Kefir administration reduced progression of renal injury in STZ-diabetic
rats by lowering oxidative stress
Giovana R. Punaro
a, Fabiane R. Maciel
a, Adelson M. Rodrigues
a, Marcelo M. Rogero
c,
Cristina S.B. Bogsan
d, Marice N. Oliveira
d, Silvia S.M. Ihara
b, Sergio R.R. Araujo
b,
Talita R.C. Sanches
e, Lucia C. Andrade
e, Elisa M.S. Higa
a,⇑aDepartment of Medicine, Universidade Federal de Sao Paulo, Sao Paulo, Brazil bDepartment of Pathology, Universidade Federal de Sao Paulo, Sao Paulo, Brazil
cDepartment of Nutrition, Public Health College, Universidade de Sao Paulo, Sao Paulo, Brazil
dDepartment of Biochemical and Pharmaceutical Technology, Universidade de Sao Paulo, Sao Paulo, Brazil eDepartment of Nephrology, Universidade de Sao Paulo, Sao Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 31 July 2013 Revised 15 December 2013 Available online 6 January 2014
Keywords: Kefir
Oxidative stress Nitric oxide
Diabetes and renal function
a b s t r a c t
This study aimed at assessing the effects of Kefir, a probiotic fermented milk, on oxidative stress in dia-betic animals. The induction of diabetes was achieved in adult male Wistar rats using streptozotocin (STZ). The animals were distributed into four groups as follows: control (CTL); control Kefir (CTLK); dia-betic (DM) and diadia-betic Kefir (DMK). Starting on the 5th day of diabetes, Kefir was administered by daily gavage at a dose of 1.8 mL/day for 8 weeks. Before and after Kefir treatment, the rats were placed in indi-vidual metabolic cages to obtain blood and urine samples to evaluate urea, creatinine, proteinuria, nitric oxide (NO), thiobarbituric acid reactive substances (TBARS) and C-reactive protein (CRP). After sacrificing the animals, the renal cortex was removed for histology, oxidative stress and NOS evaluation. When com-pared to CTL rats, DM rats showed increased levels of glycemia, plasmatic urea, proteinuria, renal NO, superoxide anion, TBARS, and plasmatic CRP; also demonstrated a reduction in urinary urea, creatinine, and NO. However, DMK rats showed a significant improvement in most of these parameters. Despite the lack of differences observed in the expression of endothelial NO synthase (eNOS), the expression of induc-ible NO synthase (iNOS) was significantly lower in the DMK group when compared to DM rats, as assessed by Western blot analysis. Moreover, the DMK group presented a significant reduction of glyco-gen accumulation within the renal tubules when compared to the DM group. These results indicate that Kefir treatment may contribute to better control of glycemia and oxidative stress, which is associated with the amelioration of renal function, suggesting its use as a non-pharmacological adjuvant to delay the progression of diabetic complications.
Ó2014 Elsevier Inc. All rights reserved.
Introduction
Diabetes mellitus has become a serious public health problem that affects millions of individuals worldwide. The World Health Organization predicts that 439 million people will have this disease in 2030, and Brazil was listed 5th of 10 countries estimated to have the highest number of people with diabetes, affecting approximately 12.7 million Brazilians in 2030[1].
Hyperglycemia and oxidative stress have been closely linked to diabetic complications, such as neuropathy, retinopathy and nephropathy. Additionally, excessively high blood glucose levels
lead to the increased production of reactive oxygen species (ROS), such as hydrogen peroxide and superoxide radicals [2]. A chronic hyperglycemic state may also cause ROS increases via glu-cose auto-oxidation in various tissues, leading to high oxidative/ nitrosative stress with subsequent impaired nitric oxide (NO) bio-availability. NO is a potent, endogenous vasodilator that modulates renal function and plays a key role in endothelial dysfunction[3]. High levels of ROS contribute to lipid peroxidation (LPO) in cellular membranes, increasing their fluidity and permeability. Specifically, high levels of ROS generate malondialdehyde (MDA), a highly toxic molecule, and its secondary product, thiobarbituric acid reactive substances (TBARS), which is used as marker of LPO
[4]. ROS are also responsible for the activation of nuclear factor-kappa B (NF-
j
B), which increases the expression of pro-inflamma-tory biomarkers, such as tumor necrosis factor-alpha (TNF-a
), and interleukin-6 (IL-6), which augments the expression of C-reactive protein (CRP)[5].1089-8603/$ - see front matterÓ2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.niox.2013.12.012
⇑ Corresponding author. Address: Universidade Federal de Sao Paulo – Escola Paulista de Medicina, Rua Botucatu #740, Vila Clementino, 04023-900 Sao Paulo, SP, Brazil.
E-mail address:emshiga@unifesp.br(E.M.S. Higa).
Contents lists available atScienceDirect
Nitric Oxide
Kefir is a beverage made from milk that is fermented by a com-plex mixture of bacteria, including various species of lactobacilli and yeasts. It has been considered a probiotic due to its antioxidant and anti-inflammatory properties[6,7]. In this study, we investi-gated the effects of Kefir on the production of nitric oxide and oxidative stress and renal damage in STZ-induced diabetic rats.
Material and methods
Animals
Male Wistar rats, 8 weeks of age, weighing ±250 g, were obtained from Central Animal Housing, Sao Paulo, Brazil. All protocols were approved by the Ethics Committee in Research of Universidade Federal de Sao Paulo (protocol #1335/09). The animals were maintained in the Animal Housing of Nephrology Division at a tem-perature of 22 ± 1°C and at a light–dark cycle of 12/12 h, beginning
at 6:00 am. The animals were given free access to standard chow (Nuvital CR-1, Nuvilab, Colombo, PR, Brazil) and water. The rats were allocated into the four following groups: CTL (n= 9, control group); CTLK (n= 9, control group that received Kefir); DM (n= 12, diabetic group) and DMK (n= 12, diabetic group treated with Kefir).
Induction of type 1 diabetes
After seven days of adaptation, 9-week-old animals received a single intravenous administration of STZ 45 mg/kg dissolved in 0.1 M cold citrate buffer, pH 4.5. The controls rats only received citrate buffer. After 72 h of induction, glycemia was measured in blood samples collected from the tail vein and the values were deter-mined using a glucometer. DM was defined in this study as fasting blood glucoseP200 mg/dL and animals that failed to meet this
criteria were excluded. The fasting blood glucose (3 h) of each animal was measured during 8 weeks protocol for monitoring diabetes.
Production and preparation of Kefir
The ingredients used were as follows: milk: skim milk powder Molico (Nestle, Sao Paulo, Brazil); lactic culture: Kefir (1010CFU/g
of lactic acid bacteria and 104–107CFU/g of yeast), containing
Lacto-coccus lactis subsp., Leuconostoc sp., Streptococcus termophilus,
Lactobacillussp.,Kefiryeast, andKefirgrains microflora that are not genetically modified, according to European Parliament (Danisco Biolacta, Olstyn, Poland). The preparation of the matrix was carried out as follows: skim milk powder was reconstituted to 10% with distilled water and was incubated at 85°C for 15 min (Lauda Wobser GMBH & CO. KG type A100, Lauda-Königshofen, Germany) using a mechanical shaker (Quimis Q250M1, Diadema, SP, Brazil). Subse-quently, 20 mg freeze-dried Kefir culture was added to 100 mL of the treated milk and fermentation was carried out at 23°C for 16 h. This was monitored by the Cinac System (Cynetique d´acidifi-cation, Ysebaert, Frépillon, France)[8]. When the desired pH of 4.6 was reached, the fermentation was stopped by cooling the flasks in ice bath, and storing them under refrigeration at 4°C until utiliza-tion. Kefir was prepared as described above, once a week, thus ensuring freshness of the product. All procedures were performed at the Laboratory of Food Technology, Faculty of Pharmaceutical Sciences, Universidade de Sao Paulo, Sao Paulo, SP, Brazil.
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 treat-ment protocol, with water and foodad 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
l
m 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 400magnification. This was carried out under blindedcondi-tions 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 dam-age 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-con-verted 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
rabbit polyclonal iNOS (1:200) primary antibody, followed by anti-mouse (1:1000) or anti-rabbit (1:5000) secondary antibody, respectively. The bands were visualized using a chemilumines-cence 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 de-tected 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 1.56105mol 1cm 1 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 (xyla-zine 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). Trichloroace-tic was obtained from LabSynth (Sao Paulo, SP, Brazil) and thiobar-bituric 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).
Statistical analysis
The results were expressed as the mean ± standard error media (SEM). The differences among the four groups were examined for statistical significance using one-way analysis of variance (ANOVA) followed by Newman–Keuls Multiple Comparison post test or by Kruskal–Wallis followed by Dunn’s Multiple Comparison post test. The differences between two groups were analyzed by unpairedt
or Mann Whitney tests, as appropriate. The correlation was analyzed by Sperman rank coefficient. Values were considered statistically significant whenp< 0.05.
Results
Metabolic profile, renal function, and oxidative stress before Kefir treatment
The parameters of the rats after the 5th day of diabetes induc-tion are shown inTable 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 de-crease 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 oxida-tive stress after 8 weeks of Kefir treatment are shown inTable 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 com-pared 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 inFig. 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 inTable 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. Unpairedtor Mann Whitney test. Control (CTL); diabetic (DM).
CTL group, while in the DMK group, it was 2.8 times lower than in the CTLK group, demonstrating that this parameter was increased in the DMK group when compared to the DM group. Moreover, plasmatic creatinine was not different among control and diabetic animals, and urinary creatinine and proteinuria were not different between DMK and DM groups.
In relation to renal histology, the diabetic groups presented with an accumulation of glycogen in the renal tubules. This was re-duced in the DMK group when compared to the DM group (9 ± 1 vs 13 ± 1,p< 0.001), as demonstrated inFig. 2).
Oxidative stress and inflammatory state
According toTable 2, there was no difference in plasmatic NO levels in the animals studied. However, the excretion of NO in
DM rats was significantly reduced by 7 times when compared to CTL rats and in DMK rats, this parameter was higher compared to DM rats, but did not show any difference when compared to CTLK rats. Interestingly, the data demonstrated a strong and in-verse correlation between NO and proteinuria excretion, which is an early marker of renal lesion, among the control and diabetic groups (r= 0.83,p< 0.001), as shown inFig. 3A.
Table 2
Metabolic profile, renal function, and oxidative stress of the groups after Kefir treatment.
Variables CTL CTLK DM DMK
Water intake (mL/24 h) 24.8 ± 1.8 25.8 ± 3.7 124.4 ± 12.4a 94.1 ± 14.4b,c
Chow intake (g/24 h) 17.3 ± 0.6 19.1 ± 1.0 36.9 ± 2.0a 30.6 ± 2.3b,c
Diuresis (mL/24 h) 13.1 ± 1.0 13.7 ± 0.7 90.9 ± 9.0a 70.9 ± 9.4b,c
Weight (D) 67.5 ± 4.4 69.3 ± 1.6 25.3 ± 4.5a 35.3 ± 5.9b
Plasmatic urea (mg/dL) 31.9 ± 1.4 36.2 ± 2.5 58.5 ± 3.6a 47.6 ± 2.4b,c
Urinary urea (mg/dL) 8,691 ± 343 8,423 ± 229 2,006 ± 142a 2,996 ± 322b,c
Plasmatic creatinine (mg/dL) 0.71 ± 0.05 0.72 ± 0.04 0.75 ± 0.03 0.78 ± 0.05
Urinary creatinine (mg/dL) 131.7 ± 9.2 127.9 ± 4.5 33.2 ± 5.6a 34.8 ± 7.8b
Proteinuria (nmol/24 h) 10.4 ± 0.8 11.2 ± 0.7 25.5 ± 3.7a 21.0 ± 2.8b
Plasmatic NO (lM) 66.6 ± 4.3 77.8 ± 6.6 79.2 ± 5.0 76.5 ± 5.4
NO excretion (lmol/24 h) 14.9 ± 3.6 17.5 ± 3.8 2.1 ± 0.7a 16.4 ± 4.9
Plasmatic TBARS (nmol/mL) 3.32 ± 0.06 3.16 ± 0.08 3.79 ± 0.10a 3.58 ± 0.17
TBARS excretion (nmol/24 h) 81.6 ± 2.1 84.4 ± 4.3 300.4 ± 18.9a 248.9 ± 19.2b,c Values are expressed as mean ± SEM. One-way ANOVA followed by Newman–Keuls Multiple Comparison post test. Control (CTL); control Kefir (CTLK); diabetic (DM); diabetic Kefir (DMK);n= 9–12/group.
ap< 0.001 vs CTL. b p< 0.01 vs CTLK. c p< 0.05 vs DM.
0 2 4 8
0 200 400 600
CTL
CTLK
DM
DMK
&&&&&& &&&
&&&
***
***
***
***
,###Treatment (weeks)
Glycemia (mg/dL)
0 30 60 90 120 0
150 300 450
CTL CTLK DM DMK
&
*
&&& &&&
&&&
***
** *,#
Time (minutes)
Glycemia (mg/dL)
A
B
Fig. 1.(A) Glycemia levels after 0, 2, 4 and 8 weeks during Kefir treatment,n= 9– 12. (B) Glycemia levels during oral glucose tolerance test (OGTT),n= 4–6/group. Control (CTL); control Kefir (CTLK); diabetic (DM); diabetic Kefir (DMK). Values are expressed as means ± SEM. One-way ANOVA followed by Newman–Keuls Multiple Comparison post test. &p< 0.05, &&&p< 0.001 vs CTL; ⁄p< 0.05, ⁄⁄p< 0.01, ⁄⁄⁄-p< 0.001 vs CTLK;#p< 0.05,###p< 0.001 vs DM.
CTL CTLK DM DMK
0 7 14 21
&&&
***
, ###Average Glycogenated Tubules
A
B
The level of NO in the renal cortex was higher in DM rats when compared to CTL rats (13 ± 2 vs 3 ± 0.4,p< 0.05), but reduced in DMK rats compared to DM rats (5 ± 2,p< 0.05). This is shown in
Fig. 3B. The Western Blot analysis of the renal cortex showed that there was no difference in eNOS expression in the DM and DMK rats. However, there was a significant reduction in the expression of iNOS in the DMK group when compared to the DM group (101 ± 2 vs 113 ± 5,p< 0.05), as shown inFigs. 4A and B.
Likewise, a similar increase in superoxide anion (OD) was observed in the renal cortex of DM rats when compared to CTL rats (0.107 ± 0.012 vs 0.023 ± 0.007,p< 0.001). However, DMK rats presented with lower values in this parameter than DM rats (0.047 ± 0.019, p< 0.01), and in the DMK rats, this value approached that of the CTLK rats (0.021 ± 0.002, NS), as seen in
Fig. 5A.
InTable 2, we observed that plasmatic TBARS was higher in the DM group however, not demonstrated difference between DMK and CTLK; otherwise, the excretion of TBARS at the end of Kefir treatment was different among the groups, and the main differ-ences were observed between the control and diabetic animals. The excretion of TBARS was three times higher in the diabetic groups when compared to the respective control groups. Notably, a significant reduction was observed in the DMK group when compared to DM and these values approached of the CTLK group (Table 2). There was also an increase in TBARS in the renal cortex of diabetic animals. However, as shown inFig. 5B, this elevation was more pronounced in the DM group compared to the CTL group (0.65 ± 0.06 vs 0.22 ± 0.04,p< 0.001).
The CRP concentration in the DM group was significantly in-creased when compared to the CTL group (0.28 ± 0.01 vs 0.18 ± 0.04, p< 0.05). The DMK group showed a reduction of this parameter compared to the DM group (NS) and an increase when compared to the CTLK group (0.23 ± 0.03 vs 0.16 ± 0.01, NS) (Fig. 5C).
Discussion
STZ-induced hyperglycemia is a recognized experimental model that is used to study both type 1 and type 2 diabetes[14]. In this study, a lower dose of STZ (45 mg/kg) was used and the rats became permanently diabetic. This allowed the production of a diabetic state with moderate to severe hyperglycemia throughout the protocol. Among the most important findings in this study, Kefir treatment in diabetic rats resulted in better glycemic control with reduced polyuria, polydipsia and polyphagia, a partial improvement in renal function, increased NO excretion and reduc-tion of superoxide anion, LPO and inflammareduc-tion.
The mechanisms by which probiotic bacteria modulate hyper-glycemia are not fully understood, although previous studies have reported that supplementation with a strain ofLactobacillus signif-icantly delayed the onset of glucose intolerance, hyperglycemia, hyperinsulinemia, and oxidative stress in rats with type 2 diabetes and also reduced the risk of the development of associated complications[15,16].
Additionally, studies that utilized animals fed with Kefiran, an exopolysaccharide isolated from Kefir grains, demonstrated that it reduced blood pressure and serum cholesterol in stroke-prone spontaneously hypertensive rats (SHR-SP)[17] and reduced ath-erosclerosis in rabbits with type 2 diabetes[18]. It also decreased the blood glucose levels in a genetic diabetic animal model (KKAy) that showed impaired glucose tolerance to an intraperitoneal glucose administration[19].
r = - 0.83
0 10 20 30 40
0 20 40 60
NO (μmol/24h)
Proteinuria (mg/24h)
CTL
CTLK
DM
DMK p<0.001
CTL CTLK DM DMK
0 7 14 21
&
#
NO Renal Cortex (nmol/mg)
A
B
Fig. 3.(A) Linear correlation of nitric oxide (NO) and proteinuria excretion after Kefir treatment. Sperman rank coefficientr= 0.83,p< 0.001. (B) NO in renal cortex. Control (CTL); control Kefir (CTLK); diabetic (DM); diabetic Kefir (DMK), n= 6–7 per group. Values are expressed as means ± SEM. Kruskal–Wallis followed by Dunn’s Multiple Comparison post test.&p< 0.05 vs CTL;#p< 0.05 vs DM.
CTL CTK DM DMK
0 50 100 150
NS
eNOS (% of control)
CTL CTLK DM DMK
0 50 100 150
#
iNOS (% of control)
CTL CTL CTLK CTLK DM DM DMK DMK
eNOS
acƟn iNOS
A
B
C
Fig. 4.(A) Representative image of Western blot analysis of eNOS and iNOS in renal cortex of control and diabetic rats after Kefir treatment. (B) NOS expression represented graphically after actin housekeeping normalization;n= 4–5/group. Diabetic (DM); diabetic Kefir (DMK). Values are expressed as means ± SEM. One-way ANOVA followed by Newman–Keuls Multiple Comparison post test. NS: non significant.#p< 0.05 vs DM.
It is known that probiotics can modulate the microbiota, and/or the intestinal immune system and influence intestinal physiology. This was observed in anin vivostudy using healthy female rats that consumed a Kefir-supplemented diet. Kefir supplementation was shown to have an effect on enzymes and proteins, increase the activity of the intestinal dipeptidase and decrease the Na+
-depen-dent uptake of intestinal sugar, which contributes to protein digestion and glycemia reduction, respectively[20].
Hyperglycemia is the main factor in the progression of DN, leading to the dysfunction of multiple organs and increasing the morbidity and mortality in patients with diabetes [21]. In our study, the kidneys of the diabetic animals were injured when com-pared to control animals, but after Kefir administration, this was partially improved. This is in agreement with the literature, where it has been previously reported that probiotic bacteria benefitted both human and animals with kidney failure by increasing gut bacterial metabolism for the excretion of ammonia, thus reducing blood urea-nitrogen levels[22,23].
In the present investigation, we observed significant proteinuria in diabetic rats, suggesting increased permeability of the glomeru-lar basement membrane [24], but this was not significantly reduced in the Kefir-supplemented group. Nevertheless, we ob-served a strong and inverse correlation between proteinuria and NO among control and diabetic animals. This shows the impor-tance of NO on this parameter, a matter which is still controversial
[3]but has been reproduced in our previous study[9]. Moreover, glycogen accumulation was observed in the kidney tubules of DM rats and this alteration was significantly reduced after Kefir treatment, most likely as a consequence of better glycemic control in these animals.
The bioavailability of vascular NO is decreased in diabetes, both in human and animals, possibly due to elevated oxidative stress stimulated by the chronic hyperglycemic state[25]. This is because NO can be inactivated by reaction with superoxide, resulting in peroxynitrite, or may be transformed into the more stable hydro-gen peroxide radical[3,14]. In this study, superoxide anion, one of the main types of ROS, was higher in the renal tissue of the DM group and was significantly reduced in the DMK group. These findings agree with that reported by Ishii et al., who showed that NOS activity is augmented in the kidney cortex of diabetic rats and as such, larger quantities of superoxide are produced[26].
In the present investigation, NO excretion was reduced in diabetic animals in comparison to their respective control and was increased after Kefir treatment to control levels. Another study showed the role of some probiotic bacteria in the generation of local NO in the intestinal lumen by nitrate reduction or acid depen-dent mechanisms, which may be counteracted by rapid NO consumption by other strains or diffused into the surrounding tissues. This may explain some of the health promoting effects of these bacteria, for example, by potentiating the antimicrobial property of NO[27,28].
Moreover, the increase in blood glucose concentration activates high expression of iNOS through NF-kB. This is followed by the increased production of NO, which influences several signal path-ways[29]. This up regulation of iNOS under conditions of oxidative stress may be a compensatory mechanism for reduced NO availability, indicating roles for NO, ROS, and peroxynitrite in the development of early diabetic damage prior to the development of complications[30].
Furthermore, researchers have shown that the uncoupling of eNOS by peroxynitrite may be a secondary mechanism by which hyperglycemia can quench and/or inactivate NO and consequently, lead to endothelial dysfunction[30,31]. However, in this study, there was a reduction of eNOS expression in both groups, DM and DMK, when compared to CTL and CTLK, although there was no difference between them. Nevertheless, in DM animals, higher iNOS expression was observed, and this was accompanied by elevated oxidative stress in the renal cortex, as reflected by an increase in NO-scavenging superoxide anions and MDA. This sug-gests that in the diabetic kidney, NO is deficient and consequently, its bioavailability is reduced [3,32]. This status was reversed in DMK animals, which showed reduced oxidative stress and iNOS and the restoration of NO excretion.
The measurement of LPO was based on the extinction coeffi-cient of MDA, which is the end product of fatty acid peroxidation that reacts with thiobarbituric acid (TBA). This is accepted as an in-dex of LPO and is widely utilized in studies relating to diabetes[4]. It has also been demonstrated that MDA in the urine is a more sen-sitive measure than that in the plasma[33], a finding that supports previous published results of our research group[9].
In the present study, there was a significant increase of LPO in diabetic rats, most likely due in part to the elevation in blood glucose levels that generate ROS upon auto oxidation [2]. This indicates that peroxidative injury may be involved in the reduction
CTL CTLK DM DMK
0.00 0.05 0.10 0.15
&&&
# #
Superoxide Anion Renal Cortex
(OD)
CTL CTLK DM DMK
0.0 0.3 0.6 0.9
&&&
# #
TBARS Renal Cortex (nmol/mg)
CTL CTLK DM DMK
0.00 0.15 0.30 0.45
&
Plasma CRP (mg/L)
A
B
C
Fig. 5.(A) Optical density (OD) of superoxide anion in renal cortex after Kefir
of antioxidant defense mechanisms and the development of diabetic complications. In our study, Kefir treatment significantly decreased LPO, and this was accompanied by a reduction in hyper-glycemia, perhaps by the preservation of pancreaticb-cells, which are vulnerable to oxidative damage [14], or by the antioxidant effect of fermented milk. It is known that the bioactive peptides released during fermentation by proteolytic lactic acid bacteria can scavenge the ROS and inhibit LPO[34].
In contrast, CRP, a recognized marker of inflammation associated with endothelial dysfunction, is elevated in the blood of patients with both type 1 [35] and type 2 diabetes [36]. Researchers have reported no change[37]or an increase[21]in plasmatic CRP levels in STZ-treated diabetic rats. In this study, a significant increase in plasmatic CRP was observed in diabetic rats; however, this parameter was decreased after Kefir treatment, per-haps due to the better control of hyperglycemia in these animals, which could modulate the activation of NF-
j
B and reduce the iNOS expression. It was demonstrated that Kefir could inhibit the innate response of intestinal epithelial cells, suggesting the modulation of the NF-j
B pathway[38].Furthermore, other studies have shown that altered bowel function contributes to the development of diabetes mellitus. In particular, the intestinal barrier has become a focus of much research because increased intestinal permeability has been asso-ciated with type 1 and type 2 diabetes; suggesting that this would precede the onset of clinical diabetes in humans and rats and that enhancing intestinal barrier function may be important for pre-venting pathophysiological mechanisms in diabetic patients[39,40]. Studies with probiotics could be used to design clinical plans for prevention-based treatment in which microbiota could be benefi-cial in the attenuation or delay of disease progression[41]. While the effects and the precise mechanisms of probiotics involved in the diabetes remain unknown, they are likely related to a decrease in oxidative stress that is associated with the immune-modulatory and anti-inflammatory properties of these bacteria, the modifica-tion of intestinal microbiota and maintenance of gut integrity. Further studies are necessary to provide new insights into the effective therapeutic approaches for type 1 diabetes mellitus.
Conclusion
The results obtained in this study show that Kefir treatment sig-nificantly reduced the progression of STZ-induced hyperglycemia and oxidative stress in rats. Thus, Kefir may play a role in slowing the metabolic changes that contribute to DN.
Acknowledgments
The authors acknowledge Margaret Gori Mouro, Guilherme Baia Nogueira, Thamires Oliveira Fernandes and Deborah Chianelli for technical assistance and to all who contributed to the development of this manuscript.
This work was supported by Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), Fundacao de Apoio a Univer-sidade Federal de Sao Paulo (FAP-Unifesp), and Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP).
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