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Effect of mononuclear cells versus pioglitazone on streptozotocin-induced diabetic nephropathy in rats
Pharmacological Reports 2012, 64, 12231233 ISSN 1734-1140
Copyright © 2012 by Institute of Pharmacology Polish Academy of Sciences
Effect of mononuclear cells versus pioglitazone on streptozotocin-induced diabetic nephropathy in rats Riham E. Masoad1, Mohamed M. S. Ewais1, Mona K. Tawfik1, Hwayda S. Abd El-All2
Departments of Pharmacology, Pathology, Faculty of Medicine, Suez Canal University, Ismailia 41522, Egypt Correspondence: Mona K. Tawfik, e-mail: dmon_kamal@ yahoo.com
Abstract: Background: Diabetic nephropathy is a serious diabetic complication that leads to end stage renal disease. Cell therapies with human embryonic and specific adult stem cells have emerged as an alternative management for various diseases. Methods: To test this hypothesis, the present study was conducted to compare effect of MNCs treatment (iv injection once in the tail vein for diabetic rats in a dose of 150 × 106 MNCs cells/rat) versus pioglitazone (10 mg/kg, for eight weeks) on improving the renal structure and function changes and reducing laminin deposition associated with STZ-induced diabetic nephropathy in rats. Results: Treatment with pioglitazone or MNCs, demonstrated a significant improvement in the STZ-induced renal functional and structural changes in comparison with diabetic control group. Additionally, our histopathological and immunohistochemical studies confirm these results. Meanwhile, MNCs treated group exhibited more improvement in all studied parameters as compared to pioglitazone treated group. Conclusion: These data indicate that MNCs treatment was superior to pioglitazone in controlling hyperglycemia, improving the renal structure and function changes and reducing renal laminin expression associated with STZ-induced diabetic nephropathy in rats. Key words: diabetic nephropathy, laminin, MNCs, pioglitazone, type 1 diabetes
Abbreviations: ECM – extracellular matrix, ESRD – end stage renal disease, FCS – fetal calf serum, GFR – glomerular filtration rate, HUCB – human umbilical cord blood, ip – intraperitoneal, MNCs – mononuclear stem cells, PBS – phosphate buffered saline, PPARs – peroxisome proliferatoractivated receptors, RPM – rich placental media , STZ – streptozotocin
Introduction Diabetes mellitus is a chronic metabolic syndrome that can result in serious complications as neuropathy, retinopathy and nephropathy [5]. Diabetic nephropathy is a leading cause of end stage renal disease
(ESRD) [43]. It is typically defined by albuminuria and abnormal renal function as represented by an abnormality in serum creatinine, calculated creatinine clearance, or glomerular filtration rate (GFR) [8]. Laminin is implicated in cell migration, remodeling of the extracellular matrix (ECM) and basement membrane. Therefore, laminin deposition could be of concern in the progression of renal diseases [2]. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily [12, 33]. PPARs regulate diverse cell functions including fatty acid metabolism, adipocyte differentiation, inflammation, atherosclerosis, and cell cycle [12]; PPAR-g plays a vital role in adipogenesis and its activation by thiazolidinediones improves insulin sensitivity via Pharmacological Reports, 2012, 64, 12231233
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this role. In addition to the demonstrated physiological roles, several clinical and experimental studies have implicated PPAR-g in the pathogenesis and the course of diabetic nephropathy [21, 29]. Cell therapies with human embryonic and specific adult stem cells have emerged as an alternative management for various diseases [48, 53]. These cells were able to proliferate and differentiate into various cell types including those bearing a phenotype of insulin-secreting b-cells [19]. Human umbilical cord blood (HUCB) as a source of stem cells has a number of significant advantages over other stem cell sources [35]. It has advantages of easy procurement, no risk to donors, low risk of transmitting infections, immediate availability and immune tolerance allowing successful transplantation despite HLA disparity and it could be cryopreserved. Moreover, cord blood can be obtained non-invasively and frequently in contrast to invasive bone marrow aspiration [35, 52]. Stem cells are attractive candidates for renal repair leading to differentiation of both nephrons and collecting ducts. Indeed, they have been used in experimental acute renal failure, which could lower renal injury, accelerate tubular proliferation and improve renal function with a dramatic repopulation of the mesangium [19, 39]. So, the present study was conducted to compare the role of pioglitazone and HUCB mononuclear stem cells (MNCs) in improving the renal structure and function changes associated with streptozotocin (STZ)-induced diabetic nephropathy in rats. As ECM accumulation is a hallmark of diabetic nephropathy and its accumulation contribute to organ dysfunction and disease progression, our study was extended to evaluate the effect of pioglitazone and MNCs on the expression of renal laminin.
Materials and Methods Experimental animals
Forty (n = 40) male adult albino rats weighing 170 ± 10 g were used in this study. They were purchased from the Egyptian Organization for Biological Products and Vaccines (Egypt), and allowed free access to food and water ad libitum. They were kept under constant conditions with 12/12 h light/dark cycles and left for acclimatization for one week before the start of the 1224
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study. The care and handling of the animals were approved by the Animal Care and Use Committee at the Suez Canal University and were in accordance with the National Institutes of Health guide for the care and use of laboratory animals (Maryland, USA). All efforts were made to minimize animal suffering and to reduce the number of animals used. Drugs and chemicals
STZ and pioglitazone HCl (Sigma Chemical Co., Egypt) were used. Isolation buffer which consists of 100 ml phosphate buffered saline (PBS) + 10 ml rich placental media (RPM ) + 1 ml 10% fetal calf serum (FCS) and lymphocyte separation medium, ficoll hypaque (Sigma Chemical Co., Egypt) were used for the separation of MNCs. Pioglitazone was suspended in distilled water and given for diabetic rats orally once a day by gastric tube at doses of 10 mg/kg [30] for eight weeks [37]. MNCs were injected iv once in the tail vein of diabetic rats in a dose of 150 × 10$ MNCs/rat [9]. Experimental induction of type 1 diabetes in rats
Type 1 diabetes (T DM) in rats was induced by single intraperitoneal (ip) injection of STZ (65 mg/ kg) dissolved in disodium citrate buffer (pH 4.5) in a dose volume of 1 ml/kg body weight [37]. After 72 h following STZ administration, blood was collected from tail vein after overnight fasting rats for 16 h, and serum samples were analyzed for blood glucose. Animals showing fasting blood glucose higher than 250 mg/dl were considered as diabetic and used for further study [23]. MNCs isolation according to Jaatinen and Laine [22]
UCB was collected in the presence of anticoagulant and was diluted 1:1 in isolation buffer. Seven ml of diluted cord blood was transferred onto 3 ml ficoll and was centrifuged 20 min at 800 × g. The interface which contains the low density mononuclear cells was collected and suspended in equal volume of isolation buffer and was centrifuged 20 min at 500 × g. Then, the pellet was resuspended in isolation buffer again and was centrifuged 20 min at 300 × g and maintained at 2–8°C.
Pioglitazone and MNCs reduces STZ-induced diabetic nephropathy
Riham E. Masoad et al.
Experimental protocol
Rats were divided into four groups each consisting of ten animals: Group 1: Normal control group: they were nondiabetic group received no medication. So they served as normal control group. Group 2: Diabetic control group: diabetic rats received distilled water given orally once a day by gastric tube for eight weeks. So they served as diabetic control group. Group 3: Diabetic treated group with pioglitazone: diabetic rats received pioglitazone given orally once a day by gastric tube for eight weeks. Group 4: Diabetic treated group with MNCs: diabetic rats received iv injection of MNCs. Investigations done at the beginning of the study, the 4th week and the 8th week after induction of diabetes
Systolic blood pressuse was measured by using tailcuff plethysmograph in conscious prewarmed rats [55]. Then, blood samples were collected via the tail veins after overnight fasting rats for 16 h to determine blood glucose level enzymatically according to the principle of Trinder [47] using Spin react diagnostics kits (Santa Coloma, Spain), and measuring serum levels of blood urea nitrogen and creatinine by standard urease assays and picric acid reactions by colorimetric methods [34], using Bioclin kit (Santa Coloma, Spain). Urine samples were evaluated for proteinuria using albumin/creatinine ratio. These urine samples were obtained by palpation and gentle pressing on urinary bladder [1]. The urinary albumin concentration was measured by using microalbumin kits strip [44]. Investigation done at the beginning of the study and the 8th week after induction of diabetes
Glycosated hemoglobin using HPLC method [6]
By the end of eight weeks after induction of diabetes, all rats in all groups were sacrificed. Right kidney was removed, weighed to calculate kidney weight/body weight ratio, and then fixed in formalin for histopathological studies. The left kidney was removed and fixed in formalin for immunohistochemical studies.
Histopathological examination
Severity of glomerular lesions was graded on scale based on Gellman criteria: D , all glomeruli normal; D , focal lesions on glomeruli; D , mesangial thickening presents diffusely throughout the kidney; D!, capillary lumina narrowed and obliterated; D", glomerulus appear hylanized [16]. Tubulointerstitial damage was graded as follows: 0 – no lesions showing cell infiltration and fibrosis; 1 – minimal injury (single focus of lesion); 2 – mild injury (more than two isolated foci); 3 – moderate injury (more than five isolated foci); and 4 – severe injury (more than ten isolated foci or diffuse infiltration and fibrosis) [44]. Vascular lesions were graded as follows: 0 – normal; 1 – focal thickening of the walls; 2 – diffuse thickening of the walls; 3 – obliterated lumen. Interstitial inflammation was graded as follows: 0 – no cell infiltration; 1 – minimal cell infiltration; 2 – mild to moderate cell infiltration; and 3 – severe diffuse infiltration [44].
Immunohistochemistry for expression of renal laminin
Laminin was detected by rabbit polyclonal antibody anti-rat laminin (Dako Company, Egypt) using the heat-induced antigen retrieval technique. Intensity of laminin stain was performed on scale based on Taneda scale: 0 – no staining; 1 – mesangial staining involving less than 25% of the area examined; 2 – segmental mesangial staining involving 25 to 50% of mesangial areas present; 3 – mesangial staining involving 50 to 75% of the areas examined; 4 – diffuse mesangial staining involving more than 75% of areas examined [44].
Statistical analysis
Results were collected and expressed as the mean ± SD. Results were analyzed using The Statistical Package for the Social Sciences, version 15 (SPSS Software, SPSS Inc., Chicago, USA). One-way analysis of variance (ANOVA) followed by Duncan’s post-hoc test were used to test the significance of the difference between quantitative variables; p value < 0.05 was considered to be statistically significant. Pharmacological Reports, 2012, 64, 12231233
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Results Diabetic control group showed significant (p < 0.05) increase in fasting blood glucose level in comparison with the untreated control group throughout the entire course of the experiment (Tab. 1). These elevations in fasting blood glucose levels were accompanied with
gradual significant (p < 0.05) increase in serum creatinine (Tab. 2), blood urea levels (Tab. 3) and urinary albumin/creatinine ratio (p < 0.05) (Tab. 4) compared with untreated control group, suggesting a significant degree of glomerular dysfunction. These functional changes were associated with a significant elevation in systolic arterial blood pressure (p < 0.05) in comparison with the untreated control group especially in
Tab. 1. Effect of pioglitazone and MNCs on fasting blood sugar (mg/dl) in study groups at different time intervals
Study groups
0 week
After 1 week
After 4 weeks
Normal control
100 ± 7
105 ± 15
100 ± 11
Diabetic control
103 ± 7
443 ± 22**
431 ± 22**
After 8 weeks 100 ± 15 451 ± 26** ##
Pioglitazone-treated
100 ± 7
429 ± 22**
258 ± 17 **
Mononuclear cells-treated
100 ± 13
421 ± 22**
220 ± 10** ## †
265 ± 15** ## 120 ± 8## †
n = 10. Values are the mean ± SD. *, ** Significantly different from normal control group at p < 0.05 and 0.001, respectively. , Significantly different from diabetic control group at p < 0.05 and 0.001, respectively. V Significantly different from pioglitazone treated group at p < 0.05
Tab. 2. Effect of pioglitazone and MNCs on serum creatinine (mg/dl) in study groups at different time intervals
Study groups
0 week
After 1 week
After 4 weeks
After 8 weeks
Normal control
0.72 ± 0.07
0.71 ± 0.08
0.68 ± 0.08
0.67 ± 0.07
Diabetic control
0.72 ± 0.05
0. 0.68 ± 0.04
Pioglitazone-treated
0.72 ± 0.09
#
0.79 ± 0.09*
0.91 ± 0.06*
Mononuclear cells-treated
0.72 ± 0.03
0.75 ± 0.03#
0.78 ± 0.02 ## †
1.2 ± 0.11** #
1.36 ± 0.05** 0.89 ± 0.09*# 0.74 ± 0.03 ## †
n = 10. Values are the mean ± SD. *, ** Significantly different from normal control group at p < 0.05 and 0.001, respectively. , Significantly different from diabetic control group at p < 0.05 and 0.001, respectively. V Significantly different from pioglitazone treated group at p < 0.05
Tab. 3. Effect of pioglitazone and MNCs on blood urea (mg/dl) in study groups at different time intervals
Study groups
0 week
After 1 week
After 4 weeks
After 8 weeks
Normal control
12.5 ± 0.78
12.7 ± 0.95
13.0 ± 0.14
12.4 ± 0.78
Diabetic control
12.7 ± 0.75
13.1 ± 2.8
36.0 ± 0.95*
47.7 ± 0.75**
Pioglitazone-treated
12.5 ± 0.78
12.8 ± 1.77
25.6 ± 0.62#
23.5 ± 0.78#
Mononuclear cells-treated
12.3 ± 0.95
13.0 ± 0.49
16.0 ± 0.24## †
16.3 ± 0.95## †
n = 10. Values are the mean ± SD. *, ** Significantly different from normal control group at p < 0.05 and 0.001, respectively. , Significantly different from diabetic control group at p < 0.05 and 0.001, respectively V Significantly different from pioglitazone treated group at p < 0.05
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Pioglitazone and MNCs reduces STZ-induced diabetic nephropathy
Riham E. Masoad et al.
Tab. 4. Effect of pioglitazone and MNCs on urinary albumin/creatinine ratio (μg/mg) in study groups at different time intervals
Study groups
0 week
After 1 week
After 4 weeks
After 8 weeks
Normal control
33.0 ± 1.38
31.4 ± 0.81
34.0 ± 1.2
30.2 ± 1.38
Diabetic control
30.2 ± 1.34
34.8 ± 7.5
90 ± 5.3**
115.2 ± 1.34** #
Pioglitazone-treated
30.5 ± 0.97
33.7 ± 3.3
68.7 ± 1.7*
50.5 ± 0.97##
Mononuclear cells-treated
30.2 ± 0.75
32.9 ± 2.1
42.2 ± 1.3## †
34.2 ± 0.75## †
n = 10. Values are the mean ± SD. *, ** Significantly different from normal control group at p < 0.05 and 0.001, respectively. , Significantly different from diabetic control group at p < 0.05 and 0.001 respectively. V Significantly different from pioglitazone-treated group at p < 0.05
Tab. 5. Effect of pioglitazone and MNCs on systolic blood pressure (mm/Hg) in study groups at different time intervals
Study groups
0 week
After 1 week
After 4 weeks
After 8 weeks
Normal control
100.1 ± 5
103 ± 4
104 ± 3
100 ± 2
Diabetic control
100 ± 4
107 ± 5
123 ± 4* #
133 ± 3**
Pioglitazone-treated
101 ± 4
102 ± 3
117 ± 3*
111 ± 2#
Mononuclear cells-treated
103 ± 5
101 ± 4
105 ± 3# †
103 ± 3## †
n = 10. Values are the mean ± SD. *, ** Significantly different from normal control group at p < 0.05 and 0.001 respectively. , Significantly different from diabetic control group at p < 0.05 and 0.001, respectively. V Significantly different from pioglitazone treated group at p < 0.05
the last four weeks (Tab. 5). Additionally, Figure 1A shows that glycosated hemoglobin increased significantly (p < 0.05) in diabetic rats at the end of 8 weeks compared to untreated control group. Moreover, the increase in fasting blood glucose level elicited significant (p < 0.05) increase in kidney weight/body weight ratio in comparison with the untreated control group (Fig. 1B). These deleterious effects associated with STZ injection were significantly improved by treatment with pioglitazone and MNCs in comparison with diabetic control group (p < 0.05). Meanwhile, MNCs treated group exhibited more improvement in all studied parameters (p < 0.05) as compared to pioglitazone treated group, indicating that MNCs offered more corrective effect than pioglitazone (Tab. 1–5, Fig. 1). Renal histopathological results and immunohistological staining verified the serological results. Fig. 2-Ib showed that diabetic control group exhibited hylanosis, hypertrophy and distortion of glomeruli, dilated tubules and thick basement membranes, with significant (p < 0.05) elevation in mean histopa-
thological score in comparison with the untreated group (Fig. 2-II). These histopathological results were associated with significant (p < 0.05) increase in the expression of renal laminin in comparison with the untreated control group indicating excessive deposition and accumulation of extracellular matrix (Fig. 3-Ib, II). These deleterious effects associated with STZ injection were ameliorated by treatment with either pioglitazone or MNCs (Fig. 2-Ic,d). It was obvious that treatment with either pioglitazone or MNCs was associated with significant (p < 0.05) reduction in mean histopathological score in comparison with diabetic control group (Fig. 2-II). These histopathological improvements were associated with significant (p < 0.05) reduction in renal laminin expression and accumulation (Fig. 3-Ic,d, II). It was obvious that MNCs administration was associated with significant reduction in the mean histopathological (Fig. 2-II) and stain intensity scores (Fig. 3-II) in comparison to that afforded by pioglitazone treatment (p < 0.05); indicating that MNCs offered more corrective effects than pioglitazone. Pharmacological Reports, 2012, 64, 12231233
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Fig. 1. Effect of pioglitazone and MNCs on (A) glycosated hemoglobin (mg/dl), (B) kidney weight/body weight (mg/g). Values are expressed as the mean ± SD (n = 10), analyzed by one-way ANOVA followed by Duncan’s multiple comparisons test. *, #, † p < 0.05; **, ## p < 0.001; * compared with normal control group, # compared with diabetic control group, † compared with pioglitazone treated group
Discussion
Diabetic nephropathy is the leading cause of endstage renal disease. To prevent the development and progression of diabetic nephropathy, a comprehensive understanding of the pathophysiology of diabetic nephropathy is needed [43]. It was initially thought 1228
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that renal injury in diabetic nephropathy is caused primarily by hemodynamic alterations, such as hyperfiltration and hyperperfusion. There is now a clear evidence that these changes are only one aspect of a complex series of pathophysiological alterations caused by disturbed glucose homeostasis. This exerts hemodynamic, inflammatory and trophic effects on renal cells [57].
Pioglitazone and MNCs reduces STZ-induced diabetic nephropathy
Riham E. Masoad et al.
Fig. 2. I: (a) A photomicrograph of renal sections stained with hematoxylin and eosin (H & E) showed no histopathological changes in kidney of normal control group. (b) Kidney sections of diabetic control group showed hypertrophied distorted glomeruli and dilated tubules with thick basement membranes. (c) Pioglitazone-treated group showed some interstitial fibrosis and inflammation with slightly dilated glomeruli. Treatment with MNCs was nearly similar to normal control group except that there was minimal focal tubular lesion (d). (H & E 40´). II: The histopathological scores in the experimental groups. Values are expressed as the mean ± SD (n = 10), analyzed by one-way ANOVA followed by Duncan’s multiple comparisons test. *, #, † p < 0.05; **, ## p < 0.001; * compared with normal control group, # compared with diabetic control group, † compared with pioglitazone-treated group
There is a strong evidence that poor glycemic control is necessary in the pathogenesis of diabetic nephropathy, and some of the mechanisms that link hyperglycemia to the functional/structural abnormalities of diabetic kidney disease have been elucidated [57]. Extracellularly, glucose reacts nonenzymatically with primary amines of proteins, forming glycated compounds. Furthermore, it has become increasingly clear that excess glucose can directly exert toxic effects by altering intracellular signaling pathways, and today this is believed to be a major mechanism by which hyperglycemia results in kidney damage manifested by increased urinary protein, increased serum creatinine
and blood urea [14, 57]. Consistently with this hypothesis, it has been demonstrated that diabetic rats exhibited significant increase in fasting blood glucose levels throughout the entire experiment accompanied with elevation of glycosated hemoglobin. Additionally, diabetic rats showed evidence of renal dysfunction in the form of significant increase in serum creatinine, urea levels associated with increased urinary albumin/creatinine ratio. Zelmanovitz et al. [57] emphasized that, hyperglycemia and glycosated hemoglobin is a significant risk factor for the development of micro and macro albuminuria; both in type 1 and in type 2 DM as the early defect in diabetic nephropathy Pharmacological Reports, 2012, 64, 12231233
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Fig. 3. I: Photomicrograph of renal sections showed the intensity of laminin stain in different studied groups. (a) Normal control group. (b) Diabetic control group. (c) Pioglitazone-treated group. (d) MNCs treated group. II: The stain intensity scores in the experimental groups. Values are expressed as the mean ± SD (n = 10), analyzed by one-way ANOVA followed by Duncan’s multiple comparisons test. *, #, † p < 0.05; **, ## p < 0.001; * compared with normal control group, # compared with diabetic control group, † compared with pioglitazone-treated group
is impairment of renal perfusion [30] which makes it easier for albumin to leak from capillaries to renal glomerulus [4]. In addition, our results confirmed the findings of Kuhad and Chopra [28], who found that diabetic rats exhibited elevated blood pressure and this is considered as an indicator of deteriorated renal function. There is an evidence that hypertension plays a critical role in the progression of diabetic nephropathy. Indeed the development of proteinuria is paralleled in most cases with a gradual increase in systemic blood pressure, and there is a significant correlation between 1230
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the blood pressure levels and the rate of decline in glomerular filtration rate [57]. This correlation between hyperglycemia and elevated blood pressure could be explained by Peterson et al. [40], who documented that under normal conditions, intraglomerular capillary pressure is tightly regulated by precise adjustments in afferent and efferent arteriolar resistance. Hyperglycemia induces vasodilatation with marked reduction in afferent and a lesser reduction in efferent arteriolar resistance. This leads to an increase in glomerular capillary pressure levels and allows increase in systemic blood pressure [57].
Pioglitazone and MNCs reduces STZ-induced diabetic nephropathy
Riham E. Masoad et al.
Our histopathological results clearly demonstrated that STZ injection was associated with hypertrophied distorted glomeruli, interstitial fibrosis, tubular atrophy and vascular lesions. Our results were in line with previous studies [31, 44, 57] which emphasized that tubulointerstitial injury is a feature of diabetic nephropathy and a predictor of renal dysfunction. Pathologic changes that have been described in association with diabetic nephropathy include thickening of the tubular basement membrane, tubular atrophy, interstitial fibrosis, and arteriosclerosis and correlate closely with renal dysfunction, albuminuria, and mesangial expansion [45]. ECM accumulation is a hallmark of diabetic nephropathy [7]; yet the mechanisms responsible for altered accumulation of matrix as well as the functional consequences of matrix accumulation remain to be elucidated. It was previously demonstrated that not only the amount of ECM in each of the renal compartments, glomerular basement membrane, tubular basement membrane, and mesangial matrix increase, but the composition of each compartment is also altered [42]. Furthermore, their accumulation contributes to organ dysfunction and disease progression [31]. In keeping with this postulate, we found that there was significant increase in expression of renal laminin indicating accumulation of extracellular matrix and increased kidney weight indicating renal hypertrophy. Under normal physiologic conditions, laminins expressed primarily in basement membrane, which provide the structural support and communication necessary between resident cells and their environment for homeostasis, but under the chronic influence of diabetes, their altered expression becomes obvious [25]. It was reported that, mesangial expansion is due to both increased production and reduced degradation of ECM proteins such as type IV and I collagen, laminin, and fibronectin [ 25, 44]. High glucose level is the main initiator of ECM deposition as it was not only associated with cell hypertrophy and increases gene expression and protein secretion of ECM components, such as collagen, laminin, and fibronectin [11, 42], but also reduced the activity of metalloproteases enzymes responsible for ECM degradation leading to accumulation of ECM component as laminin [44]. Pioglitazone was effective in correcting not only glucose metabolism, but also cardiovascular and renal complications in rats [24]. In accordance with this notion, the results of this study demonstrated that treatment with pioglitazone not only reduced fasting
blood glucose level throughout the entire course of the experiment, but also resulted in improvement in functional renal parameters, reduction in glycosated hemoglobin level, renal injury and dysfunction, and kidney weight/body weight ratio. These were associated with marked improvement of urinary albumin excretion and amelioration in the level of blood pressure. Additionally, pioglitazone treatment improved glomerular hypertrophy and ameliorated glomerulosclerosis in these animals, with significant reduction of laminin synthesis in the diabetic kidney. These results are consistent with many previous studies [20, 49, 51], which emphasized that PPAR-g agonists have been considered to have direct beneficial effects on the diabetic kidney disease both in animal and human diabetic nephropathy studies. They have been shown to not only improve lipid and glucose metabolism and glycosated hemoglobin [18], but also lowered blood pressure [41, 46] improved glomerular hypertrophy [13, 38, 46], decreased proteinuria and prevented glomerular injury, renal arteriolosclerosis and ECM deposition [30, 37]. Pioglitazone also improved the urinary albumin/creatinine ratio and the glomerular and Bowman’s capsule volume ratios [56]. Multiple mechanisms have been suggested for the renoprotective effect of PPAR-g including; inhibition of reactive oxygen species [15, 17] and biomarkers of inflammation that are crucial in the course of renal disease [3, 24, 26, 37]. Additionally, Xu et al. [54] concluded that, in a rat model, pioglitazone ameliorated many of the physiological, cellular, and molecular processes associated with diabetic nephropathy. Moreover, our results demonstrated that, MNCs afford more corrective effects on all studied parameters in comparison to pioglitazone. Injection of stem cells caused improvement not only in blood glucose levels but also normalization of glomerular hypertrophy and tubular dilatation [9]. Stem cells mediated reversal of diabetic complications was due to decrease in blood glucose levels [10] and due to their trophic influence [27, 36]. After the stem cells transplanted into the body, they can transfer to kidney lesion automatically through the automatic homing of stem cells, differentiate kinds of desired cells, and then repair the kidney inherent cell, restore the kidney function, block and reverse the kidney fibrosis process and enhance their generation [32, 39, 50]. In conclusion, MNCs may be useful to treat both the hyperglycemia and the accompanying renal damage seen in diabetic patients. It seems that MNCs ,
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treatment was superior to pioglitazone in controlling hyperglycemia, improving the renal structure and function changes and reducing renal laminin expression associated with STZ- induced diabetic nephropathy in rats.
Conflict of interest: There is no conflict of interest or funding agencies in this work.
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14. 15. 16.
References: 1. Aaltonen P, Luimula P, Tuula P, Ahola H, Palmén T, Holthöfer H: Changes in the expression of nephrin gene and protein in experimental diabetic nephropathy. Lab Invest, 2001, 81, 1185–1190. 2. Aumaille M: A simplified laminin nomenclature. Matrix Biol, 2005, 24, 326–332. 3. Bao Y, Jia R, Yuan J, Li J: Rosiglitazone ameliorates diabetic nephropathy by inhibiting reactive oxygen species and its downstream-signaling pathways. Pharmacol J, 2007, 80, 57–64. 4. Brosius F: New insights into the mechanisms of fibrosis and sclerosis in diabetic nephropathy. Rev Endocr Metab Disord, 2008, 9, 245–254. 5. Carvalho E, Ferreira L: Experimental models of induction of diabetes mellitus in rats. Acta Cir Bras, 2003, 18, 60–64. 6. Detata V, Novelli M, Bombara M: Determination of glycated hemoglobins in the rat: comparison between two different chromatographic methods and application in experimental diabetology. Res Exp Med (Berl), 1996, 196, 9–16. 7. Du J, Wang L, Liu L, Fan Q, Yao L, Cui Y, Kang P et al.: IFN-g suppresses the high glucose-induced increase in TGF-b1 and CTGF synthesis in mesangial cells. Pharmacol Rep, 2011, 63, 1137–1144. 8. Earle K: Variation of progression of diabetic nephropathy according to racial origin. Nephrol Dial Transplant, 2007, 16, 286–290. 9. Ende N, Chen R, Reddi A: Transplantation of human umbilical cord blood cells improves glycemia and glomerular hypertrophy in type 2 diabetic mice. Biochem Biophys Res Commun, 2004, 321, 168–171. 10. Ende N, Chen R, Reddi A: Effect of human umbilical cord blood cells on glycemia and insulitis in type 1 diabetic mice. Biochem Biophys Res Commun, 2004, 325, 665–669. 11. Erensoy N, Yýlmazer S, Öztürk M, Tunçdemir M, Uysal O, Hatemi H: Effects of ACE inhibition on the expression of type IV collagen and laminin in renal glomeruli in experimental diabetes. Acta Histochem, 2004, 106, 279–287. 12. Feige J, Gelman L, Michalik L, Desvergne B, Wahli W: From molecular action to physiological outputs: peroxisome proliferator-activated receptors are nuclear recep-
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17.
18.
19. 20. 21.
22. 23. 24. 25.
26.
27. 28. 29.
tors at the crossroads of key cellular functions. Prog Lipid Res, 2006, 45, 120–159. Feng-qin D, Hong L, Wei-min C, Jun T, Qun L, Yu R: Effects of pioglitazone on expressions of matrix metalloproteinases 2 and 9 in kidneys of diabetic rats. Chin Med J, 2004, 117, 1040–1044. Gabriella G, Luigi G, Gian V: Diabetes Mellitus: a Fundamental and Clinical Text, 3rd edn. Lippincott Williams & Wilkins, Philadelphia, 2004. Gang J, Young S, Sang Y, Mi H: pioglitazone attenuates diabetin nephropathy through anti-inflamatory mechanism. Nephrol Dial Transplant, 2007, 23, 750–760. Gellman D, Pirani C, Soothill G, Muethrcke R, Kark R: Diabetic nephropathy: clinical and pathologic studies based on renal biopsy. Med J, 1959, 38, 312–317. Gumieniczek A: Effect of the new thiazolidinedionepioglitazone on the development of oxidative stress in liver and kidney of diabetic rabbits. Life Sci, 2003, 74, 553–562. Hirasawa Y, Matsui Y, Yamane K, Yabuki SY, Kawasaki Y, Toyoshi T, Kyuki K, et al.: Pioglitazone improves obesity type diabetic nephropathy: relation to the mitigation of renal oxidative reaction. Exp Anim, 2008, 57, 423–432. Hussain M, Theise N: Stem-cell therapy for diabetes mellitus. Lancet, 2004, 364, 203–205. Iglesias P. Díez J: Peroxisome proliferator-activated receptor gamma agonists in renal disease. Eur J Endocrinol, 2006, 154, 613–621. Isshiki K, Haneda M, Koya D, Maeda S, Sugimoto T, Kikkawa R: Thiazolidinedione compounds ameliorate glomerular dysfunction independent of their insulinsensitizing action in diabetic rats. Diabetes, 2000, 49, 1022–1032. Jaatinen T, Laine J: Isolation of mononuclear cells from human cord blood by Ficoll Paque density gradient. Curr Prot Stem Cell Biol, 2007, 2A.1. Kakadiya J, Brambhatt J, Shah N: Renoprotective activity of pioglitazone on ischemia/reperfusion induced renal damage in diabetic rats. Rec Res Sci Tech, 2010, 2, 92–97. Kan C, Wu C, Zhang M, Wang L: Pioglitazone renal cortex of diabetic vascular endothelial growth factor expression. Diabetes, 2011, 48, 2229–2239. Katavetin P, Eiam-Ong S, Suwanwalaikorn S: Pioglitazone reduces urinary protein and urinary transforming growth factor-b excretion in patients with type 2 diabetes and overt nephropathy. J Med Assoc Thai, 2006, 89, 170–177. Ko GJ, Kang YS, Han SY, Lee MH, Song HK, Han KH, Kim HK et al.: Pioglitazone attenuates diabetic nephropathy through an anti-inflammatory mechanism in type 2 diabetic rats. Nephrol Dial Transplant, 2008, 23, 2750–2760. Koblas T, Harman S, Saudek F: The application of umbilical cord blood in treatment of diabetes mellitus. Rev Diabetic Stud, 2005, 2, 228–234. Kuhad A, Chopra K: Attenuation of diabetic nephropathy by tocotrienol: involvement of NF-kB signaling pathway. Life Sci, 2009, 84, 296–301. Lee G, Olson P, Hevener A: PPARd regulates glucose metabolism and insulin sensitivity. Proc Natl Acad Sci USA, 2006, 103, 3444–3449.
Pioglitazone and MNCs reduces STZ-induced diabetic nephropathy
Riham E. Masoad et al.
30. Lee R, Seo M, Reger R, Spees J, Pulin A, Prockop D: Multipotent stromal cells promote repair of pancreatic and renal glomeruli in diabetic NOD/SICD mice. Proc Natl Acad Sci USA, 2007, 103, 17438–17443. 31. Li J, Kwak SJ, Jung DS, Kim J, Yoo T, Ryu D: Podocyte biology in diabetic nephropathy. Kidney Int, 2007, 72, S36–S42. 32. Lim J, Jang J, Kim J, Moon S, Lee C, Lee K: Human umbilical cord blood mononuclear cell transplantation in rats with intrinsic sphincter deficiency. J Korean Med Sci, 2010, 25, 663–670. 33. Michalik L, Auwerx J, Berger J, Chatterjee V, Wahli W: International union of pharmacology. Peroxisome proliferator-activated receptors. Pharmacol Rev, 2006, 58, 726– 741. 34. Mizutani A, Okajima K, Uchiba M, Noguchi T: Activated protein C reduces ischemia/reperfusion-induced renal injury in rats by inhibiting leukocyte activation. Blood, 2000, 95, 3781–3787. 35. Neil H, Kyle C, Annette M, Thomas E: Cord blood in regenerative medicine: do we need immune suppression. J Transl Med, 2007, 26, 5–8. 36. Ende N, Ruifeng Chen R, Reddi AS: Transplantation of human umbilical cord blood cells improves glycemia and glomerular hypertrophy in type 2 diabetic mice. Biochem Biophys Res Commu, 2004, 321, 168–171. 37. Ohga S, Shikata K, Yozai K: Thiazolidinedione ameliorates renal injury in experimental diabetic rats through anti-inflammatory effects mediated by inhibition of NF-kB activation. Am J Physiol Renal Physiol, 2007, 292, F1141–F1150. 38. Okada T, Wada J, Hida K, Eguchi J, Hashimoto I, Baba M, Yasuhara A et al.: Thiazolidinediones ameliorate diabetic nephropathy via cell cycle–dependent mechanisms. Diabetes, 2006, 55, 1666–1677. 39. Parker J: Can stem cell transplant treat diabetes and kidney disease? Stem Cells J, 2011, 29, 38–42. 40. Peterson JC, Adler S, Burkart J: Blood pressure control, proteinuria and the progression of renal disease: the modification of diet in renal disease study. Ann Int Med, 1995, 123,754–762. 41. Sarafidis PA, Lasaridis AN, Nilsson PM, Pagkalos EM, Hitoglou-Makedou AD, Pliakos A: The effect of rosiglitazone on urine albumin excretion in patients with type 2 diabetes mellitus and hypertension. Am J Hypertens, 2005, 18, 227–234. 42. Schaeffer V, Hansen K, Morris D, Abrass C: Reduction in laminin b2 mRNA translation is responsible for impaired IGFBP-5-mediated mesangial cell migration in the presence of high glucose. Am J Physiol Renal Physiol, 2010, 298, F314–F322. 43. Sevensson M, Eriksson J, Dahlquist G: Early glycemic control, age at onset, and development of microvascular complications in childhood-onset type 1 diabetes. Diabetes Care, 2004, 27, 955–962.
44. Taneda S, Jeffery W, Pippin B: Amelioration of diabetic nephropathy in SPARC-null mice. J Am Soc Nephrol, 2003, 14, 968–980. 45. Thallas-Bonke V, Lindschau C, Rizkalla B, Bach LA, Boner G, Meier M, Haller H et al.: Attenuation of extracellular matrix accumulation in diabetic nephropathy by the advanced glycation end product cross-link breaker ALT-711 via a protein kinase C-a-dependent pathway. Diabetes, 2004, 53, 2921–2930. 46. Toblli J, Cao G, Giani J, Angerosa M, Dominici F, Gonzalez-Cadavid N: Antifibrotic effects of pioglitazone at low doses on the diabetic rat kidney are associated with the improvement of markers of cell turnover, tubular and endothelial integrity, and angiogenesis. Kidney Blood Press Res, 2011, 34, 20–33. 47. Trinder P: Determination of blood glucose using 4-aminophenazone as oxygen acceptor. J Clin Pathol, 1969, 222, 46–50. 48. Tuch B: Stem cells – a clinical update. Aust Family Phys, 2006, 35, 719–721. 49. Vijay S, Mishra M, Kumar H, Tripathi K: Effect of pioglitazone and rosiglitazone on mediators of endothelial dysfunction, markers of angiogenesis and inflammatory cytokines in type-2 diabetes. Acta Diabetol, 2006, 46, 27–33. 50. Volarevicn V, Arsenijevic E, Miodrag L: Concise review: Mesenchymal stem cell treatment of the complications of diabetes mellitus. Stem Cells J, 2011, 29, 5–10. 51. Wang X, Jiang T, Levi M: Nuclear hormone receptors in diabetic nephropathy. Nature Rev Nephrol, 2010, 6, 342–351. 52. Tse W, Laughlin M: Umbilical cord blood transplantation: A new alternative option. Hematol J, 2005, 377-383. 53. Wobus A, Boheler K: Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol Rev, 2005, 85, 635–678. 54. Xu X, Chen P, Zheng Q, Wang Y, Chen W: Effect of pioglitazone on diabetic nephropathy and expression of HIF-1a and VEGF in the renal tissues of type 2 diabetic rats. Diabetes Res Clin Pract, 2011, 93, 63–69. 55. Yozai K, Shikata K, Sasaki M, Tone A, Ohga S, Usui H, Okada S et al.: Methotrexate prevents renal injury in experimental diabetic rats via anti-inflammatory actions. J Am Soc Nephrol, 2005, 16, 3326–3338. 56. Zafiriou S, Stanner S, Saad S, Polhill T, Poronnik P, Pollock C: Pioglitazone inhibits cell growth and reduces matrix production in human kidney fibroblasts. J Am Soc Nephrol, 2005, 6, 638–645. 57. Zelmanovitz T, Gerchman F, Balthazar A, Thomazelli F, Matos J, Canani L: Diabetic nephropathy. Diabetol Metab Syndr, 2009, 1, 1–10.
Received: November 9, 2011; in the revised form: May 25, 2012; accepted: June 20, 2012.
Pharmacological Reports, 2012, 64, 12231233
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Copyright © 2012 by Institute of Pharmacology Polish Academy of Sciences
Effect of mononuclear cells versus pioglitazone on streptozotocin-induced diabetic nephropathy in rats Riham E. Masoad1, Mohamed M. S. Ewais1, Mona K. Tawfik1, Hwayda S. Abd El-All2
Departments of Pharmacology, Pathology, Faculty of Medicine, Suez Canal University, Ismailia 41522, Egypt Correspondence: Mona K. Tawfik, e-mail: dmon_kamal@ yahoo.com
Abstract: Background: Diabetic nephropathy is a serious diabetic complication that leads to end stage renal disease. Cell therapies with human embryonic and specific adult stem cells have emerged as an alternative management for various diseases. Methods: To test this hypothesis, the present study was conducted to compare effect of MNCs treatment (iv injection once in the tail vein for diabetic rats in a dose of 150 × 106 MNCs cells/rat) versus pioglitazone (10 mg/kg, for eight weeks) on improving the renal structure and function changes and reducing laminin deposition associated with STZ-induced diabetic nephropathy in rats. Results: Treatment with pioglitazone or MNCs, demonstrated a significant improvement in the STZ-induced renal functional and structural changes in comparison with diabetic control group. Additionally, our histopathological and immunohistochemical studies confirm these results. Meanwhile, MNCs treated group exhibited more improvement in all studied parameters as compared to pioglitazone treated group. Conclusion: These data indicate that MNCs treatment was superior to pioglitazone in controlling hyperglycemia, improving the renal structure and function changes and reducing renal laminin expression associated with STZ-induced diabetic nephropathy in rats. Key words: diabetic nephropathy, laminin, MNCs, pioglitazone, type 1 diabetes
Abbreviations: ECM – extracellular matrix, ESRD – end stage renal disease, FCS – fetal calf serum, GFR – glomerular filtration rate, HUCB – human umbilical cord blood, ip – intraperitoneal, MNCs – mononuclear stem cells, PBS – phosphate buffered saline, PPARs – peroxisome proliferatoractivated receptors, RPM – rich placental media , STZ – streptozotocin
Introduction Diabetes mellitus is a chronic metabolic syndrome that can result in serious complications as neuropathy, retinopathy and nephropathy [5]. Diabetic nephropathy is a leading cause of end stage renal disease
(ESRD) [43]. It is typically defined by albuminuria and abnormal renal function as represented by an abnormality in serum creatinine, calculated creatinine clearance, or glomerular filtration rate (GFR) [8]. Laminin is implicated in cell migration, remodeling of the extracellular matrix (ECM) and basement membrane. Therefore, laminin deposition could be of concern in the progression of renal diseases [2]. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily [12, 33]. PPARs regulate diverse cell functions including fatty acid metabolism, adipocyte differentiation, inflammation, atherosclerosis, and cell cycle [12]; PPAR-g plays a vital role in adipogenesis and its activation by thiazolidinediones improves insulin sensitivity via Pharmacological Reports, 2012, 64, 12231233
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this role. In addition to the demonstrated physiological roles, several clinical and experimental studies have implicated PPAR-g in the pathogenesis and the course of diabetic nephropathy [21, 29]. Cell therapies with human embryonic and specific adult stem cells have emerged as an alternative management for various diseases [48, 53]. These cells were able to proliferate and differentiate into various cell types including those bearing a phenotype of insulin-secreting b-cells [19]. Human umbilical cord blood (HUCB) as a source of stem cells has a number of significant advantages over other stem cell sources [35]. It has advantages of easy procurement, no risk to donors, low risk of transmitting infections, immediate availability and immune tolerance allowing successful transplantation despite HLA disparity and it could be cryopreserved. Moreover, cord blood can be obtained non-invasively and frequently in contrast to invasive bone marrow aspiration [35, 52]. Stem cells are attractive candidates for renal repair leading to differentiation of both nephrons and collecting ducts. Indeed, they have been used in experimental acute renal failure, which could lower renal injury, accelerate tubular proliferation and improve renal function with a dramatic repopulation of the mesangium [19, 39]. So, the present study was conducted to compare the role of pioglitazone and HUCB mononuclear stem cells (MNCs) in improving the renal structure and function changes associated with streptozotocin (STZ)-induced diabetic nephropathy in rats. As ECM accumulation is a hallmark of diabetic nephropathy and its accumulation contribute to organ dysfunction and disease progression, our study was extended to evaluate the effect of pioglitazone and MNCs on the expression of renal laminin.
Materials and Methods Experimental animals
Forty (n = 40) male adult albino rats weighing 170 ± 10 g were used in this study. They were purchased from the Egyptian Organization for Biological Products and Vaccines (Egypt), and allowed free access to food and water ad libitum. They were kept under constant conditions with 12/12 h light/dark cycles and left for acclimatization for one week before the start of the 1224
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study. The care and handling of the animals were approved by the Animal Care and Use Committee at the Suez Canal University and were in accordance with the National Institutes of Health guide for the care and use of laboratory animals (Maryland, USA). All efforts were made to minimize animal suffering and to reduce the number of animals used. Drugs and chemicals
STZ and pioglitazone HCl (Sigma Chemical Co., Egypt) were used. Isolation buffer which consists of 100 ml phosphate buffered saline (PBS) + 10 ml rich placental media (RPM ) + 1 ml 10% fetal calf serum (FCS) and lymphocyte separation medium, ficoll hypaque (Sigma Chemical Co., Egypt) were used for the separation of MNCs. Pioglitazone was suspended in distilled water and given for diabetic rats orally once a day by gastric tube at doses of 10 mg/kg [30] for eight weeks [37]. MNCs were injected iv once in the tail vein of diabetic rats in a dose of 150 × 10$ MNCs/rat [9]. Experimental induction of type 1 diabetes in rats
Type 1 diabetes (T DM) in rats was induced by single intraperitoneal (ip) injection of STZ (65 mg/ kg) dissolved in disodium citrate buffer (pH 4.5) in a dose volume of 1 ml/kg body weight [37]. After 72 h following STZ administration, blood was collected from tail vein after overnight fasting rats for 16 h, and serum samples were analyzed for blood glucose. Animals showing fasting blood glucose higher than 250 mg/dl were considered as diabetic and used for further study [23]. MNCs isolation according to Jaatinen and Laine [22]
UCB was collected in the presence of anticoagulant and was diluted 1:1 in isolation buffer. Seven ml of diluted cord blood was transferred onto 3 ml ficoll and was centrifuged 20 min at 800 × g. The interface which contains the low density mononuclear cells was collected and suspended in equal volume of isolation buffer and was centrifuged 20 min at 500 × g. Then, the pellet was resuspended in isolation buffer again and was centrifuged 20 min at 300 × g and maintained at 2–8°C.
Pioglitazone and MNCs reduces STZ-induced diabetic nephropathy
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Experimental protocol
Rats were divided into four groups each consisting of ten animals: Group 1: Normal control group: they were nondiabetic group received no medication. So they served as normal control group. Group 2: Diabetic control group: diabetic rats received distilled water given orally once a day by gastric tube for eight weeks. So they served as diabetic control group. Group 3: Diabetic treated group with pioglitazone: diabetic rats received pioglitazone given orally once a day by gastric tube for eight weeks. Group 4: Diabetic treated group with MNCs: diabetic rats received iv injection of MNCs. Investigations done at the beginning of the study, the 4th week and the 8th week after induction of diabetes
Systolic blood pressuse was measured by using tailcuff plethysmograph in conscious prewarmed rats [55]. Then, blood samples were collected via the tail veins after overnight fasting rats for 16 h to determine blood glucose level enzymatically according to the principle of Trinder [47] using Spin react diagnostics kits (Santa Coloma, Spain), and measuring serum levels of blood urea nitrogen and creatinine by standard urease assays and picric acid reactions by colorimetric methods [34], using Bioclin kit (Santa Coloma, Spain). Urine samples were evaluated for proteinuria using albumin/creatinine ratio. These urine samples were obtained by palpation and gentle pressing on urinary bladder [1]. The urinary albumin concentration was measured by using microalbumin kits strip [44]. Investigation done at the beginning of the study and the 8th week after induction of diabetes
Glycosated hemoglobin using HPLC method [6]
By the end of eight weeks after induction of diabetes, all rats in all groups were sacrificed. Right kidney was removed, weighed to calculate kidney weight/body weight ratio, and then fixed in formalin for histopathological studies. The left kidney was removed and fixed in formalin for immunohistochemical studies.
Histopathological examination
Severity of glomerular lesions was graded on scale based on Gellman criteria: D , all glomeruli normal; D , focal lesions on glomeruli; D , mesangial thickening presents diffusely throughout the kidney; D!, capillary lumina narrowed and obliterated; D", glomerulus appear hylanized [16]. Tubulointerstitial damage was graded as follows: 0 – no lesions showing cell infiltration and fibrosis; 1 – minimal injury (single focus of lesion); 2 – mild injury (more than two isolated foci); 3 – moderate injury (more than five isolated foci); and 4 – severe injury (more than ten isolated foci or diffuse infiltration and fibrosis) [44]. Vascular lesions were graded as follows: 0 – normal; 1 – focal thickening of the walls; 2 – diffuse thickening of the walls; 3 – obliterated lumen. Interstitial inflammation was graded as follows: 0 – no cell infiltration; 1 – minimal cell infiltration; 2 – mild to moderate cell infiltration; and 3 – severe diffuse infiltration [44].
Immunohistochemistry for expression of renal laminin
Laminin was detected by rabbit polyclonal antibody anti-rat laminin (Dako Company, Egypt) using the heat-induced antigen retrieval technique. Intensity of laminin stain was performed on scale based on Taneda scale: 0 – no staining; 1 – mesangial staining involving less than 25% of the area examined; 2 – segmental mesangial staining involving 25 to 50% of mesangial areas present; 3 – mesangial staining involving 50 to 75% of the areas examined; 4 – diffuse mesangial staining involving more than 75% of areas examined [44].
Statistical analysis
Results were collected and expressed as the mean ± SD. Results were analyzed using The Statistical Package for the Social Sciences, version 15 (SPSS Software, SPSS Inc., Chicago, USA). One-way analysis of variance (ANOVA) followed by Duncan’s post-hoc test were used to test the significance of the difference between quantitative variables; p value < 0.05 was considered to be statistically significant. Pharmacological Reports, 2012, 64, 12231233
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Results Diabetic control group showed significant (p < 0.05) increase in fasting blood glucose level in comparison with the untreated control group throughout the entire course of the experiment (Tab. 1). These elevations in fasting blood glucose levels were accompanied with
gradual significant (p < 0.05) increase in serum creatinine (Tab. 2), blood urea levels (Tab. 3) and urinary albumin/creatinine ratio (p < 0.05) (Tab. 4) compared with untreated control group, suggesting a significant degree of glomerular dysfunction. These functional changes were associated with a significant elevation in systolic arterial blood pressure (p < 0.05) in comparison with the untreated control group especially in
Tab. 1. Effect of pioglitazone and MNCs on fasting blood sugar (mg/dl) in study groups at different time intervals
Study groups
0 week
After 1 week
After 4 weeks
Normal control
100 ± 7
105 ± 15
100 ± 11
Diabetic control
103 ± 7
443 ± 22**
431 ± 22**
After 8 weeks 100 ± 15 451 ± 26** ##
Pioglitazone-treated
100 ± 7
429 ± 22**
258 ± 17 **
Mononuclear cells-treated
100 ± 13
421 ± 22**
220 ± 10** ## †
265 ± 15** ## 120 ± 8## †
n = 10. Values are the mean ± SD. *, ** Significantly different from normal control group at p < 0.05 and 0.001, respectively. , Significantly different from diabetic control group at p < 0.05 and 0.001, respectively. V Significantly different from pioglitazone treated group at p < 0.05
Tab. 2. Effect of pioglitazone and MNCs on serum creatinine (mg/dl) in study groups at different time intervals
Study groups
0 week
After 1 week
After 4 weeks
After 8 weeks
Normal control
0.72 ± 0.07
0.71 ± 0.08
0.68 ± 0.08
0.67 ± 0.07
Diabetic control
0.72 ± 0.05
0. 0.68 ± 0.04
Pioglitazone-treated
0.72 ± 0.09
#
0.79 ± 0.09*
0.91 ± 0.06*
Mononuclear cells-treated
0.72 ± 0.03
0.75 ± 0.03#
0.78 ± 0.02 ## †
1.2 ± 0.11** #
1.36 ± 0.05** 0.89 ± 0.09*# 0.74 ± 0.03 ## †
n = 10. Values are the mean ± SD. *, ** Significantly different from normal control group at p < 0.05 and 0.001, respectively. , Significantly different from diabetic control group at p < 0.05 and 0.001, respectively. V Significantly different from pioglitazone treated group at p < 0.05
Tab. 3. Effect of pioglitazone and MNCs on blood urea (mg/dl) in study groups at different time intervals
Study groups
0 week
After 1 week
After 4 weeks
After 8 weeks
Normal control
12.5 ± 0.78
12.7 ± 0.95
13.0 ± 0.14
12.4 ± 0.78
Diabetic control
12.7 ± 0.75
13.1 ± 2.8
36.0 ± 0.95*
47.7 ± 0.75**
Pioglitazone-treated
12.5 ± 0.78
12.8 ± 1.77
25.6 ± 0.62#
23.5 ± 0.78#
Mononuclear cells-treated
12.3 ± 0.95
13.0 ± 0.49
16.0 ± 0.24## †
16.3 ± 0.95## †
n = 10. Values are the mean ± SD. *, ** Significantly different from normal control group at p < 0.05 and 0.001, respectively. , Significantly different from diabetic control group at p < 0.05 and 0.001, respectively V Significantly different from pioglitazone treated group at p < 0.05
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Pioglitazone and MNCs reduces STZ-induced diabetic nephropathy
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Tab. 4. Effect of pioglitazone and MNCs on urinary albumin/creatinine ratio (μg/mg) in study groups at different time intervals
Study groups
0 week
After 1 week
After 4 weeks
After 8 weeks
Normal control
33.0 ± 1.38
31.4 ± 0.81
34.0 ± 1.2
30.2 ± 1.38
Diabetic control
30.2 ± 1.34
34.8 ± 7.5
90 ± 5.3**
115.2 ± 1.34** #
Pioglitazone-treated
30.5 ± 0.97
33.7 ± 3.3
68.7 ± 1.7*
50.5 ± 0.97##
Mononuclear cells-treated
30.2 ± 0.75
32.9 ± 2.1
42.2 ± 1.3## †
34.2 ± 0.75## †
n = 10. Values are the mean ± SD. *, ** Significantly different from normal control group at p < 0.05 and 0.001, respectively. , Significantly different from diabetic control group at p < 0.05 and 0.001 respectively. V Significantly different from pioglitazone-treated group at p < 0.05
Tab. 5. Effect of pioglitazone and MNCs on systolic blood pressure (mm/Hg) in study groups at different time intervals
Study groups
0 week
After 1 week
After 4 weeks
After 8 weeks
Normal control
100.1 ± 5
103 ± 4
104 ± 3
100 ± 2
Diabetic control
100 ± 4
107 ± 5
123 ± 4* #
133 ± 3**
Pioglitazone-treated
101 ± 4
102 ± 3
117 ± 3*
111 ± 2#
Mononuclear cells-treated
103 ± 5
101 ± 4
105 ± 3# †
103 ± 3## †
n = 10. Values are the mean ± SD. *, ** Significantly different from normal control group at p < 0.05 and 0.001 respectively. , Significantly different from diabetic control group at p < 0.05 and 0.001, respectively. V Significantly different from pioglitazone treated group at p < 0.05
the last four weeks (Tab. 5). Additionally, Figure 1A shows that glycosated hemoglobin increased significantly (p < 0.05) in diabetic rats at the end of 8 weeks compared to untreated control group. Moreover, the increase in fasting blood glucose level elicited significant (p < 0.05) increase in kidney weight/body weight ratio in comparison with the untreated control group (Fig. 1B). These deleterious effects associated with STZ injection were significantly improved by treatment with pioglitazone and MNCs in comparison with diabetic control group (p < 0.05). Meanwhile, MNCs treated group exhibited more improvement in all studied parameters (p < 0.05) as compared to pioglitazone treated group, indicating that MNCs offered more corrective effect than pioglitazone (Tab. 1–5, Fig. 1). Renal histopathological results and immunohistological staining verified the serological results. Fig. 2-Ib showed that diabetic control group exhibited hylanosis, hypertrophy and distortion of glomeruli, dilated tubules and thick basement membranes, with significant (p < 0.05) elevation in mean histopa-
thological score in comparison with the untreated group (Fig. 2-II). These histopathological results were associated with significant (p < 0.05) increase in the expression of renal laminin in comparison with the untreated control group indicating excessive deposition and accumulation of extracellular matrix (Fig. 3-Ib, II). These deleterious effects associated with STZ injection were ameliorated by treatment with either pioglitazone or MNCs (Fig. 2-Ic,d). It was obvious that treatment with either pioglitazone or MNCs was associated with significant (p < 0.05) reduction in mean histopathological score in comparison with diabetic control group (Fig. 2-II). These histopathological improvements were associated with significant (p < 0.05) reduction in renal laminin expression and accumulation (Fig. 3-Ic,d, II). It was obvious that MNCs administration was associated with significant reduction in the mean histopathological (Fig. 2-II) and stain intensity scores (Fig. 3-II) in comparison to that afforded by pioglitazone treatment (p < 0.05); indicating that MNCs offered more corrective effects than pioglitazone. Pharmacological Reports, 2012, 64, 12231233
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Fig. 1. Effect of pioglitazone and MNCs on (A) glycosated hemoglobin (mg/dl), (B) kidney weight/body weight (mg/g). Values are expressed as the mean ± SD (n = 10), analyzed by one-way ANOVA followed by Duncan’s multiple comparisons test. *, #, † p < 0.05; **, ## p < 0.001; * compared with normal control group, # compared with diabetic control group, † compared with pioglitazone treated group
Discussion
Diabetic nephropathy is the leading cause of endstage renal disease. To prevent the development and progression of diabetic nephropathy, a comprehensive understanding of the pathophysiology of diabetic nephropathy is needed [43]. It was initially thought 1228
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that renal injury in diabetic nephropathy is caused primarily by hemodynamic alterations, such as hyperfiltration and hyperperfusion. There is now a clear evidence that these changes are only one aspect of a complex series of pathophysiological alterations caused by disturbed glucose homeostasis. This exerts hemodynamic, inflammatory and trophic effects on renal cells [57].
Pioglitazone and MNCs reduces STZ-induced diabetic nephropathy
Riham E. Masoad et al.
Fig. 2. I: (a) A photomicrograph of renal sections stained with hematoxylin and eosin (H & E) showed no histopathological changes in kidney of normal control group. (b) Kidney sections of diabetic control group showed hypertrophied distorted glomeruli and dilated tubules with thick basement membranes. (c) Pioglitazone-treated group showed some interstitial fibrosis and inflammation with slightly dilated glomeruli. Treatment with MNCs was nearly similar to normal control group except that there was minimal focal tubular lesion (d). (H & E 40´). II: The histopathological scores in the experimental groups. Values are expressed as the mean ± SD (n = 10), analyzed by one-way ANOVA followed by Duncan’s multiple comparisons test. *, #, † p < 0.05; **, ## p < 0.001; * compared with normal control group, # compared with diabetic control group, † compared with pioglitazone-treated group
There is a strong evidence that poor glycemic control is necessary in the pathogenesis of diabetic nephropathy, and some of the mechanisms that link hyperglycemia to the functional/structural abnormalities of diabetic kidney disease have been elucidated [57]. Extracellularly, glucose reacts nonenzymatically with primary amines of proteins, forming glycated compounds. Furthermore, it has become increasingly clear that excess glucose can directly exert toxic effects by altering intracellular signaling pathways, and today this is believed to be a major mechanism by which hyperglycemia results in kidney damage manifested by increased urinary protein, increased serum creatinine
and blood urea [14, 57]. Consistently with this hypothesis, it has been demonstrated that diabetic rats exhibited significant increase in fasting blood glucose levels throughout the entire experiment accompanied with elevation of glycosated hemoglobin. Additionally, diabetic rats showed evidence of renal dysfunction in the form of significant increase in serum creatinine, urea levels associated with increased urinary albumin/creatinine ratio. Zelmanovitz et al. [57] emphasized that, hyperglycemia and glycosated hemoglobin is a significant risk factor for the development of micro and macro albuminuria; both in type 1 and in type 2 DM as the early defect in diabetic nephropathy Pharmacological Reports, 2012, 64, 12231233
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Fig. 3. I: Photomicrograph of renal sections showed the intensity of laminin stain in different studied groups. (a) Normal control group. (b) Diabetic control group. (c) Pioglitazone-treated group. (d) MNCs treated group. II: The stain intensity scores in the experimental groups. Values are expressed as the mean ± SD (n = 10), analyzed by one-way ANOVA followed by Duncan’s multiple comparisons test. *, #, † p < 0.05; **, ## p < 0.001; * compared with normal control group, # compared with diabetic control group, † compared with pioglitazone-treated group
is impairment of renal perfusion [30] which makes it easier for albumin to leak from capillaries to renal glomerulus [4]. In addition, our results confirmed the findings of Kuhad and Chopra [28], who found that diabetic rats exhibited elevated blood pressure and this is considered as an indicator of deteriorated renal function. There is an evidence that hypertension plays a critical role in the progression of diabetic nephropathy. Indeed the development of proteinuria is paralleled in most cases with a gradual increase in systemic blood pressure, and there is a significant correlation between 1230
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the blood pressure levels and the rate of decline in glomerular filtration rate [57]. This correlation between hyperglycemia and elevated blood pressure could be explained by Peterson et al. [40], who documented that under normal conditions, intraglomerular capillary pressure is tightly regulated by precise adjustments in afferent and efferent arteriolar resistance. Hyperglycemia induces vasodilatation with marked reduction in afferent and a lesser reduction in efferent arteriolar resistance. This leads to an increase in glomerular capillary pressure levels and allows increase in systemic blood pressure [57].
Pioglitazone and MNCs reduces STZ-induced diabetic nephropathy
Riham E. Masoad et al.
Our histopathological results clearly demonstrated that STZ injection was associated with hypertrophied distorted glomeruli, interstitial fibrosis, tubular atrophy and vascular lesions. Our results were in line with previous studies [31, 44, 57] which emphasized that tubulointerstitial injury is a feature of diabetic nephropathy and a predictor of renal dysfunction. Pathologic changes that have been described in association with diabetic nephropathy include thickening of the tubular basement membrane, tubular atrophy, interstitial fibrosis, and arteriosclerosis and correlate closely with renal dysfunction, albuminuria, and mesangial expansion [45]. ECM accumulation is a hallmark of diabetic nephropathy [7]; yet the mechanisms responsible for altered accumulation of matrix as well as the functional consequences of matrix accumulation remain to be elucidated. It was previously demonstrated that not only the amount of ECM in each of the renal compartments, glomerular basement membrane, tubular basement membrane, and mesangial matrix increase, but the composition of each compartment is also altered [42]. Furthermore, their accumulation contributes to organ dysfunction and disease progression [31]. In keeping with this postulate, we found that there was significant increase in expression of renal laminin indicating accumulation of extracellular matrix and increased kidney weight indicating renal hypertrophy. Under normal physiologic conditions, laminins expressed primarily in basement membrane, which provide the structural support and communication necessary between resident cells and their environment for homeostasis, but under the chronic influence of diabetes, their altered expression becomes obvious [25]. It was reported that, mesangial expansion is due to both increased production and reduced degradation of ECM proteins such as type IV and I collagen, laminin, and fibronectin [ 25, 44]. High glucose level is the main initiator of ECM deposition as it was not only associated with cell hypertrophy and increases gene expression and protein secretion of ECM components, such as collagen, laminin, and fibronectin [11, 42], but also reduced the activity of metalloproteases enzymes responsible for ECM degradation leading to accumulation of ECM component as laminin [44]. Pioglitazone was effective in correcting not only glucose metabolism, but also cardiovascular and renal complications in rats [24]. In accordance with this notion, the results of this study demonstrated that treatment with pioglitazone not only reduced fasting
blood glucose level throughout the entire course of the experiment, but also resulted in improvement in functional renal parameters, reduction in glycosated hemoglobin level, renal injury and dysfunction, and kidney weight/body weight ratio. These were associated with marked improvement of urinary albumin excretion and amelioration in the level of blood pressure. Additionally, pioglitazone treatment improved glomerular hypertrophy and ameliorated glomerulosclerosis in these animals, with significant reduction of laminin synthesis in the diabetic kidney. These results are consistent with many previous studies [20, 49, 51], which emphasized that PPAR-g agonists have been considered to have direct beneficial effects on the diabetic kidney disease both in animal and human diabetic nephropathy studies. They have been shown to not only improve lipid and glucose metabolism and glycosated hemoglobin [18], but also lowered blood pressure [41, 46] improved glomerular hypertrophy [13, 38, 46], decreased proteinuria and prevented glomerular injury, renal arteriolosclerosis and ECM deposition [30, 37]. Pioglitazone also improved the urinary albumin/creatinine ratio and the glomerular and Bowman’s capsule volume ratios [56]. Multiple mechanisms have been suggested for the renoprotective effect of PPAR-g including; inhibition of reactive oxygen species [15, 17] and biomarkers of inflammation that are crucial in the course of renal disease [3, 24, 26, 37]. Additionally, Xu et al. [54] concluded that, in a rat model, pioglitazone ameliorated many of the physiological, cellular, and molecular processes associated with diabetic nephropathy. Moreover, our results demonstrated that, MNCs afford more corrective effects on all studied parameters in comparison to pioglitazone. Injection of stem cells caused improvement not only in blood glucose levels but also normalization of glomerular hypertrophy and tubular dilatation [9]. Stem cells mediated reversal of diabetic complications was due to decrease in blood glucose levels [10] and due to their trophic influence [27, 36]. After the stem cells transplanted into the body, they can transfer to kidney lesion automatically through the automatic homing of stem cells, differentiate kinds of desired cells, and then repair the kidney inherent cell, restore the kidney function, block and reverse the kidney fibrosis process and enhance their generation [32, 39, 50]. In conclusion, MNCs may be useful to treat both the hyperglycemia and the accompanying renal damage seen in diabetic patients. It seems that MNCs ,
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treatment was superior to pioglitazone in controlling hyperglycemia, improving the renal structure and function changes and reducing renal laminin expression associated with STZ- induced diabetic nephropathy in rats.
Conflict of interest: There is no conflict of interest or funding agencies in this work.
13.
14. 15. 16.
References: 1. Aaltonen P, Luimula P, Tuula P, Ahola H, Palmén T, Holthöfer H: Changes in the expression of nephrin gene and protein in experimental diabetic nephropathy. Lab Invest, 2001, 81, 1185–1190. 2. Aumaille M: A simplified laminin nomenclature. Matrix Biol, 2005, 24, 326–332. 3. Bao Y, Jia R, Yuan J, Li J: Rosiglitazone ameliorates diabetic nephropathy by inhibiting reactive oxygen species and its downstream-signaling pathways. Pharmacol J, 2007, 80, 57–64. 4. Brosius F: New insights into the mechanisms of fibrosis and sclerosis in diabetic nephropathy. Rev Endocr Metab Disord, 2008, 9, 245–254. 5. Carvalho E, Ferreira L: Experimental models of induction of diabetes mellitus in rats. Acta Cir Bras, 2003, 18, 60–64. 6. Detata V, Novelli M, Bombara M: Determination of glycated hemoglobins in the rat: comparison between two different chromatographic methods and application in experimental diabetology. Res Exp Med (Berl), 1996, 196, 9–16. 7. Du J, Wang L, Liu L, Fan Q, Yao L, Cui Y, Kang P et al.: IFN-g suppresses the high glucose-induced increase in TGF-b1 and CTGF synthesis in mesangial cells. Pharmacol Rep, 2011, 63, 1137–1144. 8. Earle K: Variation of progression of diabetic nephropathy according to racial origin. Nephrol Dial Transplant, 2007, 16, 286–290. 9. Ende N, Chen R, Reddi A: Transplantation of human umbilical cord blood cells improves glycemia and glomerular hypertrophy in type 2 diabetic mice. Biochem Biophys Res Commun, 2004, 321, 168–171. 10. Ende N, Chen R, Reddi A: Effect of human umbilical cord blood cells on glycemia and insulitis in type 1 diabetic mice. Biochem Biophys Res Commun, 2004, 325, 665–669. 11. Erensoy N, Yýlmazer S, Öztürk M, Tunçdemir M, Uysal O, Hatemi H: Effects of ACE inhibition on the expression of type IV collagen and laminin in renal glomeruli in experimental diabetes. Acta Histochem, 2004, 106, 279–287. 12. Feige J, Gelman L, Michalik L, Desvergne B, Wahli W: From molecular action to physiological outputs: peroxisome proliferator-activated receptors are nuclear recep-
1232
Pharmacological Reports, 2012, 64, 12231233
17.
18.
19. 20. 21.
22. 23. 24. 25.
26.
27. 28. 29.
tors at the crossroads of key cellular functions. Prog Lipid Res, 2006, 45, 120–159. Feng-qin D, Hong L, Wei-min C, Jun T, Qun L, Yu R: Effects of pioglitazone on expressions of matrix metalloproteinases 2 and 9 in kidneys of diabetic rats. Chin Med J, 2004, 117, 1040–1044. Gabriella G, Luigi G, Gian V: Diabetes Mellitus: a Fundamental and Clinical Text, 3rd edn. Lippincott Williams & Wilkins, Philadelphia, 2004. Gang J, Young S, Sang Y, Mi H: pioglitazone attenuates diabetin nephropathy through anti-inflamatory mechanism. Nephrol Dial Transplant, 2007, 23, 750–760. Gellman D, Pirani C, Soothill G, Muethrcke R, Kark R: Diabetic nephropathy: clinical and pathologic studies based on renal biopsy. Med J, 1959, 38, 312–317. Gumieniczek A: Effect of the new thiazolidinedionepioglitazone on the development of oxidative stress in liver and kidney of diabetic rabbits. Life Sci, 2003, 74, 553–562. Hirasawa Y, Matsui Y, Yamane K, Yabuki SY, Kawasaki Y, Toyoshi T, Kyuki K, et al.: Pioglitazone improves obesity type diabetic nephropathy: relation to the mitigation of renal oxidative reaction. Exp Anim, 2008, 57, 423–432. Hussain M, Theise N: Stem-cell therapy for diabetes mellitus. Lancet, 2004, 364, 203–205. Iglesias P. Díez J: Peroxisome proliferator-activated receptor gamma agonists in renal disease. Eur J Endocrinol, 2006, 154, 613–621. Isshiki K, Haneda M, Koya D, Maeda S, Sugimoto T, Kikkawa R: Thiazolidinedione compounds ameliorate glomerular dysfunction independent of their insulinsensitizing action in diabetic rats. Diabetes, 2000, 49, 1022–1032. Jaatinen T, Laine J: Isolation of mononuclear cells from human cord blood by Ficoll Paque density gradient. Curr Prot Stem Cell Biol, 2007, 2A.1. Kakadiya J, Brambhatt J, Shah N: Renoprotective activity of pioglitazone on ischemia/reperfusion induced renal damage in diabetic rats. Rec Res Sci Tech, 2010, 2, 92–97. Kan C, Wu C, Zhang M, Wang L: Pioglitazone renal cortex of diabetic vascular endothelial growth factor expression. Diabetes, 2011, 48, 2229–2239. Katavetin P, Eiam-Ong S, Suwanwalaikorn S: Pioglitazone reduces urinary protein and urinary transforming growth factor-b excretion in patients with type 2 diabetes and overt nephropathy. J Med Assoc Thai, 2006, 89, 170–177. Ko GJ, Kang YS, Han SY, Lee MH, Song HK, Han KH, Kim HK et al.: Pioglitazone attenuates diabetic nephropathy through an anti-inflammatory mechanism in type 2 diabetic rats. Nephrol Dial Transplant, 2008, 23, 2750–2760. Koblas T, Harman S, Saudek F: The application of umbilical cord blood in treatment of diabetes mellitus. Rev Diabetic Stud, 2005, 2, 228–234. Kuhad A, Chopra K: Attenuation of diabetic nephropathy by tocotrienol: involvement of NF-kB signaling pathway. Life Sci, 2009, 84, 296–301. Lee G, Olson P, Hevener A: PPARd regulates glucose metabolism and insulin sensitivity. Proc Natl Acad Sci USA, 2006, 103, 3444–3449.
Pioglitazone and MNCs reduces STZ-induced diabetic nephropathy
Riham E. Masoad et al.
30. Lee R, Seo M, Reger R, Spees J, Pulin A, Prockop D: Multipotent stromal cells promote repair of pancreatic and renal glomeruli in diabetic NOD/SICD mice. Proc Natl Acad Sci USA, 2007, 103, 17438–17443. 31. Li J, Kwak SJ, Jung DS, Kim J, Yoo T, Ryu D: Podocyte biology in diabetic nephropathy. Kidney Int, 2007, 72, S36–S42. 32. Lim J, Jang J, Kim J, Moon S, Lee C, Lee K: Human umbilical cord blood mononuclear cell transplantation in rats with intrinsic sphincter deficiency. J Korean Med Sci, 2010, 25, 663–670. 33. Michalik L, Auwerx J, Berger J, Chatterjee V, Wahli W: International union of pharmacology. Peroxisome proliferator-activated receptors. Pharmacol Rev, 2006, 58, 726– 741. 34. Mizutani A, Okajima K, Uchiba M, Noguchi T: Activated protein C reduces ischemia/reperfusion-induced renal injury in rats by inhibiting leukocyte activation. Blood, 2000, 95, 3781–3787. 35. Neil H, Kyle C, Annette M, Thomas E: Cord blood in regenerative medicine: do we need immune suppression. J Transl Med, 2007, 26, 5–8. 36. Ende N, Ruifeng Chen R, Reddi AS: Transplantation of human umbilical cord blood cells improves glycemia and glomerular hypertrophy in type 2 diabetic mice. Biochem Biophys Res Commu, 2004, 321, 168–171. 37. Ohga S, Shikata K, Yozai K: Thiazolidinedione ameliorates renal injury in experimental diabetic rats through anti-inflammatory effects mediated by inhibition of NF-kB activation. Am J Physiol Renal Physiol, 2007, 292, F1141–F1150. 38. Okada T, Wada J, Hida K, Eguchi J, Hashimoto I, Baba M, Yasuhara A et al.: Thiazolidinediones ameliorate diabetic nephropathy via cell cycle–dependent mechanisms. Diabetes, 2006, 55, 1666–1677. 39. Parker J: Can stem cell transplant treat diabetes and kidney disease? Stem Cells J, 2011, 29, 38–42. 40. Peterson JC, Adler S, Burkart J: Blood pressure control, proteinuria and the progression of renal disease: the modification of diet in renal disease study. Ann Int Med, 1995, 123,754–762. 41. Sarafidis PA, Lasaridis AN, Nilsson PM, Pagkalos EM, Hitoglou-Makedou AD, Pliakos A: The effect of rosiglitazone on urine albumin excretion in patients with type 2 diabetes mellitus and hypertension. Am J Hypertens, 2005, 18, 227–234. 42. Schaeffer V, Hansen K, Morris D, Abrass C: Reduction in laminin b2 mRNA translation is responsible for impaired IGFBP-5-mediated mesangial cell migration in the presence of high glucose. Am J Physiol Renal Physiol, 2010, 298, F314–F322. 43. Sevensson M, Eriksson J, Dahlquist G: Early glycemic control, age at onset, and development of microvascular complications in childhood-onset type 1 diabetes. Diabetes Care, 2004, 27, 955–962.
44. Taneda S, Jeffery W, Pippin B: Amelioration of diabetic nephropathy in SPARC-null mice. J Am Soc Nephrol, 2003, 14, 968–980. 45. Thallas-Bonke V, Lindschau C, Rizkalla B, Bach LA, Boner G, Meier M, Haller H et al.: Attenuation of extracellular matrix accumulation in diabetic nephropathy by the advanced glycation end product cross-link breaker ALT-711 via a protein kinase C-a-dependent pathway. Diabetes, 2004, 53, 2921–2930. 46. Toblli J, Cao G, Giani J, Angerosa M, Dominici F, Gonzalez-Cadavid N: Antifibrotic effects of pioglitazone at low doses on the diabetic rat kidney are associated with the improvement of markers of cell turnover, tubular and endothelial integrity, and angiogenesis. Kidney Blood Press Res, 2011, 34, 20–33. 47. Trinder P: Determination of blood glucose using 4-aminophenazone as oxygen acceptor. J Clin Pathol, 1969, 222, 46–50. 48. Tuch B: Stem cells – a clinical update. Aust Family Phys, 2006, 35, 719–721. 49. Vijay S, Mishra M, Kumar H, Tripathi K: Effect of pioglitazone and rosiglitazone on mediators of endothelial dysfunction, markers of angiogenesis and inflammatory cytokines in type-2 diabetes. Acta Diabetol, 2006, 46, 27–33. 50. Volarevicn V, Arsenijevic E, Miodrag L: Concise review: Mesenchymal stem cell treatment of the complications of diabetes mellitus. Stem Cells J, 2011, 29, 5–10. 51. Wang X, Jiang T, Levi M: Nuclear hormone receptors in diabetic nephropathy. Nature Rev Nephrol, 2010, 6, 342–351. 52. Tse W, Laughlin M: Umbilical cord blood transplantation: A new alternative option. Hematol J, 2005, 377-383. 53. Wobus A, Boheler K: Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol Rev, 2005, 85, 635–678. 54. Xu X, Chen P, Zheng Q, Wang Y, Chen W: Effect of pioglitazone on diabetic nephropathy and expression of HIF-1a and VEGF in the renal tissues of type 2 diabetic rats. Diabetes Res Clin Pract, 2011, 93, 63–69. 55. Yozai K, Shikata K, Sasaki M, Tone A, Ohga S, Usui H, Okada S et al.: Methotrexate prevents renal injury in experimental diabetic rats via anti-inflammatory actions. J Am Soc Nephrol, 2005, 16, 3326–3338. 56. Zafiriou S, Stanner S, Saad S, Polhill T, Poronnik P, Pollock C: Pioglitazone inhibits cell growth and reduces matrix production in human kidney fibroblasts. J Am Soc Nephrol, 2005, 6, 638–645. 57. Zelmanovitz T, Gerchman F, Balthazar A, Thomazelli F, Matos J, Canani L: Diabetic nephropathy. Diabetol Metab Syndr, 2009, 1, 1–10.
Received: November 9, 2011; in the revised form: May 25, 2012; accepted: June 20, 2012.
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