chronic renal failure, glomerular injury and obstructive
nephropathy [4,5]. Various studies have demonstrated that the sup-
plementation of antioxidants such as vitamin E, -lipoic acid and
plant polyphenols are known to attenuate free radical mediated
diabetic nephropathy [69]. Thus, the agent that restrains the det-
onated production of reactive oxygen species might alleviate the
oxidative stress thereby protecting the kidney tissues.
d-Pinitol, 3-methoxy analogue of d-chiroinositol, is one of the
most abundant cyclitol present in soybean seeds, legumes and soy
food [10,11]. The role of d-pinitol in plants is often associated
with salt and drought stress [12], osmoprotectant [13], embryo
development [14] and nodulation [15]. In addition, d-pinitol has
been suggested to possess multifunctional properties including
anti-inammatory [16], antihyperlipidemic [17], cardioprotective
[18] and inhibition of ovalbumin-induced airway inammation

0009-2797/$ see front matter 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2010.07.014
238 S. Sivakumar et al. / Chemico-Biological Interactions 188 (2010) 237245
[19]. It also modulates the immune response by interacting with
maturation of dendritic cells [20]. Recently, we have reported the
antihyperglycemic potential of d-pinitol by assessing the activi-
ties of hepatic key carbohydrate metabolizing enzymes [21] and
the pancreatic and hepatic tissue protective nature by assessing
the markers of oxidative stress and proinammatory cytokines
in streptozotocin-induced diabetic rats. However, none of the
study reported the benecial effect of d-pinitol on the protection
of diabetic kidney in experimental animals. In this context, the
present study was designed to elucidate the renoprotective nature
of d-pinitol by assessing the markers of oxidative stress as well
as proinammatory cytokines in streptozotocin-induced diabetic
rats.
2. Materials and methods
2.1. Chemicals
d-Pinitol and streptozotocin were purchased from Sigma Chem-
icals Co., St Louis, MO, USA. All other chemicals used in the present
study were of analytical grade available commercially.
2.2. Animals and diet
All the animal experiments compiled with the ethical norms
approved by Ministry of Social Justices and Empowerment, Govern-
ment of India and Institutional Animal Ethics Committee Guidelines
(approval no. 01/017/08). Male, Wistar rats weighing 160180 g,
procured from Tamil Nadu Veterinary and Animal Sciences Uni-
versity, Chennai, India were used in this study. The rats were
housed in standard laboratory condition under a 12 h light/dark
cycle at an ambient temperature of 2025 C with relative humid-
ity of 50 15%. The animals were acclimatized to the laboratory
conditions 2 week prior to the commencement of experiments.
Throughout the experimental period, the rats were fed with bal-
anced commercial pellet diet (Hindustan Lever Ltd., Bangalore,
India) with a composition of 5% fat, 21% protein, 55% nitrogen-free
extract, and 4% ber (w/w) with adequate mineral and vitamins to
the animals. Diet and water were provided ad libitum.
2.3. Induction of experimental diabetes
The overnight fasted rats received a single intraperitoneal injec-
tion of streptozotocin (50 mg/kg body weight) dissolved in freshly
prepared 0.1 M cold citrate buffer, pH 4.5 [24]. As streptozotocin
is capable of inducing fatal hypoglycemia as a result of massive
pancreatic insulin release, the streptozotocin-treated rats were
provided with 10% glucose solution after 6 h for the next 24 h to
prevent hypoglycemia [25]. After a week in time for the devel-
opment and aggravation of diabetes, rats with moderate diabetes
(i.e. blood glucose concentration, >14 mM) were selected for the
experiment.
2.4. Experimental design
The rats were divided into four groups, each group comprised of
six rats, as follows; Group 1 served as control rats; Group 2 served
as streptozotocin-induced diabetic rats; Group 3 served as diabetic
rats orally treated with d-pinitol (50 mg/kg body weight/day) for
30 days; and Group 4 served as diabetic rats orally treated with
glyclazide (5 mg/kg body weight/day) for 30 days.
During the experimental period, body weight, respiratory
changes, distress, abnormal locomotion and catalepsy were mon-
itored at regular intervals in all the rats and the blood glucose
level was estimated twice a week. At the end of the experimental
period, the rats were fasted overnight, anaesthetized and sacriced
by cervical decapitation. The blood was collected with or without
anticoagulant for plasma or serum separation, respectively. Fasting
blood glucose, glycosylated hemoglobin and plasma insulin lev-
els were determined to conrm the antihyperglycemic property of
d-pinitol [2123].
2.5. Biochemical estimations
Blood urea was determined by the method of Natelson et al.
[26]. Serum uric acid was estimated according to the method of Car-
away [27] and creatinine was estimated according to the method
of Brod and Sirota [28]. The concentration of serum AGEs was mea-
sured by ELISA (Abcam, Cambridge, UK) as per the manufacturers
instructions.
2.6. Preparation of kidney tissue homogenate
A portion of the kidney tissue was dissected out, rinsed with ice-
cold saline and homogenized in 0.1 M TrisHCl buffer (pH 7.4) with
a Teon homogenizer at 4 C. The homogenate was centrifuged at
13,000 g to remove the debris and the supernatant was used for
the estimations. The protein content in the tissue homogenate was
estimated by the method of Lowry et al. [29].
2.7. Determination of proinammatory cytokines
The levels of proinammatory cytokines such as TNF-, IL-1
and IL-6 in kidney tissue homogenate were determined by using
specic ELISA kits (Biosource, California, US). The analyses were
performed according to instructions of the manufacturers. Stan-
dard plots were constructed by using standard cytokines and the
concentrations for unknown samples were calculated from the
standard plot. The level of NF-B p65 unit was determined in the
nuclear fraction of kidney tissues by using ActivELISA (Imgenex,
San Diego, USA) kit. The kidney nitrite level was indirectly esti-
mated by determining the nitrite levels in the kidney tissue
homogenate using a colorimetric method based on the Griess reac-
tion [30].
2.8. Assessment of antioxidant status
The levels of kidney non-enzymatic antioxidants such as vita-
min C [31], vitamin E [32] and reduced glutathione (GSH) [33] were
estimated in the control and experimental groups of rats. Further,
the levels of lipid peroxides [34], hydroperoxides [35] and pro-
tein carbonyls [36] were determined in the kidney homogenate.
The activities of kidney enzymatic antioxidants such as superoxide
dismutase (SOD) [37], catalase [38], glutathione peroxidase (GPx)
[39], glutathione-S-transferase (GST) [40] and glutathione reduc-
tase (GR) [41] were determined in the control and experimental
groups of rats.
2.9. Histological study
A portion of the kidney tissue was xed in 10% formalin for a
week at room temperature. Then the specimens were dehydrated
in a graded series of ethanol, cleared in xylene and embedded
in parafn wax. The blocks were then sectioned into 5 m thick
using a rotary microtome. Sections were stained with hematoxylin
and eosin and photomicrographs were obtained under light micro-
scope.
2.10. Transmission electron microscopic study
A portion of the kidney (about 1 mm3 ) were excised from the
control and experimental groups of rats and xed in 3% glutaralde-
 S. Sivakumar et al. / Chemico-Biological Interactions 188 (2010) 237245 239

Table 1 3.2. Effect of d-pinitol on the level of serum AGEs levels

Levels of urea, uric acid and creatinine in control and experimental groups of rats.

Groups Urea (mg/dl) Creatinine Uric acid The effect of d-pinitol on the level of AGEs in control and exper-
(mg/dl) (mg/dl) imental groups of rats is depicted in Fig. 1. The serum AGEs levels
Control 22.77 0.78 0.41 0.31 2.62 0.19 in streptozotocin-induced diabetic group of rats were signicantly
Diabetic control 45.18 1.34a 1.23 0.61a 7.06 0.28a (p < 0.05) increased, when compared with control group of rats. The
Diabetic + d-pinitol 26.25 0.41b 0.57 0.25b 2.98 0.11b oral administration of d-pinitol as well as glyclazide signicantly
Diabetic + glyclazide 25.64 0.22b 0.63 0.19b 3.31 0.14b
(p < 0.05) decreased serum AGEs when compared with diabetic
Results are mean SEM (n = 6). One way ANOVA followed by post hoc test LSD. Val- group of rats.
ues are statistically signicant at p < 0.05, when compared with (a) control rats, (b)
diabetic control rats.
3.3. Effect of d-pinitol on the attenuation of kidney
proinammatory cytokines
hyde in sodium phosphate buffer (0.2 M, pH 7.4) for 3 h at 4 C.
Tissue samples were washed with the same buffer, post-xed in
Fig. 2AC summarizes the levels of kidney proinammatory
1% osmium tetroxide and sodium phosphate buffer (0.2 M, pH 7.4)
cytokines such as TNF-, IL-1 and IL-6 of control and experimental
for 1 h at 4 C. The samples were again washed with the same
groups of rats. The levels of TNF-, IL-1 and IL-6 were signicantly
buffer for 3 h at 4 C, dehydrated with graded series of ethanol
(p < 0.05) elevated in streptozotocin-induced diabetic group of rats
and embedded in Araldite. Thin sections were cut with LKBUM4
when compared to control group of rats. A signicant (p < 0.05)
ultramicrotome using a diamond knife, mounted on a copper grid
decline in the levels of TNF-, IL-1 and IL-6 was observed on oral
and stained with 2% uranyl acetate and Reynolds lead citrate. The
administration of d-pinitol as well as glyclazide.
grids were examined under a Philips EM201C transmission electron
The level of kidney NF-B p65 subunit and renal nitrite in con-
microscope.
trol and experimental groups of rats is depicted in Fig. 3A and B. The
levels of kidney NF-B p65 subunit and renal nitrite were signi-
2.11. Statistical analysis cantly (p < 0.05) escalated in diabetic group of rats when compared
to control group of rats. However the diabetic rats administered
The results were expressed as mean SEM of six rats per group with d-pinitol as well as glyclazide for 30 days showed a marked
and the statistical signicance was evaluated by one-way analysis decline in the levels of kidney NF-B p65 subunit and renal nitrite.
of variance (ANOVA) using the SPSS/16.0 software followed by the
post hoc test LSD. Values were considered statistically signicant at
3.4. Effect of d-pinitol on the augmentation of antioxidant
p < 0.05.
competence

3. Results
3.1. Effect of d-pinitol on the levels of blood urea, serum uric acid
and creatinine levels
Table 1 shows the effect of d-pinitol on the blood urea, serum
uric acid and creatinine in control and experimental groups of
rats. The blood urea, serum uric acid and creatinine levels in
streptozotocin-induced diabetic group of rats were signicantly
(p < 0.05) increased, when compared with control group of rats. The
oral administration of d-pinitol as well as glyclazide signicantly
(p < 0.05) decreased blood urea, serum uric acid and creatinine
when compared with diabetic group of rats.
Fig. 4AC depicts the levels of LPO, hydroperoxides and protein
carbonyls in the kidney tissues of the control and experimental
groups of rats. The diabetic group of rats showed a signicant
(p < 0.05) increase in the levels of LPO, hydroperoxides and pro-
tein carbonyls when compared to the control group of rats. These
elevated levels were signicantly (p < 0.05) declined in the diabetic
rats administered with d-pinitol as well as glyclazide.
Table 2 shows the levels of kidney non-enzymatic antioxidants
such as vitamin C, vitamin E and reduced glutathione in con-
trol and experimental groups of rats. The levels of non-enzymatic
antioxidants were signicantly (p < 0.05) declined in diabetic rats
as compared with control rats and oral administration of d-pinitol
as well as glyclazide elevated these levels to near normalcy.

Fig. 1. Levels of AGEs in control and experimental groups of rats. Results are mean SEM (n = 6). One way ANOVA followed by post hoc test LSD. Values are statistically
signicant at p < 0.05, when compared with (a) control rats, (b) diabetic control rats.
240 S. Sivakumar et al. / Chemico-Biological Interactions 188 (2010) 237245

Fig. 2. Levels of (A) TNF-, (B) IL-1 and (C) IL-6 in the renal tissues of control and experimental groups of rats. Results are mean SEM (n = 6). One way ANOVA followed by
post hoc test LSD. Values are statistically signicant at p < 0.05, when compared with (a) control rats, (b) diabetic control rats, (c) diabetic + glyclazide.

Table 2
Levels of non-enzymatic antioxidants such as vitamin C, vitamin E and reduced glutathione in kidney tissue of control and experimental groups of rats.

Groups Vitamin C (g/mg of protein) Vitamin E (g/mg of protein) Reduced glutathione (mg/100 g of wet tissue)

Control 1.46 0.51 1.11 0.29 37.26 1.14

Diabetic control 0.49 0.33a 0.44 0.21a 20.75 0.88a
Diabetic + d-pinitol 0.95 0.36b 0.88 0.37b 30.83 0.69b
Diabetic + glyclazide 1.01 0.35b 0.94 0.45b 31.09 0.59b

Results are mean SEM (n = 6). One way ANOVA followed by post hoc test LSD. Values are statistically signicant at p < 0.05, when compared with (a) control rats, (b) diabetic
control rats.
 S. Sivakumar et al. / Chemico-Biological Interactions 188 (2010) 237245 241

Fig. 3. Levels of (A) renal p65 unit of NF-B and (B) renal NO in control and experimental groups of rats. Results are mean SEM (n = 6). One way ANOVA followed by post
hoc test LSD. Values are statistically signicant at p < 0.05, when compared with (a) control rats, (b) diabetic control rats, (c) diabetic + glyclazide.

The activities of kidney enzymatic antioxidants such as SOD,
catalase, GPx, GST and GR in control and experimental groups
of rats are depicted in Table 3. There was a signicant (p < 0.05)
decline in the activities of enzymatic antioxidants in kidney tis-
sues of streptozotocin-induced diabetic rats when compared to the
control group of rats. Conversely, these activities were signicantly
(p < 0.05) elevated in diabetic rats treated with d-pinitol as well as
glyclazide.
3.5. Effect of d-pinitol on the normalization of renal histology and
ultrastructure
The photomicrograph of hematoxylin-eosin stained kidney tis-
sues of control and experimental groups of rats are represented in
Fig. 5AD. Fig. 5A illustrates the section of kidney tissue of control
rats showing normal convoluted tubules, corticomedullary junc-
tion, renal hilum and glomeruli within the cortex. The diabetic
group of rats (Fig. 5B) shows the enlargement of lining cells of
tubules, fatty inltration, capillary tufts and large area of hemor-
rhage and lymphocyte inltration. These changes were normalized
notably in the diabetic group of rats treated with d-pinitol as well
as glyclazide (Fig. 5C and D).
The ultrastructural changes occurred in the kidney tissues
of control and experimental groups of rats are exemplied in
Fig. 6AD. The electron micrograph of control rats kidney shows a
normal ultrastructure (Fig. 6A). In the diabetic rats, the glomeruli
appears larger and dilated, Bowmans capsule is partially thickened,
swollen mitochondria, broadened podocyte, and vacuolization
(Fig. 6B). Oral administration of d-pinitol as well as glyclazide nor-
malized the above said alterations signicantly (Fig. 6C and D).

Table 3
Activities of enzymatic antioxidants such as SOD, catalase, GPx, GST and GR in kidney tissue of control and experimental groups of rats.

Groups SOD Catalase GPx GST GR

Control 17.75 0.71 44.40 1.65 8.57 0.34 7.20 0.28 33.76 1.01
Diabetic control 9.44 0.73a 20.93 1.03a 4.04 0.21a 2.44 0.17a 11.21 1.07a
Diabetic + d-pinitol 14.34 0.43b 34.49 1.10b 6.93 0.09b 6.20 0.13b 26.72 1.63b
Diabetic + glyclazide 14.88 0.44b 30.70 0.83bc 7.22 0.15b 6.31 0.27b 26.87 1.49b

Results are mean SEM (n = 6). One way ANOVA followed by post hoc test LSD. Values are statistically signicant at p < 0.05, when compared with (a) control rats, (b) diabetic
control rats, (c) diabetic + glyclazide. Activity is expressed as: 50% of inhibition of epinephrine autoxidation/min for SOD; mol of hydrogen peroxide decomposed/min/mg
of protein for catalase; mol of glutathione oxidized/min/mg of protein for GPx; units/min/mg of protein for GST; mol of DTNB-GSH conjugate formed/min/mg of protein
for GR.
242 S. Sivakumar et al. / Chemico-Biological Interactions 188 (2010) 237245

Fig. 4. Levels of (A) lipid peroxide, (B) hydroperoxide and (C) protein carbonyls in kidney tissues of control and experimental groups of rats. Results are mean SEM (n = 6).
One way ANOVA followed by post hoc test LSD. Values are statistically signicant at p < 0.05, when compared with (a) control rats, (b) diabetic control rats.

4. Discussion followed by nicotinamide adenine dinucleotide depletion resulting

Streptozotocin is a glucopyranose derivative of l-methyl-l-
nitrosourea and the existence of 2-deoxy-d-glucose in its structure
expedites the preferential uptake of streptozotocin through GLUT2
into the pancreatic -cells [42]. The nitrosourea moiety of strepto-
zotocin is known to be involved in the alkylation of DNA, especially
at the O6 position of guanine [43]. This induces a chain of cel-
lular processes including poly-ADP-ribose synthetase activation
in the necrosis of pancreatic -cells [44]. Further, streptozotocin
is also known to provoke reactive oxygen species especially nitric
oxide along with superoxide anion, hydroxyl radicals and hydrogen
peroxide [45] thereby exerts its toxic effects on the pancreas, liver
and kidneys [46].
Pancreatic -cells are one among the least endowed cells
because of their limited antioxidant competence than that of other
tissues and hence they are selectively destroyed by streptozotocin.
 S. Sivakumar et al. / Chemico-Biological Interactions 188 (2010) 237245 243

Fig. 5. Light micrographs of hematoxylin and eosin staining of kidney tissues of control and experimental groups of rats. Photomicrographs of (A) control, (B) diabetic control,
(C) diabetic + d-pinitol and (D) diabetic + glyclazide stained by hematoxylin and eosin at 100 magnication, scale bar: 0.1 mm.

As a result, the expression and secretion of insulin is declined
notably thereby leading to hyperglycemia, a clinical hallmark of
diabetes. The declined level of plasma insulin also reects its neg-
ative impact on hepatic tissue leading to the overproduction of
hepatic glucose through accelerated glycogenolysis and gluconeo-
genesis. This supraphysiologic glucose non-enzymatically reacts
with the free amino groups of globulin component of hemoglobin
leading to the increased formation of glycosylated hemoglobin
[47]. However, oral administration of d-pinitol to diabetic rats is
reported to increase the plasma insulin level and normalizes the
chronic hyperglycemia thereby reduces the formation of glycosy-
lated hemoglobin in streptozotocin-induced experimental diabetic
rats [2123].
The ambient blood glucose level in diabetics induces notable
decline in the total proteins and signicant elevation in the levels
of blood urea, serum creatinine and uric acid which are standard
markers of renal dysfunction [48]. These alterations might be due
to the metabolic disturbances during diabetes by elevation in the
activities of xanthine oxidase, increased levels of lipid peroxides,
triglycerides and cholesterol. One of the major metabolic products
of protein metabolism is urea. The protein glycation during diabetes
is associated with muscle wasting and thereby, an increased release
of purines. The elevated levels of purine nucleotides are the main
source of uric acid, a water soluble antioxidant, by the increased
activity of xanthine oxidase [49]. The creatinine is a byproduct of
creatine and phosphocreatine catabolism, which is an energy stor-
age compound in muscles. Creatinine is endogenously produced
and released into body uids and its clearance measured as an
indicator of glomerular ltration rate. The oral administration of
d-pinitol decreased the levels of blood urea, serum creatinine and

Fig. 6. Transmission electron micrographs of kidney tissues of control and experimental groups of rats. Transmission electron micrographs of (A) Control (10,000), (B)
diabetic control (20,000), (C) diabetic + d-pinitol (20,000) and (D) diabetic + glyclazide (10,000). Brush border (BB), basement membrane (BM), capillary loop (CL),
endothelial fenestrations (EF), mesangial cell (MC), mitochondria (M), podocytes (P), urinary space (US), scale bar: 1 m.
244 S. Sivakumar et al. / Chemico-Biological Interactions 188 (2010) 237245
uric acid in streptozotocin-induced diabetic group of rats, thereby
elicits the renoprotective nature of d-pinitol.
Chronic hyperglycemia is one of the chief causative factors
involved in the irreversible formation of AGEs through a reac-
tion between sugars and the free amino groups on proteins, lipids,
and nucleic acids [50]. Various studies reported that the AGEs
are known to possess a variety of chemical, cellular and tissue
damaging potential implicated in the development and progres-
sion of diabetic nephropathy [51]. However, oral administration
of d-pinitol to diabetic group of rats showed a signicant reduc-
tion in the formation of AGEs. The observed decrease in the
levels of AGEs in diabetic rats might be one of the ameliorative
ways by which d-pinitol recovers diabetic kidney from oxidative
glycation.
Moreover, AGEs are also involved in the activation of various
transcription factors including NF-B which are implicated in the
development of diabetic nephropathy [50]. The activation of NF-B
in diabetic state mediates a cascade of signaling pathway leading to
the renal dysfunction which is positively correlated with elevated
oxidative-nitrosative stress and inammation. Moreover, NF-B
enhances the production of nitric oxide, which is reported to be
involved in the pathogenesis of diabetic nephropathy, especially,
cell destruction and progression of kidney tubular damage [52].
During hyperglycemia-mediated oxidative stress, NF-B activa-
tion obviously provokes the elevation of various proinammatory
cytokines such as TNF-, IL-1 and IL-6, which signify its role in
diabetic nephropathy [53].
TNF- is a pleiotropic proinammatory cytokine produced by
mesangial, glomerular, endothelial, dendritic and renal tubular
cells [54]. TNF- induced cytotoxicity occurs through varying
mechanisms including the overproduction of reactive oxygen
species which in turn damages the cellular components such as
protein, lipids and DNA. Elevated production of TNF- along with
several inammatory cytokines including IL-1 and IL-6 has been
revealed to play a central role in the development and progression
of diabetic nephropathy. IL-1 is able to stimulate the produc-
tion of prostaglandins and nitric oxide in mesangial cells [55]. IL-6
disturbs extracellular matrix dynamics at mesangial and podocyte
levels, stimulates mesangial cell proliferation, increases bronectin
expression and enhances endothelial permeability [56]. The over-
production of IL-6 and TNF- are associated with deterioration
of glycemic control, increased insulin resistance and dyslipidemia,
which contributes to the dysfunction of the metabolic status of dia-
betics [57]. However, the oral administration of d-pinitol to diabetic
group of rats reduced the TNF-, IL-1 and IL-6 levels suggesting
its anti-inammatory role in the attenuation of proinammatory
cytokines mediated toxicity.
The elevated level of nitric oxide reacts with superoxide anion
to produce peroxynitrite, a strong oxidant that in turn hastens the
elevation of lipid peroxidation, which is one of the crucial cellu-
lar complications of chronic diabetes [58]. Hydroperoxides also
causes cellular damage by itself or by disintegrating to highly toxic
hydroxyl radicals. Thus prevention of the formation of hydroxyl
radicals reduces the hydroxyl radical induced damage. Protein car-
bonyls are the secondary products of oxidative stress which are
presumably formed in response to metal catalyzed Fenton reac-
tion on proline, arginine, lysine or threonine residues [59]. The
elevated level of protein carbonyls in addition with lipid perox-
ides and hydroperoxides resulted in the functional loss of specic
membrane proteins and membrane permeability or destruction of
cell or whole cell system [60]. The elevated levels of lipid peroxides,
hydroperoxides and protein carbonyls in the diabetic group of rats
are signicantly reduced by the oral administration of d-pinitol by
protecting the cells from peroxidative stress and lipid peroxida-
tion. These results signify the free radical scavenging potential and
antioxidant nature of d-pinitol.
The extent of oxidative stress is measured by elevation in the
levels of lipid peroxidation, diminution of non-enzymatic antiox-
idant and enzymatic antioxidant activities in tissues and these
modication leads to the increased susceptibility of tissues to
oxidative damage. The GSH acts as a rst line defense against the
proxidant status [61]. It is a major intracellular redox system, acts
as a direct scavenger as well as a co-substrate for GPx [62]. Vitamin
E, a fat soluble vitamin, is regenerated from its oxidized form and its
level is preserved in the presence of vitamin C. Vitamin E plays a piv-
otal role in the suppression of the propagation of lipid peroxidation.
It inhibits the hydroperoxide formation along with vitamin C [63].
The levels of non-enzymatic antioxidants such as vitamin E, vitamin
C and GSH are signicantly decreased in streptozotocin-induced
diabetic rats. However, oral administration of d-pinitol elevated
the levels of these non-enzymatic antioxidants to near normalcy in
streptozotocin-induced diabetic rats, thus signifying its role in the
free radical scavenging property, which in turn readily accounts for
its antidiabetic nature.
The elevated level of oxidative stress leads to damage of mem-
brane lipids, cellular proteins and nucleic acids which eventually
leads to cell death. One of the sources of free radicals is oxida-
tion of glucose in a transition-metal dependant reaction to enediol
radicals; this is further converted into reactive ketoaldehydes and
superoxide anion. The superoxide dismutase converts superox-
ide radical into hydrogen peroxide, subsequently catalase and GPx
convert hydrogen peroxide into water [64]. Catalase catalyzes the
reduction of hydroperoxide, that in turn protects the tissue from
hydroxyl radical mediated damage [65]. Oxidized glutathione is
recycled back to glutathione by GR, through an NADPH consuming
process [66]. The observed decline in these enzymatic antioxidants
in streptozotocin-induced diabetic rat is normalized by the admin-
istration of d-pinitol signifying its role in free radical quenching and
antioxidant defense.
Further, the renoprotective nature of d-pinitol was demon-
strated by the hampering of oxidative stress, brought about by the
amelioration of antioxidant defense and restraining peroxides in
the streptozotocin-induced diabetic group of rats. The histologi-
cal observation made on the kidney tissues further substantiates
the claim that d-pinitol has renoprotective nature. Thus it may
be concluded that d-pinitol possesses antioxidant activity against
hyperglycemia-mediated oxidative stress in kidney.
Conict of interest
The authors declare that there are no conicts of interest.
Acknowledgements
The Research Fellowship of the University Grant Commission
(UGC), New Delhi, India, to the rst author, Mr. S. Sivakumar is
gratefully acknowledged. The authors wish to record sincere thanks
to Ms. B. Rita and Mr. P. Srinivasan, The Wellcome Trust Research
Laboratory, Department of Gastrointestinal Sciences, Christian
Medical College and Hospital, Vellore-632004, India, for their help
in transmission electron microscopic and histopathological studies.
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