Table Of ContentPtilovancivetal.Diabetology&MetabolicSyndrome2013,5:3
DIABETOLOGY &
http://www.dmsjournal.com/content/5/1/3
METABOLIC SYNDROME
RESEARCH Open Access
Heme oxygenase 1 improves glucoses
metabolism and kidney histological alterations in
diabetic rats
Ellen ON Ptilovanciv1, Gabryelle S Fernandes1, Luciana C Teixeira1, Luciana A Reis1, Edson A Pessoa1,
Marcia B Convento1, Manuel J Simões2, Guilherme A Albertoni1, Nestor Schor1 and Fernanda T Borges1*
Abstract
One important concern in thetreatmentof diabetes is the maintenance of glycemic levels and the prevention of
diabetic nephropathy. Inducible heme oxygenase 1 (HO-1) is a rate-limiting enzyme thought to have antioxidant
and cytoprotective roles. The goal of the present study was to analyze theeffect of HO-1 induction inchronically
hyperglycemic rats. The hyperglycemic rats were divided intotwo groups: one group, called STZ, was givena
single injection of streptozotocin; and the other group was given a single streptozotocininjection as well as daily
injections of hemin, an HO-1 inducer, over 60 days (STZ+HEME). A group ofnormoglycemic, untreated rats was
used as thecontrol (CTL).
Body weight, diuresis, serum glucose levels,microalbuminuria, creatinine clearance rate, urea levels, sodium
excretion, and lipid peroxidationwere analyzed. Histological alterations and immunohistochemistry for HO-1 and
inducible nitric oxide synthase (iNOS) were assessed. After 60 days, theSTZ group exhibited anincreasein blood
glucose, diuresis, urea, microalbuminuria, and sodium excretion. There was no weight gain, and there was a
decrease in creatinine clearance incomparison to theCTL group. Inthe STZ+HEME group therewas an
improvement inthe metabolic parameters and kidney function,a decrease inblood glucose, serum urea,and
microalbuminuria, and an increase ofcreatinine clearance, in comparison to the STZgroup.
There was glomerulosclerosis, collagen deposition inthe STZrats and increase iniNOS and HO-1 expression. Inthe
STZ+HEME group, the glomerulosclerosis and fibrosis was prevented and therewas anincreasein theexpression
ofHO-1, but decrease in iNOS expression and lipid peroxidation. In conclusion, our data suggest that chronic
induction ofHO-1 reduces hyperglycemia, improves glucose metabolism and, atleast in part,protects the renal
tissue from hyperglycemic injury, possibly through the antioxidant activityof HO-1.
Keywords: Heme oxygenase 1, Nitric oxide synthase, Renal function,Diabetic nephropathy, Glucose metabolism
Introduction According to clinical studies, the comorbidities of
Diabetic nephropathy is one of the most serious compli- hypertension, glucose intolerance, and insulin resistance
cations of diabetes and the most common cause of end- are considered the major contributors to the develop-
stage renal failure. At present, diabetic kidney disease mentofmetabolicsyndrome anddiabetes[3-5].
affects about 15%–25% of type I diabetes patients [1] Reactive oxygen species (ROS) play an important role
and 30%–40% of patients with type II diabetes [2]. Dia- in diabetes and its complications [6,7]. Experimental
betic nephropathy is characterized by specific renal mor- studies demonstrated the involvement of ROS in the
phologicalandfunctionalalterations. pathogenesis of diabetes [1] and, more importantly, in
the development of diabetic nephropathy [8-10]. Free
radicals are capable of damaging cellular molecules,
DNA, proteins, and lipids, leading to altered cellular
*Correspondence:[email protected]
1NephrologyDivision,DepartmentofMedicine,UniversidadeFederaldeSão functions. Antioxidants capable of neutralizing free radi-
Paulo(UNIFESP),SãoPaulo,Brazil cals are effective in preventing experimentally induced
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CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,
distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
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diabetes in animal models [11,12] as well as in reducing rats in the second group, the STZ+HEME group, were
the severity of diabetic complications [13]. ROS are gen- treated with tail injections of streptozotocin as well as
erated by the electron transport system in mitochondrial daily intraperitoneal injections of hemin [0.1 mg/kg]
respiration and increase in conditions associated with over 60 days; and the rats in the third group, the control
enhanced oxidation of substrate such as glucose or free (CTL) group, received tail injection of citrate buffer and
fatty acids. Other ROS-generating agents of relevance to nohemin.
insulin-resistant diabetes include uncoupled endothelial The animals were allowed free access to standard rat
nitric oxide [3,7]. food and tap water during the experimental protocol. At
Generally, insulin resistance is accompanied by ele- 0 (baseline), 30, and 60 days after the beginning of ex-
vated hyperglycemia, free fatty acids, and adipokines, all perimental protocols, blood samples were collected from
of which are factors known to increase oxidative stress the lateral tail vein, and the animals were maintained in
[14].Thereisaconsensusthatelevatedoxidative/inflam- metabolic cages over 24 h for urine collection. The ani-
matory stimuli unleash the cascade of events that mals were killed 60 days after the beginning of the ex-
impairs insulin signaling [14]. As such, insulin resistance perimental protocol, and both the right and left kidneys
may be regarded asa state ofincreased exposureto ROS were removed for histological analysis. Biochemical
[14], and thus, strategies that concomitantly reduce oxi- parameters in the plasma and urine samples were deter-
dative stress and glucose/insulin intolerance may retard mined. The experimental protocol was approved by the
and/orannulthe complicationsobservedindiabetes. ethicscommitteeoftheUniversidadeFederaldeSãoPaulo.
Among a panel of potential candidate genes related to
oxidative stress, the gene that encodes inducible heme Biochemicalanalysis
oxygenase (HO-1) has attracted much attention, and Blood samples were taken from the tails of all the rats
HO-1 is considered an “emerging molecule” with potent and plasma glucose concentrations were determined
antioxidant, anti-inflammatory, and anti-proliferative (Accuchek, Boehringer Mannheim, Indianapolis, Ind.,
effects[15,16]. USA). The levels of blood creatinine and blood urea
In a physiological system, many cytoprotective were assayed spectrophotometrically according to stand-
mechanisms are triggered during tissue insult in an ard procedures by using commercially available diagnos-
attempt to limit injury. For example, the cellular redox tic kits (Labtest Diagnostica, Minas Gerais, Brazil).
state modulates the expression of stress proteins such as Creatinine was determined by a colorimetric method
hypoxia-inducible factor and heme oxygenase (HO) dur- based on the Jafé reaction [25] and the creatinine clear-
ing cellular defense [17,18]. HO is a microsomal enzyme ance was assessed. Urea was determined using a colori-
with inducible (HO-1) and constitutive (HO-2) isoforms metric assay based on urease activity [26]. Levels of
[19]. The HO-catalyzed breakdown of heme yields cyto- creatinine and urea were expressed in mg/dL. Urine
protective products including bilirubin, ferritin, and sodium concentrationswere determined withaMicronal
carbon monoxide [19]. HO-1 is strongly induced by oxi- B462 flame photometer (São Paulo, Brazil). Sodium
dative stress and it shows cytoprotective effects by the (ENa)excretionswereexpressedasmEq/24 h.
anti-inflammatory, anti-apoptotic, and anti-proliferative The concentrations of albumin in 24-h urine sam-
action of its end by-products [19]. Recent studies ples were assessed using commercially available
showed that HO-1 induction is protective in many acute enzyme-linked immunosorbent assays (ELISA) (Bethyl
andchronic renalinsults[20-24]. laboratories, USA). The optical density of each sample
The present study observed that the induction of was determined using an Ultra Microplate Reader
HO-1 with hemin improved glucose metabolism, as seen (EL808; Bio-Tek Instruments, Winooski, VT, USA)
by a decrease in serum glucose and urea, prevented and expressed as mg/24 h for urine concentrations.
histological changes observed in diabetic animals, and
induced an increase in creatinine clearance. The Lipidperoxidation
improvement in glucose metabolism may result, in part, Lipid peroxidation was determined in urine samples by
from theantioxidanteffectofHO-1. quantifying thiobarbituric acid reactive substances
(TBARS).Thereactive substances combinewith thiobar-
Material and methods bituric acid, forming a red compound whose concentra-
Animaltreatment tion can be assessed spectrophotometrically at an
Male Wistar rats weighing 200–250 g, 10 per group, absorbance of 535 nm [27]. Malondialdehyde (MDA)
were divided into three groups: the rats in the first was used as the standard curve, and the results were
group, the STZ group, were treated with tail injections expressed as nM of MDA/mg creatinine. Urine samples
of streptozotocin [from Sigma (St. Louis, Mo., USA), were added to a solution of 0.375% thiobarbituric acid,
60 mg/kg dissolved in sodium citrate buffer, pH 4.5]; the 15% trichloroacetic acid, and 0.25 N HCl (Sigma-
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Aldrich, Saint Louis, USA), after which they were obliteration and collapse. Results were reported as the
continuously agitated while being heated to 95°C for mean±standard deviation and expressed as arbitrary
20 min. Subsequently, the samples were allowed to cool units(AUs).
to room temperature. The assessment of the creatinine
level in the urine samples was performed as previously Immunohistochemistry
describedandusedtonormalizetheresultsofMDA. The kidney slices were deparaffinized and rehydrated.
Endogenous peroxidase activity was blocked with 5%
Urinaryhydroperoxide H O in absolute methanol for 10 min at room
2 2
Urinary levels of hydroperoxides were measured by the temperature. To expose the antigens, kidney sections
ferrous oxidation with Xylenol Orange, FOX2 assay for were boiled in a target retrieval solution [1 mmol/L tris
lipid hydroperoxide [28]. Briefly, 100 μl aliquots of urine (hydroxymethyl)aminomethane (Tris), pH 9.0, with
were transferred into microcentrifuge vials with the 0.5 mM ethylene glycol tetraacetic acid (EGTA)] for
addition of 900 μl FOX2 reagent. After incubation at 10 min. Nonspecific binding was prevented by incubat-
room temperature for a further 30 min, the vials were ing the sections in phosphate buffered saline (PBS) con-
centrifuged at 12,000 rpm at 25°C for 10 min. Absorb- taining 1% bovine serum albumin (BSA), 0.05% saponin,
ance of the supernatant was then determined at 560 nm. and 0.2% gelatin. Sections were incubated with primary
Lipid hydroperoxide content in urine samples was deter- antibodies against HO-1 (1: 200, rabbit anti-human;
mined according absorption molar coefficient (3 10 [4] Enzo Life Sciences, Michigan, USA) and iNOS (1: 200,
M-1 cm-1). The assessment of the creatinine level in the rabbit anti-rat IgG; ABCAM, MA). Protein expression
urine samples was performed as previously described was measured using a streptavidin peroxidase kit (Dako,
and used to normalize the results of urinary California, USA). Sections were stained with diamino-
hydroperoxides. benzidine for HO-1 and iNOS detection and then coun-
terstained with hematoxylin. Signals in negative control
Nitricoxidedetermination sections were absent. Digital photomicrographs were
Nitric oxide was determined in the 24-h urine samples taken through a Nikon Eclipse E600 upright microscope
by the Griess method. Briefly, a mixture of 1% sulfanila- equipped with Plan Apo objectives and connected to a
W
mide (in 5% H3PO4) and 0.1% naphthylethylenediamine Dell workstation computer through the PixeLINK
solution (Sigma, Saint Louis, USA) was added to the Microscope CameraSoftware (Ottawa,Canada).Eight or
samples and the absorbance at 546 nm was measured nine photomicrographs along the kidney cortex were
using a GENESYS 2 spectrophotometer (Spectronic taken and the light brown staining was quantified
Instruments, Rochester, USA). Nitrite, one of the stable (Axionvision microscope program) and averaged for
metabolites of NO, was then estimated from a standard each rat. The values obtained were expressed as pixels
curve constructed using NaNO2. The urine volume was perfield.
usedtonormalizethequantification.
Statisticalanalysis
Histologicalanalysis The results are expressed as a mean±SE. The data were
The kidneys were dissected along the non-hilar axis and analyzed by one-way analysis of variance (ANOVA)
fixed in 10% phosphate-buffered formalin (Erviegas, São followed by the Tukey tests. Nonparametric data were
Paulo, Brazil). Kidney sections were fixed with 4% buf- analyzed by the Dunn tests and p<0.05 was considered
fered paraformaldehyde and then embedded in paraffin statisticallysignificant.
(Erviegas, São Paulo, Brazil). Next, 4-μm thick sections
were prepared for hematoxylin and eosin (HE; Erviegas, Results
São Paulo, Brazil) staining, and picro-sirius red staining Table 1 shows the biochemical parameters observed in
(PC; Erviegas), and then examined by light microscopy the three study groups, (STZ), (STZ+HEME), and
inablinded manner. (CTL).
Glomerulosclerosis was semiquantitatively evaluated Long-term diabetes induced by streptozotocin was
on HE-stained paraffin sections as follows: grade 0, nor- associated with significant increase in the blood glucose
mal glomerulus; grade 1, beginning mesangial expan- level after 30 (STZ: 470±24 versus CTL: 113±12 mg/
sion/thickening of the basement membrane and/or dL, p<0.01) and 60 (STZ: 554±29 versus CTL: 109±
irregular lumina of capillaries; grade 2, mild/moderate 2 mg/dL, p<0.01) days of treatment, and progressive
segmental hyalinosis/sclerosis involving less than 50% of body weight loss after 30 (STZ: 241±15 versus CTL:
the glomerular tuft; grade 3, diffuse glomerular hyalino- 364±24 g, p<0.01) and 60 (STZ: 255±20 versus CTL:
sis/sclerosis involving more than 50% of the tuft; and 436±17 g, p<0.01) days of treatment in comparison to
grade 4, diffuse glomerulosclerosis with total tuft CTL. Hemin significantly decreased the blood glucose
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Table1Physiologicalparameters
Groups CTL STZ STZ+HEME
day0 day30 day60 day60 day30 day60 day60 day30 day60
BW,g 278±14 364±24 436±17 264±10 241±15* 255±20* 247±5 213±18* 226±18*
Uv,ml 13±2 14±1 12±1 13.1±1 69±11* 57±18* 16±2 83±5* 86±2*
BG,mg/dl 86±4 113±12 109±2 84±4 470±24* 554±29* 80±3 557±27* 338±24*#
U,mg/dl 45±3 47±4 44±2 48±2 91±11* 88±8* 48±2 70±5* 59±6*#
ClCr,ml/min - - 1.02±0.07 - - 0.12±0.03* - - 0.28±0.02*#
UA,mg/24h 0.50±0.08 0.33±0.06 0.28±0.04 0.81±0.09 1.67±0.98 3.50±1.04* 0.42±0.05 1.05±0.63 0.85±0.42
UNaV,mEq/24h 0.7±0.1 0.8±0.1 0.7±0.1 0.7±0.1 2.8±0.8* 0.9±0.3 1.0±0.1 2.4±0.5* 1.4±0.2*
Datawereexpressedasthemean±SEM.Thesignificancelevelforanullhypothesiswassetat5%(p<0.05).BW(bodyweight),Uv(urinaryvolume),BG(blood
glucose),U(serumurea),ClCr(creatinineclearance),UA(urinaryalbumin)andUNaV(urinarysodiumexcretion)wereanalyzedinCTL,STZandSTZ+HEMEgroups.
(*)versusCTLand(#)versusSTZ.
levels (STZ+HEME: 338±24 mg/dL, p<0.01), but 24 h, p=0.01) days in the STZ+HEME group in com-
there was no difference in the body weight (STZ+ parison to the CTL group after 30 (0.8±0.1 mEq/24 h)
HEME: 226±18 g, p=0.36) in STZ+HEME animals and 60 (0.7±0.1 mEq/24 h) days, respectively (p<0.05),
after60days,whencomparedtotheSTZ group. but there wasnodifference insodium excretionbetween
Therewas a significantincrease in diuresis inSTZ and theSTZ andSTZ+HEMEgroups.
STZ+HEME groups after 30 (STZ: 69±11; STZ+ Figure 1 shows the histological sections of renal tissue
HEME: 83±5 versus CTL: 14±1 mL/24 h, p<0.01) and staining for HE, and picro-sirius red. When compared
60 (STZ: 57±18; STZ+HEME: 86±2 versus CTL: 12± with the control group, there was glomerular hyper-
1 mL/24 h, p<0.01 and p<0.05 respectively) days when trophy, collagen deposition in STZ group. There was an
comparedtoCTL. improvement in these alterations in the STZ+HEME
The glomerular filtration was assessed by microalbu- group, we observed structural collagen deposition. The
minuria, and it was observed that the STZ group had a polarized light showed the immature collagen (green)
significant increase in microalbuminuria after 30 (STZ: and intermediate in CTL and SZT+HEME groups,
1.67±0.98 versus CTL: 0.33±0.06 mg/24 h, p=0.22) while STZ groups showed mature collagen (red) prob-
and 60 (STZ: 3.50±1.04 versus CTL: 0.28±0.04 mg/ ablyaroundtubulesandvessels.The scoreofglomerulo-
24 h, p<0.01) days (Table 1). Treatment with hemin sclerosis was significantly increased in the STZ group
significantly inhibited the increase in microalbuminuria (0.90±0.01 AU) compared with the CTL group (0.19±
when compared to the STZ group (STZ: 3.50±1.04 0.03 AU), whereas hemin treatment (STZ+HEME)
versus STZ+HEME: 0.85±0.42mg/24h,p<0.01). reduced the glomerulosclerosis (0.55±0.02 AU) com-
Renal function was assessed by creatinine clearance pared withthe STZ group.
and urea levels. Table 1 shows that there was a signifi- HO-1 and iNOS staining were examined by immuno-
cant decrease in creatinine clearance in STZ (0.12± histochemistry. There was a small amount of staining
0.03 mL/min, p<0.01) compared to CTL (1.02± for HO-1 (STZ: 32.1±8.4 versus CTL: 11.6±1.9 pixels
0.07 mL/min) after 60 days of treatment. There was a per field) and a significant increase for iNOS (STZ:
small but significant increase in creatinine clearance in 122±13.2 versus CTL: 1.6±0.4 pixels per field) staining
the STZ+HEME (0.28±0.02 mL/min, p<0.01) group in the STZ group in comparison with the CTL
when compared totheSTZ group (p<0.01). group. Treatment with hemin (STZ+HEME) signifi-
The plasma urea concentration significantly increased cantly increased the expression of HO-1 (STZ+HEME:
in the STZ group (30 days: 91±11, 60 days: 88±8 mg/ 162.2±63.9 pixels per field), as expected, but inhibit the
dL,p<0.01) whencomparedtotheCTL group(30days: staining for iNOS (34.6±4.8 pixels per field) (Figure 1).
47±4, 60 days: 44±2 mg/dL) (Table 1). In the STZ+ We also evaluated the urinary nitric oxide metabolites.
HEME group (30 days: 70±5, 60 days: 59±6 mg/dL), We observed an increase in urinary nitric oxide in STZ
this increase was significantly prevented after 60 days (43.7±6.8 μM/day) after 60 days of treatment compared
(p=0.03) incomparisontotheSTZ group (Table1). to the CTL (6.7±2.4 μM/day) group in 24 h urine
Tubular lesion was assessed by excretion of sodium. volume. In STZ+HEME group (11.3±2.9 μM/day)
There was a significant increase in sodium excretion preventedtheincrease ofurinarynitric oxide.
after 30 days (2.8±0.8 mEq/24 h, p=0.03) but not 60 There was no difference in the lipid peroxidation
(0.9±0.3mEq/24h,p=0.66) days inthe STZ group and between the groups at baseline; however there was a
30 (2.4±0.5 mEq/24 h, p=0.02) and 60 (1.4±0.2 mEq/ significant increase in TBARS in the STZ group (STZ:
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A
CTL STZ STZ + HEME
HE 100x
PS 200x
PS 100x
HO-1 200x
iNOS 200x
B
U)1.00 * 200 *# 200
A
erulosclerosis (000...572055 *# O-1 (pixels/field)11505000 * OS (pixels/field)11505000 * *#
m H N
o i
Gl 0 0 0
Figure1(A)Lightmicroscopyofkidneysectionsstainedwithhematoxylin-eosinat10x(HE100),Picro-siriusred20x(PC200),10x
(polarizedlight,PC100)andimmunohistochemistryforHO-1(200),iNOS(200)incontrolrats(CTL),ratstreatedwithstreptozotocin
(STZ),andratstreatedwithSTZanddailyinjectionofhemin(STZ+HEME).(B)Quantitativeanalysesofkidneysectionsstainedforheme-
oxygenase-1(HO-1),inducednitricoxidesynthase(iNOS)expressedaspixels/field,stainedwithHE(glomerulosclerosis)expressedasarbitrary
units(AU).Datawereexpressedasthemean±SEM.Thesignificancelevelforanullhypothesiswassetat5%(p<0.05)(*)significantlydifferent
incomparisontoCTLgroupand(#)significantlydifferentincomparisontoSTZgroup.
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93.5±17.0 versus CTL: 47.5±11.0 nM MDA/mg of and carbon monoxide, can be toxic at very high concen-
creatinine) after 60 days (p<0.05). Additionally, we trations. However, recent evidence indicates that they
observed a significant decrease in TBARS in the STZ+ are not toxic at physiological concentrations in normal
HEME group (STZ+HEME: 44.0±0.8 versus STZ: cells, and that they may have important antioxidant,
93.5±17.0 nM MDA/mg of creatinine) compared with anti-inflammatory, oranti-apoptoticproperties[31,32].
the STZ group in the same period (Figure 2). This result The level of HO-1 protein is increased in diabetes.
was corroborated by the hydroperoxide production. Our work demonstrated that in hyperglycemic rats trea-
There was an increase in STZ group (STZ: 2.39±0.31 ted with streptozotocin, the HO-1 expression in the
versus CTL: 0.99±0.18 μM/mg of creatinine) after kidneys was increased, and that in similar animals that
60 days that was inhibited in the STZ+HEME group were treated with hemin, the induction of HO-1
(STZ+HEME: 0.50±0.05μM/mg ofcreatinine). improvedthehistologicalalterations observed.
The hyperglycemia induced by streptozotocin pro-
Discussion moted diuresis and inhibited weight gain. In our experi-
One of the majorconcerns of current diabetes treatment ment, the treatment with hemin decreased blood
is the maintenance of glycemic levels within a narrow glucose and urea, as already observed in other studies.
range, reflecting the recommendations of the Diabetes Correa-Costa observed that hemin treatment improved
Control and Complications Trial [29]. However, such the kidney function andreversed the fibrosis observedin
tight glycemic control may not be feasible, and even chronic kidney disease induced by ureteral unilateral ob-
patients who diligently regulate their blood glucose struction [32]. Hemin improved the glucose metabolism
levels are still vulnerable to the many complications that in hyperglycemic and spontaneously hypertensive rats
characterize diabetes. [33], and type 2 diabetic subjects showed an increase in
There are obvious therapeutic benefits to studying HO-1[34].
diabetic nephropathy and evaluating novel antidiabetic In streptozotocin-induced hyperglycemic rats, glom-
strategies that are capable of improving the chronic erular alterations such as microalbuminuria, increase in
consequences of hyperglycemia. urea levels, and decrease in creatinine clearance, as well
In the present study, we provide evidence that HO-1 as tubular disorders, and increased excretion of sodium
induction could confer protection against metabolic were observed. The treatment of hyperglycemic animals
alterations and, at least in part, in kidney histological with hemin over 60 days inhibited the microalbumi-
alterationsobservedindiabeticanimals. nuria, decreased urea levels, and induced a slight
HO-1, the inducible isoform of the HO system, is a increase in creatinine clearance, but did not improve the
rate-limiting enzyme, which convertsheme into equimo- level ofsodium excretion.Theeffect of heminon micro-
lar amounts of iron, carbon monoxide, and biliverdin. albuminuriahasalsobeen shown byothers[32].
HO-1 is thought to have antioxidant and cytoprotective Our histological analysis showed that the renal tissues
roles [30]. The products of the HO reaction, biliverdin from diabetic animalstreatedwith heminwere protected
A B
e)
nin120 * mg 3 *
eati100 uM/ 2,5
nM/mg of cr 468000 # CSTTZL operoxides (creatinine) 1,125 CSTTZL
ARS ( 20 STZ+HEME Hydr 0,5 # STZ+HEME
B
T 0 0
0 30 60 0 30 60
Time (Days) Time (Days)
Figure2(A)TBARSlevels(nM/mgofcreatinine)and(B)Hydroperoxides(μM/mgofcreatinine)inthecontrolgroup(CTL),ratstreated
withstreptozotocin(STZ)andratstreatedwithstreptozotocin+hemin(STZ+HEME)at0,30and60days.Datawereexpressedasthe
mean±SEM.Thesignificancelevelforanullhypothesiswassetat5%(p<0.05)(*)significantlydifferentincomparisontoCTLgroupand
(#)significantlydifferentincomparisontoSTZgroup.
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glucosesmetabolismandkidneyhistologicalalterationsindiabeticrats.
Diabetology&MetabolicSyndrome20135:3.
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