The Effect of Lactobacillus reuteri on Bone Morphogenetic Protein-7 and Beta Transforming Growth Factor Gene Expressions in Streptozotocin-induced Diabetic Rats Kidneys
Diabetes mellitus is a serious health problem in the world and about 20 to 40% of the patients are being affected with diabetic nephropathy. The anti diabetic property of Lactobacillus reuteri (L. reuteri) has been reported. The study designed to investigate the effect of L. reuteri on the expression of BMP-7 and TGF-β genes, the two basic factors involved in diabetic nephropathy. This experimental study was carried out in two months. For this goal thirty male Wistar rats with 12 weeks old and 200±50 g weight was divided into 5 groups, each consisting six rats. (1) Non diabetic, (2) Untreated diabetic, (3) Diabetic rats fed with L. reuteri, (4) Diabetic rats treated with insulin (4-5 U/kg/day), (5) Non diabetic rat fed with L. reuteri. Diabetes in the was induced single intraperitoneal (i.p.) injection of streptozotocin (50 mg kg-1 b. wt). The L. reuteri 1x108 Colony Forming Units (CFU) were administered via oral gavages. After two months rats were anesthetized and blood samples collected. Serum nitric oxide (NO) levels were determined by a chemiluminescence method using NO analyzer and serum glucose by glucose oxidize method. The expression of BMP-7 and TGF-β genes in the rats kidneys were determined by real time PCR. Results showed that BMP-7 gene expression was increased in diabetic rats that fed with L. reuteri, while TGF-β gene expressions were decreased. Histopathological study showed that administration of L. reuteri (1x108 CFU/rat/day) significantly reduced kidney fibrosis and increased meaningfully NO levels in diabetic rats fed with L. reuteri. It was concluded that L. reuteri increase BMP-7 gene expression and may prevents from renal damage by oxidative stress by increasing antioxidant capacity.
to cite this article:
S.M. Nourazarian, G. Irajian, M. Najafi, M. Nourbakhsh, J. Maleki and M. Shabani, 2012. The Effect of Lactobacillus reuteri on Bone Morphogenetic Protein-7 and Beta Transforming Growth Factor Gene Expressions in Streptozotocin-induced Diabetic Rats Kidneys. Pakistan Journal of Biological Sciences, 15: 374-379.
June 14, 2012; Accepted: July 20, 2012;
Published: September 03, 2012
Diabetes is a lasting and common complication all over the world. Due to the
epidemiological studies, the researchers believe that 1.5 million of Iranian
population carry this disease (Akbarzadeh et al.,
2007) and about to 20 to 40% of these patient will be affected with diabetic
nephropathy (Bottinger and Bitzer, 2002). Hyperglycemia
is identified as a major factor on the extension of effects on the diabetic
individuals (Dolan et al., 2005). Diabetic Nephropathy
(DN) is the main causes for final stage of kidney debases in the USA and west.
At the initial stage of extension of (DN), kidney tubules shows the over expression
of TGF-β (Wang et al., 2001). Due to lack
of understanding of pathway, TGF-β is a mediators that causes fibrosis
of glomerulosclerosis and tubulointerstitial (Dronavalli
and Duka, 2008). On the other hand, expression of BMP-7 as an antagonist
of TGF-β could be effective on the investigation of DN (Helder
et al., 1995; Fioretto et al., 2006).
BMP-7 is a member of superfamily of TGF-β that plays an important role
on the growth of kidney (Helder et al., 1995;
Dolan et al., 2005; Fioretto
et al., 2006). Due to expression of different BMP proteins during
kidney organogenesis, the indicated evidences shows that only the BMP-7 is efficient
for formation of kidney (Simic and Vukicevic, 2005).
The tubular expression of BMP-7 in animal models with kidney deficiency is decreased
(Simon et al., 1999; Wang
et al., 2001). BMP-7 causes the deactivation of TGF-β profibrotic
activity and the findings indicate this case that the BMP-7 in the process of
the change of Epithelial to Mesangial Transition (EMT) that cause the creation
of fibroblasts, has deactivation roles (Weiskirchen et
al., 2009). In general, the metabolic activity in the kidney nephrons,
produce a large amount of Reactive Oxygen Species (ROS) that commonly balanced
with antioxidant enzymes and free radical sweeping systems. ROS creates negative
biological effects in the body, includes lipids peroxidation of cell wall, oxidation
of proteins and finally DNA defects. Unfortunately, hyperglycemia produces ROS
with unbalancing that seems it is produced in the mitochondria (Nishikawa
et al., 2007). Hyperglycemia causes increase of oxidative stress,
even before appearance of DN signs in the individuals. In the DN the concentration
of defected markers of DNA increase and tissue analysis of kidney biopsy samples
showed increase production of glycoxidation and lipid oxidation products in
matrix of mesangial and kidney glomeruli. These effects are detected in a lower
amount in the none diabetic people (Suzuki et al.,
1999; Vasavada and Agarwal, 2005). One of the mechanisms
that infused performance of endothelial is increase of oxidative stress that
interference with NO production (Rodriguez-Manas et al.,
1998). Recently studying on the HG-treated mesangial cells, the antioxidant
role of BMP-7 has been reported (Yeh et al., 2009)
and it has been suggested that probiotic supplements causes to normality of
glucose rate in the diabetic rats (Yadav et al.,
2007, 2008). The probiotic bacteria defined as live
microorganism, when are used in sufficient amount could cause the health of
host (Andreasen et al., 2010). The probiotic
bacteria produces exopolysaccharide as a layer connected to the cells surface
or feed it to the environment. Some studies has shown that these exopolysaccharide
has some roles like, stimulation of immune system, anti tumor activity and also
phosphate groups of exopolysaccharide has important roles on the activation
of macrophages and lymphocytes (Zahedi et al., 2011).
With attention to the role of hyperglycemia in increase secretion of inflammatory
factors (Pickup, 2004; Shoelson et
al., 2006), the study designed to investigate the effects of lactobacillus
reuteri PTCC1655 on serum levels of glucose, no and gene expression of BMP-7
and TGF-β in streptozotocin-induced diabetic rats.
MATERIALS AND METHODS
Preparation of L. reuteri PTCC1655: Lactobacillus reuteri PTCC1655 (DSM 20016, ATCC23272) was purchased from Persian Type Culture Collection (PTCC), Tehran, Iran. The microorganism was regenerated in to Man Rogosa Sharpe (MRS) broth from the lyophilized vial cultured in anaerobic jar Containing (80% N2, 10% CO2, 10% H2), incubated for 48 h at 37°C and preparation (1x108 CFU/rat/day).
Animal studies: Twelve week-old Male Wistar rats (200±50 g) were received from Pasteur Institute of Iran. Prior ethical permission was obtained from the animal care committee of Tehran University of medical sciences. The study comprised of five groups i.e. a non-diabetic (N), diabetic untreated (D), diabetic rat fed with L. reuteri (1x109 CFU/rat/day) (D+L), diabetic treated with Insulin (4-5 U kg day-1) (D+I), non-diabetic rats fed L. reuteri (1x109 CFU/rat/day) (N+L). Each group comprising of six (n = 6) rats. The animals were housed in individual cages, given free access to water and standard chow ad libitum. Diabetes was induced by single intraperitoneal (i.p.) injection of streptozotocin (STZ) at 50 mg kg-1 b. wt. in sodium citrate buffer (0.01 M, pH 4.5). Blood glucose levels were monitored 48 h later and periodically thereafter by ACCU-CHEK advantage blood glucose monitor (Roche Group, Indianapolis, IN, USA), by rat-tailed blood sampling. Rats with blood glucose levels above 250 mg dL-1 were considered as diabetic. All rats had unrestricted access to food/water and were maintained for 8 weeks in accordance with institutional animal care and use committee procedures of Tehran University.
Kidney preparation: After 60 days, rats were sacrificed and kidney were rapidly removed and weighed, fixed in 10% formalin, cleared in xylene embedded in paraffin and cut at 4 μm and stained with hematoxylin and eosin, Periodic Acid Shiff (PAS) and trichrome.
Measurement of glucose: All the rats were anesthetized with ether and blood was taken from the heart and serum was separated. Blood Glucose was measured by the glucose kit based on glucose-oxidize method.
Nitric oxide measurement: NO was quantified by a chemiluminescence method
using NO Analyzer (CLD88 ECO MEDICS AG Switzerland), a high-sensitive detector
for measuring NO, based on a gas phase chemiluminescent reaction between NO
The emission of a photon from electrically excited nitrogen dioxide is in the red and near-infrared region of the spectrum and it is detected by a thermoelectrically cooled red-sensitive photomultiplier tube.
Total RNA extraction: Total RNA were extracted from kidney tissues by
miRCURY RNA isolation kit (Exiqon, Vedbaek, Denmark) according to the
|| Primer used for real-time PCR
Extraction procedure were applied to 50-70 mg tissue, that yield near 500
μg μL-1 purified cellular total RNA after DNase I (RNase-free)
(Fermentas GmbH, Leon-Rot, Germany) treat of RNA trapped on column.
Quality and amount of total RNA measured by Nanodrop 1000 spectrophotometer (Thermo scientific, Wilmington, DE 19810 USA).
cDNA synthesized real time PCR: For cDNA synthesis form mRNA, 3 μg of total RNA was reverse transcribed by Revert Aid Premium Reverse Transcriptase (Fermentas GmbH, Leon-Rot, Germany) according to manufacture instructions.
Real-time PCR was performed on the Bio-RadiQ 5 detection system, using SYBR
Green master mix (Exiqon, Vedbaek, Denmark) and designed primers for BMP-7 and
TGF-β and GUSB as housekeeping gene, sequences derived from (www.ncbi.nlm.nih.gov)
and primer design was performed with the Beacon designer software.
The PCR conditions for amplification of mRNA was initial denaturation and enzyme
activation at 95°C for 10 min, followed by 45 cycles of 95°C for 10
sec and combined annealing and extension 60°C for 60 sec. The Ct amount
of PCR products was normalized with housekeeping gene GUSB (Glucuronidase beta).
A standard curve was generated from dilution series constructed from housekeeping
gene pooled cDNA. Relative changes in gene expression were quantified using
ΔΔCT method (Yuan et al., 2006).
Real-time PCR assays were carried out in triplicate and each experiment was
performed at least three times. Primer (invitrogen) used for real-time PCR are
listed in Table 1.
Statistical analysis: Statistical analyses were performed using SPSS version 16.0 software. Analysis of variance (ANOVA) was used to compare means in different groups. The difference of experimental data was analyzed by using student t test. Data were expressed as the Mean±SE. p<0.05 was considered to be significant.
Effect of L. reuteri on BMP-7 and TGF-β expression: To investigate
the effect of L. reuteri on BMP-7 expression the mRNA levels were analyzed
with real-time PCR.
|Fig. 1(a, b):
||The expression levels of TGF-β and BMP-7 in kidney of
studied rats, (a) Lactobacillus reuteri reduced TGF-β mRNA expression
but in groups D and D+1 the expression of TGF-β significantly induced,
(b) In diabetic rats that administrated with Lactobacillus reuteri
the expression of BMP-7 significantly induced but in groups D and D+2 BMP-7
expression decreased (p<0.05). The TGF-β and BMP-7 levels were analyzed
to GUSB RNA levels. Values are Mean±SE from least three separate
experiments performed in triplicate, *Significance between control and other
Fig. 1 showed the effect of L. reuteri on expression
level of TGF-β and BMP-7 in kidney of studied rats.
||The effect of Lactobacillus reuteri on serum levels
of NO in studied rats. Values are Mean±SD (n = 6 per group), In each
group *p<0.05: **p<0.05 compared to value of normal rats
||Effect of L. reuteri on serum glucose, *p<0.05 vs.
normal, p<0.05 vs. diabetic groups
Following two months administration of L. reuteri (1x108
CFU/rat/day), the diabetic rats (D) were found to have an increase in TGF-β
expression (p<0.05). However, in diabetic rats that administration of L.
reuteri (1x108 CFU/rat/day) (D+L) the expression of TGF-β
significantly decreased (Fig. 1a). The BMP-7 expression level
increased in the kidney of STZ-diabetic rats that fed L. reuteri (p<0.05),
but in insulin treatment (D+I) and diabetic groups (D) BMP-7 expression significantly
decreased (Fig. 1b).
Effect of L. reuteri on NO level: Studying the effect of L. reuteri on NO level in rats serum, It was found that the NO levels in diabetic rats were lower than normal groups (N and N+L), however after administration of L. reuteri (1x108 CFU/rat/day) the increase in levels of NO were increased in (D+L) group than in the other diabetic groups(D and D+I) (p<0.05) (Fig. 2).
Effect of L. reuteri on serum glucose levels: The effect of L. reuteri on serum glucose levels in STZ-induced diabetic rats are shown in Fig. 3. Our results showed that STZ-induced diabetic rats had increased serum glucose (>250 mg mL-1) than normal rats. In diabetic groups that administration of L. reuteri (1x108 CFU/rat/day) serum glucose levels decreased but not significantly than control group. Also in insulin treatment groups the glucose levels significantly decreased.
Effect of L. reuteri on kidney fibrosis: The optical microscopy
stained with H and E, PAS and Massons trichrome showed in (Fig.
4). The renal tissue stained by Periodic Acid Schiffs (PAS) solution appeared
normal glomeruli in the kidney of non diabetic groups (N and N+L).
||Kidney structures of STZ-induced diabetic rates after 60 days
administration of L. reuteri (1x108 CFU/rat/day), (a)
Normal views of glomerulus of rats kidney that administration of L. reuteri
(1x108 CFU/rat/day) (H and E x100), (b) Hypercellularity
and congestion in glomerular in diabetic rats no fed L. reuteri (H
and E), (c) Interstitial fibrosis in diabetic rats kidney (Trichrome
x400) and (d) Basement membrane thickness increasing in diabetic rats (PAS
In diabetic groups (D and D+I) the increase in glomerular epithelial cells
and glomerular basement membrane thickening in glomerular capillary, to a severe
extent was shown. Also some glomeruli showed glomerular atrophy with thickening
of bowman capsule in tubular area, vacuolization of epithelium of proximal tubular
and interstitial edema with increase in fibroblast and macrophage cells were
observed. In Massons trichrome staining, often interspersed fibrosis seen
in the tubules, comparable to normal groups. In STZ-induced diabetic rats that
fed with L. reuteri (1x108 CFU/rat/day) the kidney sections
showed no pathological changes.
It is known that pathogenesis of DN is associated with abnormalities of renal
NO generation (Kuno et al., 2011). The inhibition
of mesangial cell growth is another effect of NO. Nitric oxide also is a strong
repressor of connective tissue growth factors that is important to development
of interstitial glomerular fibrosis. It has been shown that hyperglycemia promoted
oxidative stress in nephritic tissues, eventually leading to renal injury in
diabetes (Chang et al., 2011). Present results
suggested that when STZ-induced diabetic rats fed with L. reuteri (1x108
CFU/rat/day) for 60 days, extension of hypercellularity, congestion in glomerulus
and thickening of glomerular basement membrane did not occur, comparing to diabetic
group. We also found the normal glomerulus and no pathological changes in the
group that fed with insulin and normal groups. In diabetic rats in the absence
of excessive NO mesangial matrix production will occur associated with type
I collagen accumulation (Chiarelli et al., 2000).
In this study, comparing with the control group the plasma level of NO was significantly
decreased in diabetic group. Increased the Reactive Oxygen Molecules (ROMs)
and decrease antioxidants may be responsible for decrease of NO production (Onozato
et al., 2002). In the STZ-induced diabetic rats increase of renal
oxidative stress caused by hyperglycemia have been shown (Cheng
et al., 2010). Changes the production of NO associate with glomerular
hyper filtration and vascular permeability (Dooley et al.,
2012). A decrease in oxidative stress plays important role on the improvement
of renal function by recovering renal BMP-7 expression in STZ-DN rats. BMP-7
plays an anti oxidative role in mesangial cells (Yeh et
al., 2009). Due to the antioxidant role of BMP-7, it seems that Lactobacillus
reuteri with ability to increase expression of BMP-7, prevented the progression
of renal fibrosis in diabetic rats. TGF-β and the receptor together constitute
a major biologic signal inducing a switch toward a profibrotic cellular phenotype
(Wang et al., 2003). Our result shows the increase
of TGF-β expression in diabetic group of rats are associated with decrease
of BMP-7 gene expression. We also find that the expression of BMP-7 gene increased
in diabetic rats fed with L. reuteri 1x108 CFU/rat/day).
In this case, it seems that BMP-7 could prevent oxidative stress hyperglycemia
in the diabetic rats.
Present results showed that BMP-7 expression in kidney and serum levels of NO decreased in STZ-induced diabetic rats. But in diabetic groups that fed with L. reuteri (1x108 CFU/rat/day)the kidney expression of BMP-7 and serum levels of NO increased. Perhaps the protective role of L. reuteri on rats kidney maybe due to their antioxidant properties. So it may be suggested that L. reuteri or other beneficial bacteria increase BMP-7 gene expression and improve DN. Further studies are required to evaluate the effect of different species of probiotic bacteria on the DN.
This work was fully supported by a research grand from the Tehran University of Medical Sciences, Faculty of Medicine for which authors are thankful. We are also grateful to the members of the Department of Biochemistry, Faculty of Medicine, for their contributions.
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