Kefir treatmentThe CTLK and DMK gro

Kefir treatment
The CTLK and DMK groups received Kefir starting on the 5th day
after diabetes induction. Administration was carried out for
8 weeks by gavage, at a dose of 1.8 mL/day. The other groups,
CTL and DM, received water as a control. All animals were placed
in individual metabolic cages for 24 h prior to and after the treatment
protocol, with water and food ad libitum, for urine collection.
After this period, the rats were fasted for 3 h, after which a blood
sample was collected from the retro-orbital plexus while the rats
were under anesthesia. For anesthesia, intramuscular injection of
ketamine chloridrate (67 mg/kg) and xylazine chloridrate (8 mg/
kg) was used. All samples were stored in freezer at 20 C. At
the end of protocol, the animals were killed by CO2, followed by
exsanguination and the removal of the kidneys.
Oral glucose tolerance test (OGTT)
After 8 weeks of Kefir treatment, OGTT was performed in the
animals. After 12 h of fasting, the animals were orally fed a 20%
glucose solution (1 g/kg) and their blood glucose concentrations
were determined at 0, 30, 60 and 120 min intervals using a
glucometer.
Renal function assessment
The plasma and urinary urea concentrations were estimated by
urea kit CE. Creatinine was measured by colorimetric assay
creatinine kit and proteinuria was evaluated by sensiprot kit.
Kidney histology
The kidneys were fixed in 10% formaldehyde, embedded in
paraffin, sectioned at 4 lm thickness and stained with periodic
acid–Schiff reagent (PAS). The histological changes in the stained
sections were assessed by a nephrologist under a light microscope
at 400 magnification. This was carried out under blinded conditions
and for each kidney, 6 randomly selected areas of cortex were
photographed. The area of each renal cortex was digitalized by an
imaging program and a count was made according to changes in
the tubules.
Estimation of oxidative stress and inflammatory parameters
Kidney tissue homogenate
Homogenates of the renal cortex were prepared as previously
described [9]. The homogenates were used for NO, superoxide
anion and LPO assays and the protein content was analyzed by
Bradford assay.
NO measurement
NO was measured to evaluate the magnitude of vascular damage
and oxidative stress of diabetic rats in plasma, urine and renal
cortex. Because NO is extremely unstable, we used a method in
which the stable NO metabolites, nitrite and nitrate, were re-converted
to NO through a reaction with vanadium. The NO that was
generated was quantified by a chemiluminescence method using
the Nitric Oxide Analyzer (Sievers Instruments, Boulder, USA), a
high-sensitive detector of NO in liquid samples (1 pmol) that is
based on the gas-phase chemiluminescent reaction between NO
and ozone [10].
Western blot analysis
The protein expression of endothelial nitric oxide synthase
(eNOS) and inducible nitric oxide synthase (iNOS) were assessed
in the kidney samples that were individually homogenized with
a Polytron in K-HEPES buffer containing a mixture of protease
inhibitors. After incubation at 4 C for 15 min, the samples were
centrifuged at 2000g. The protein concentrations were quantified
using the Bradford assay method and the 80 lg of protein from
each sample was separated on an 8% polyacrylamide gel and
transferred to a nitrocellulose membrane. Subsequently, the membranes
were probed with a mouse monoclonal eNOS (1:1000) or arabbit polyclonal iNOS (1:200) primary antibody, followed by antimouse
(1:1000) or anti-rabbit (1:5000) secondary antibody,
respectively. The bands were visualized using a chemiluminescence
substrate and analyzed by gel documentation Alience 4.7
Uvitec (Cambridge, Cambs, UK). The relative expression of NOS
proteins in each kidney were normalized using actin antibody
and the values are expressed as a percentage of normal protein
expression.
Superoxide anion
The level of the superoxide anion in the renal cortex was detected
indirectly according to the adapted nitroblue tetrazolium
(NBT) protocol. The optical density (OD) was read in microplate
reader at 560 nm [11].
LPO estimation
The LPO was estimated in terms of MDA by using the TBARS
method. The MDA concentration was calculated using a molar
extinction coefficient of 1.56  105 mol1 cm1 in plasma, urine
and renal cortex [12] at the end of the Kefir treatment.
CRP measurement
To determine the plasmatic CRP, we utilized the turbidimetric
technique [13].
Reagents
Streptozotocin was purchased from Sigma Chemical (St. Louis,
MO, USA). Citric acid was acquired from LabSynth (Sao Paulo, SP,
Brazil) for preparation of the citrate buffer. For the OGTT test, the
Accu-chek advantage II glucometer strips were purchased from
Roche Diagnostics (Mannheim, Baden-Württemberg, Germany)
and glucose D anidra was obtained from LabSynth (Sao Paulo, SP,
Brazil). The Dopalen (ketamine chloridrate) and Anasedan (xylazine
chloridrate) anesthetics were obtained from Sespo (Sao Paulo,
SP, Brazil). The creatinine, urea and proteinuria assay kits were
obtained from Labtest (Lagoa Santa, MG, Brazil). Vanadium was
purchased from Sigma Chemical (St. Louis, MO, USA). Trichloroacetic
was obtained from LabSynth (Sao Paulo, SP, Brazil) and thiobarbituric
acid was purchased from J.T. Baker Chemical (Phillipsburg,
NJ, USA). Nitroblue tetrazolium chloride (NBT) was obtained from
Amresco (Solon, OH, USA). HEPES was purchased from USB
Corporation (Cleveland, OH, USA) and the K-HEPES buffer
contained 200 mM mannitol, 80 mM HEPES and 41 mM KOH, pH
7.5. Protease inhibitors cocktailI was acquired from Millipore
(Billerica, MA, USA). Mouse anti-eNOS was obtained from Abcam
(Cambridge, Cambs, UK), rabbit anti-iNOS and actin were obtained
from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The
enhanced chemiluminescence (ECL) Western blotting analysis
system was obtained from Amersham International, Plc (Little
Chalfont, Bucks, UK).


Results
Metabolic profile, renal function, and oxidative stress before Kefir
treatment
The parameters of the rats after the 5th day of diabetes induction
are shown in Table 1. DM animals demonstrated significant
differences in all parameters, except for plasmatic TBARS. These
animals showed significant increases in water and chow intake,
diuresis, fasting blood glucose, plasmatic urea and creatinine, and
excretion of proteinuria and TBARS. In contrast, DM rats had a decrease
in body weight, urinary urea and creatinine, and plasmatic
and urinary NO when compared to CTL rats.
The changes in metabolic variables, renal function, and oxidative
stress after 8 weeks of Kefir treatment are shown in Table 2.
Kefir administration did not cause difference in any of these
parameters in the CTLK group. The control and diabetic groups
showed significant differences in the following parameters: water
and chow intake, diuresis and weight gain. However, in the DMK
group, these parameters were significantly decreased when
compared to the DM group; an exception was weight gain, which
was higher in DMK animals than in DM animals.
Glycemia
The levels of glycemia after Kefir treatment are shown in
Fig. 1A. On the 5th day after diabetes induction (0), the fasting
blood glucose level was increased in DM rats when compared to
CTL rats (293 ± 21 vs 89 ± 5, p < 0.001) and in DMK rats when compared
to CTLK rats (294 ± 14 vs 95 ± 5, p < 0.001). After the 8th
week of Kefir treatment, these levels were reduced in DMK rats
when compared to DM rats (325 ± 32 vs 457 ± 31, p < 0.001). In
contrast, during OGTT, as shown in Fig. 1B, it was observed that
glycemia in the DMK group was decreased after 30, 60 and
120 min when compared to DM, although this was only significant
after 30 min (344 ± 37 vs 221 ± 51, p < 0.05).
Renal function
As shown in Table 2, the levels of plasmatic urea were increased
in DM rats when compared with CTL rats and in the DMK group
when compared to the CTLK group. However, this was reduced in
DMK rats when compared with DM rats. The concentration of
urinary urea was four times lower in the DM group than in the
Table 1
Metabolic profile, renal function, and oxidative stress in animals before Kefir
treatment.
Parameters CTL DM
n = 18 n = 24
Water intake (mL/24 h) 29.9 ± 0.9 80.2 ± 4.9b
Chow intake (g/24 h) 19.1 ± 0.4 23.9 ± 0.9b
Diuresis (mL/24 h) 13.0 ± 0.7 61.1 ± 4.6b
Weight (g) 269.8 ± 5.2 253.7 ± 4.0a
Fasting blood glucose (mg/dL) 91.8 ± 3.5 293.5 ± 12.3b
Plasmatic urea (mg/dL) 29.2 ± 1.9 55.6 ± 4.6b
Urinary urea (mg/dL) 7,556 ± 444 2,403 ± 129b
Plasmatic creatinine (mg/dL) 0.70 ± 0.04 0.90 ± 0.02b
Urinary creatinine (mg/dL) 166.8 ± 28.0 62.8 ± 14.7b
Proteinuria (nmol/24 h) 11.2 ± 0.6 21.4 ± 1.0b
Plasmatic NO (lM) 89.7 ± 12.2 58.9 ± 9.0a
NO excretion (lmol/24 h) 15.9 ± 2.7 1.4 ± 0.2b
Plasmatic TBARS (nmol/mL) 3.03 ± 0.06 3.17 ± 0.06
TBARS excretion (nmol/24 h) 86.9 ± 5.3 192.4 ± 10.4b
Values are expressed as means ± SEM. Unpaired t or Mann Whitney test. Control
(CTL); diabetic (DM).
a p < 0.05.
b p < 0.001 vs CTL.